WO2018141025A1

WO2018141025A1 - Reagents for treatment of ocular diseases and conditions associated with neovascularisation and use thereof

Info

Publication number: WO2018141025A1
Application number: PCT/AU2018/050075
Authority: WO
Inventors: David Suhy; Shih-Chu KAO; Peter ROELVINK
Original assignee: Benitec Biopharma Limited
Priority date: 2017-02-03
Filing date: 2018-02-05
Publication date: 2018-08-09

Abstract

This disclosure relates to RNA interference (RNAi) reagents for treatment or prevention of ocular diseases and conditions associated with neovascularisation, such as age-related macular degeneration (AMD), compositions comprising same, and use thereof to treat individuals suffering from, or predisposed to, ocular diseases and conditions associated with neovascularisation, such as AMD.

Description

Reagents for treatment of ocular diseases and conditions associated with neovascularisation and use thereof

Cross-Reference to Related Applications

This application claims the right of priority to US Provisional No. 62/454,449, filed

3 February 2017, the complete contents of which is incorporated by reference herein in its entirety.

Technical Field

The present disclosure relates to RNA interference (RNAi) reagents for treatment of ocular diseases and conditions associated with neovascularisation, such as age-related macular degeneration (AMD), compositions comprising same, and use thereof to treat individuals suffering from, or predisposed to, ocular diseases and conditions associated with neovascularisation, such as AMD.

Background

Age related macular degeneration (AMD) is the leading cause of irreversible vision loss in the United States and many other industrialised countries. "Dry" AMD is the most common type of macular degeneration and affects 90% of the people who have the condition. The dry form is characterized by the formation of drusen within the macula, a specialized structural region of the retina which capture the light that enters the eye.

Typically, drusen is formed under the retinal pigment epithelial (RPE) cells and its presence is thought to lead to atrophy of photoreceptors due to a breakdown or thinning of the RPE layer of that supports the photoreceptor cells. It is also thought that persistence of drusen within the retina leads to a persistent inflammatory reaction and results in a cascade of secondary responses that eventually can lead to wet AMD.

The "wet" form of AMD is characterized by an abnormal outgrowth of blood vessels from the vasculature situated behind the retina in a process that is often referred to as choroidal neovascularization (CNV). While not as prevalent as the dry form, it has a more rapid onset and is more severe phenotype, often leading to reduction of a substantial portion of the visual field. The current standard of care for wet AMD is Ranibizumab (RAN), a monoclonal antibody fragment with strong affinity to the vascular endothelial growth factor-A (VEGF- A), a molecular moiety secreted from cells and known to cause the formation or growth of nascent blood vessels. RAN binds to and inhibits the biologic activity of VEGF-A, thereby preventing the interaction of VEGF-A with its receptors (VEGFR1 and VEGFR2) on the surface of endothelial cells. This results in a reduction in endothelial cell proliferation, less vascular leakage, and a reduction in new blood vessel formation characteristic of CNV.

The ocular half-life of RAN, however, is only nine days following intravitreal injection, thus therapeutic doses must be administered monthly to patients to remain effective at suppressing vascular proliferation. Although useful at stabilizing visual acuity in nearly 95% of patients, improved vision was noted in only 29%-40% of patients. RAN acts as a molecular sponge to mop-up secreted VEGF-A. Inefficiencies in this process may be one reason why vision is only stabilized, not improved in most patients. In other words, it treats the symptoms but not the cause.

Another therapy approved for treatment of AMD in a number of industrialised countries is Aflibercept. Aflibercept is a recombinant fusion protein consisting of VEGF- binding portions from the extracellular domains of human VEGF receptors 1 and 2, that are fused to the Fc portion of the human IgGl immunoglobulin. As well as binding all isomers of the VEGF-A family, it also binds VEGF-B and placental growth factor.

Aflibercept is deleivered as an intravitreal injection, and because (like RAN) it has a relatively short half-life, it must be administered regularly (one injection per month for three consecutive months, followed by one injection every 2 months).

The principal drawback with existing wet AMD therapies is the requirement for frequent, continuous treatment, typically involving monthly injections into the eye.

Combined with a rapidly aging population and correspondingly low numbers of clinicians who are qualified to administer intravitreal injections, application of this therapy has placed enormous strain on healthcare systems. Thus there is clearly a need for longer lasting treatments and/or treatments that can reverse the symptoms. Alternative treatments for wet AMD have been similarly unsatisfactory, also as a result of their frequency of

administration, but as well as their side effects or poor efficacy.

AAV2-sFLT01 is a gene therapy vector that expresses a modified soluble Fltl receptor coupled to a human IgGl Fc. As a high affinity VEGF binding protein, AAV2-sFLT01 functions to neutralize the pro-angiogenic activities of VEGF for treatment of wet AMD via an intravitreal injection. (Wasworth et al. Molecular Therapy vol. 19 no. 2 Feb. 2011; 326- 334). The use of an AAV vector is expected to ensure long-term expression, lasting for many months or even years, from a single injection. However, in order to accommodate the sFLTOl and IgGl Heavy Chain Fc fusion protein, single stranded AAV must be used, which in turn requires high quantities of vector for efficient transduction and thus increases the risk of an immune response to the viral capsid proteins. Moreover, a high prevalence of the normal adult population has been exposed to serotype 2 variant of AAV, and may have preexisting immunity against it.

The molecule PF-04523655 is a 19 nucleotide siRNA that inhibits the expression of the hypoxia-inducible gene RTP801 (Nguyen et al. Ophthalmology. 2012 Sep;119(9): 1867- 73). In clinical studies conducted to date, it has been found to prevent neovascularization and vessel leakage, although does so via a different pathway than VEGF. It has been demonstrated that the siRNA only persists in the eye for several weeks, meaning that like so many of the other existing and developing therapies, patients will require regular intravitreal injections for treatment. A failure to do so with many treatments has seen a continued loss of visual acuity, and a progression of degeneration.

More generally, previous siRNA-based approaches for treating and managing wet AMD have failed. Although initial pre-clinical experimental results were encouraging, it was subsequently demonstrated that mode of action of these molecules was not through a sequence specific RNAi-based mechanism, but rather through induction of a non-specific interferon response mediated by the interaction of siRNAs with Toll-like receptor TLR3 (Kleinmann et al 2008). Toll-like receptors are transmembrane proteins that play a key role in the innate immune system. Often positioned on either the cell surface or on intracellular vesicles such as the endosome, some family members of this family recognize double stranded RNA, not normally present in the endogenous cell, as foreign substance and triggers a cascade of molecule responses. This leads to interferon activation, which has a transitory therapeutic effect in mouse models. However interferon has a much lower efficacy in humans which explains the poor efficacy of this treatment in human clinical testing.

Retinostat is an equine infectious anaemia virus (EIAV) based lentivirus vector expressing angiostatin and endostatin, both of which are naturally occurring angiogenesis inhibitors in the ocular compartment. Endostatin blocks VEGF signalling, reduces vascular permeability, decreases cell matrix adhesion and promotes endothelial cell apoptosis.

Angiostatin prevents endothelial cell proliferation and migration. The genes are delivered via a subretinal injection and inhibit the formation of new blood vessels. Sub-retinal delivery however requires an intensive surgical procedure, which, unlike intravitreal delivery, does not lend itself to outpatient treatments or treatment at a local doctor.

Despite the large amount of development activity in the field of AMD therapeutics, and wet AMD in particular, there remains a need to create more effective therapies that are also patient friendly with respect to side effects, the mode of treatment and the frequency thereof.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

Summary

The present disclosure is based, in part, on the recognition that existing therapeutic agents for treatment of AMD are limited in their efficacy, have undesirable side-effects and/or require frequent adminstration, typically involving monthly injections into the eye. On this basis, the inventors recognised a need for further therapeutic agents for treatment of AMD e.g., wet AMD, preferably with longer lasting effects and/or which can reverse the symptoms with greater efficacy and minimal side-effects. To this end, the present disclosure provides DNA-directed RNA interference (ddRNAi) constructs for expressing one or more short hairpin micro-RNAs (shmiRs) targeting conserved regions of RNA transcripts produced by genes associated with AMD, including vascular endothelial growth factor B (VEGFb) and placental growth factor (PGF). Exemplary shmiRs of the disclosure comprise effector sequences capable of inhibiting or reducing expression of VEGFb or PGF gene transcripts, and can be expressed the ddRNAi construct alone or together, including in combination with shmiRs targeting conserved regions of RNA transcripts produced by vascular endothelial growth factor A (VEGFa). One exemplary ddRNAi construct of the disclosure expresses three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF, respectively. For example,the inventors have shown that exemplary shmiRs of the disclosure comprise effector sequences which are capable of inhibiting or reducing expression of gene transcripts from the respective AMD-associated gene i.e., VEGFb, VEGFa or PGF, in vitro when expressed together from a ddRNAi construct. Specifcially, it has been shown that an exemplary ddRNAi construct expressing three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF respectively, is capable of inhibiting or reducing expression of gene transcripts from VEGFb and VEGFa independently when introduced to ARPE-19 cells (a human retinal pigment epithelial cell line). It has also been shown that the same exemplary ddRNAi construct expressing three shmiRs targeting conserved regions of RNA transcripts produced by VEGFb, VEGFa and PGF respectively, is capable of inhibiting or reducing expression of gene transcripts from PGF when introduced to JEG- cells (a human placental choriocarcinoma cell lined that expresses high levels of PGF). Thus, the inventors provide new compounds that inhibit or reduce expression of a nucleic acid and/or protein expressed by AMD-associated genes and uses of such compounds to treat a AMD in a subject.

Accordingly, the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising:

an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22. Preferably, the effector sequence will be less than 30 nucleotides in length. For example, a suitable effector sequence may be in the range of 17-29 nucleotides in length. Preferably, the effector sequence will be 20 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.

The effector sequence may comprise 6 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In another example, the effector sequence comprises 5 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In another example, the effector sequence comprises 4 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In another example, the effector sequence comprises 3 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In another example, the effector sequence comprises 2 base pair mismatches relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In another example, the effector sequence comprises 1 base pair mismatch relative to the region of equivalent length in the sequence set forth in any one of SEQ ID NOs: 1-22 to which the effector sequence is substantially complementary. In yet another example, the effector sequence is 100% complementary to a region of equivalent length within a sequence set forth in any one of SEQ ID NOs: 1-22. Where mismatches are present, it is preferred that they are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.

In one example, the nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:

VEGFb_shmiR- 1 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:23; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:25; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:27; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:29; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:31; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:33; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:35; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:37; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

VEGFb_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:39; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence; VEGFb_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:41; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:43 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:43; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:45 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:45; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:47 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:47; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:49 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:49; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:51 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:51; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:53 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:53; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:55 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:55; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:57 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:57; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:59 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:59; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:61 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:61; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence;

PGF_shmiR-l l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:63 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:63; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence; and

PGF_shmiR-12 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:65 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:65; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.

In another example, the nucleic acid described herein may comprise a DNA sequence encoding a shmiR selected from the group consisting of:

VEGFb_shmiR- 1 comprising an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 and capable of forming a duplex therewith;

VEGFb_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 and capable of forming a duplex therewith;

VEGFb_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 and capable of forming a duplex therewith;

VEGFb_shmiR-4 comprising an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 and capable of forming a duplex therewith;

VEGFb_shmiR-5 comprising an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 and capable of forming a duplex therewith

VEGFb_shmiR-6 comprising an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 and capable of forming a duplex therewith;

VEGFb_shmiR-7 comprising an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 and capable of forming a duplex therewith;

VEGFb_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 and capable of forming a duplex therewith;

VEGFb_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 and capable of forming a duplex therewith; VEGFb_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 and capable of forming a duplex therewith;

PGF_shmiR-l comprising an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 and capable of forming a duplex therewith;

PGF_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 and capable of forming a duplex therewith;

PGF_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 and capable of forming a duplex therewith;

PGF_shmiR-4 comprising an effector sequence set forth in SEQ ID NO: 50 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 and capable of forming a duplex therewith;

PGF_shmiR-5 comprising an effector sequence set forth in SEQ ID NO: 52 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 and capable of forming a duplex therewith;

PGF_shmiR-6 comprising an effector sequence set forth in SEQ ID NO: 54 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 and capable of forming a duplex therewith;

PGF_shmiR-7 comprising an effector sequence set forth in SEQ ID NO: 56 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 and capable of forming a duplex therewith;

PGF_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 and capable of forming a duplex therewith;

PGF_shmiR-9 comprising an effector sequence set forth in SEQ ID NO: 60 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 and capable of forming a duplex therewith; PGF_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 and capable of forming a duplex therewith;

PGF_shmiR-l l comprising an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 and capable of forming a duplex therewith; and

PGF_shmiR-12 comprising an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 and capable of forming a duplex therewith.

For example, the shmiR encoded by the nucleic acid described herein may comprise an effector complement sequence comprising 1, 2, 3, 4, 5 or 6 mismatches relative to the corresponding effector sequence, provided that the cognate effector and effector

complement sequences are capable of forming a duplex region.

VEGFb_shmiR- 1 comprising an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence set forth in SEQ ID NO:23;

VEGFb_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence set forth in SEQ ID NO:25;

VEGFb_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence set forth in SEQ ID NO:27;

VEGFb_shmiR-4 comprising an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence set forth in SEQ ID NO:29;

VEGFb_shmiR-5 comprising an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence set forth in SEQ ID NO:31;

VEGFb_shmiR-6 comprising an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence set forth in SEQ ID NO:33;

VEGFb_shmiR-7 comprising an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence set forth in SEQ ID NO:35;

VEGFb_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence set forth in SEQ ID NO:37; VEGFb_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence set forth in SEQ ID NO:39;

VEGFb_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence set forth in SEQ ID NO:41;

PGF_shmiR-l comprising an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence set forth in SEQ ID NO:43;

PGF_shmiR-2 comprising an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence set forth in SEQ ID NO:45;

PGF_shmiR-3 comprising an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence set forth in SEQ ID NO:47;

PGF_shmiR-4 comprising an effector sequence set forth in SEQ ID NO: 50 and an effector complement sequence set forth in SEQ ID NO:49;

PGF_shmiR-5 comprising an effector sequence set forth in SEQ ID NO: 52 and an effector complement sequence set forth in SEQ ID NO:51;

PGF_shmiR-6 comprising an effector sequence set forth in SEQ ID NO: 54 and an effector complement sequence set forth in SEQ ID NO:53;

PGF_shmiR-7 comprising an effector sequence set forth in SEQ ID NO: 56 and an effector complement sequence set forth in SEQ ID NO:55;

PGF_shmiR-8 comprising an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence set forth in SEQ ID NO:57;

PGF_shmiR-9 comprising an effector sequence set forth in SEQ ID NO:60 and an effector complement sequence set forth in SEQ ID NO:59;

PGF_shmiR-10 comprising an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence set forth in SEQ ID NO:61;

PGF_shmiR-l 1 comprising an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence set forth in SEQ ID NO:63; and

PGF_shmiR-12 comprising an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence set forth in SEQ ID NO:65.

The shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:

a 5' flanking sequence of the pri-miRNA backbone;

the effector complement sequence; the stemloop sequence;

the effector sequence; and

a 3' flanking sequence of the pri-miRNA backbone.

a 5' flanking sequence of the pri-miRNA backbone;

the effector sequence;

the stemloop sequence;

the effector complement sequence; and

a 3' flanking sequence of the pri-miRNA backbone.

Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.

Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art. For example, the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone. Preferably, however, the pri-miRNA backbone is a pri-miR-30a backbone. In accordance with an example in which the pri- miRNA backbone is a pri-miR-30a backbone, the 5' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 68 and the 3' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 69.

In one example, the nucleic acid described herein comprises a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-113. In accordance with this example, a shmiR encoded by the nucleic acid of the disclosure may comprise a sequence set forth in any one of SEQ ID NOs: 70-91. For example, a nucleic acid described herein which encodes a shmiR targeting VEGFb may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-101 and encode a shmiR comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 70-79. For example, a nucleic acid described herein which encodes a shmiR targeting PGF may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs:

102-113 and encode a shmiR comprising or consisting of a sequence set forth in any one SEQ ID NOs: 80-91. It will be understood by a person of skill in the art that effector/effector complement sequence combinations encoded by the nucleic acids described herein may also be expressed and thus provided in the form of a short hairpin RNA (shRNA). Accordingly, the present disclosure also provides a nucleic acid comprising a DNA sequence encoding a shRNA comprising:

an effector sequence of at least 17 nucleotides in length;

a stemloop sequence; and

an effector complement sequence;

wherein the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22. Preferably, the effector sequence will be less than 30 nucleotides in length. For example, a suitable effector sequence may be in the range of 17-29 nucleotides in length.

Exemplary effector and effector complement sequence combinations are described herein in the context of shmiRs of the disclosure targeting VEGFb and PGF and shall be taken to apply mutatis mutandis to each example in which a shRNA targeting the corresponding region of a transcript of VEGFb of PGF is described, including nucleic acids encoding such shRNAs.

According to any example in which a nucleic acid of the disclosure encodes a shRNA, the shRNA will comprise a stem loop sequence positioned between the effector sequence and the effector complement sequence.

In one example, the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:

the effector complement sequence;

the stemloop sequence; and

the effector sequence;

In another example, the shRNA encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:

the effector sequence;

the stemloop sequence; and

the effector complement sequence. Suitable loop sequences are described herein in the context of shmiRs and shall be taken to apply mutatis mutandis to each example in which a shRNA is described. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.

It will be also understood by a person of skill in the art that a plurality of nucleic acids in accordance with the present disclosure may be combined or used in conjunction with one or more agents for treating AMD. Accordingly, the present disclosure provides a plurality of nucleic acids comprising:

(a) at least one nucleic acid as described herein; and

(b) at least one further nucleic acid selected from:

(i) a nucleic acid in accordance with the nucleic acids described herein; or

(ii) a nucleic acid comprising a DNA sequence encoding a shmiR targeting VEGFa (VEGFa_shmiR) comprising:

an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence of the shmiR at (b)(ii) is substantially complementary to a region of equivalent length in the RNA transcript set forth in SEQ ID NO: 114;

wherein the shmiRs encoded by the nucleic acids at (a) and (b) comprise different effector sequences.

Preferably, the effector sequence of VEGFa_shmiR at (b)(ii) which is substantially complementary to a region of equivalent length in the RNA sequence set forth in SEQ ID NO: 114 will be less than 30 nucleotides in length. For example, a suitable effector sequence of the shRNA may be in the range of 17-29 nucleotides in length. Preferably, the effector sequence will be 20 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.

In one example, the effector sequence of the VEGFa_shmiR at (b)(ii) may comprise 6 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In another example, the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 5 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In another example, the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 4 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In another example, the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 3 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In another example, the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 2 base pair mismatches relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In another example, the effector sequence of VEGFa_shmiR at (b)(ii) may comprise 1 base pair mismatch relative to the sequence set forth in SEQ ID NO: 114 to which the effector sequence is substantially complementary. In yet another example, the effector sequence of VEGFa_shmiR is 100% complementary to a region of equivalent length within the sequence set forth in SEQ ID NO: 114. Where mismatches are present, it is preferred that they are not located within the region

corresponding to the seed region of VEGFa_shmiR i.e., nucleotides 2-8 of the effector sequence.

In one example, VEGFa_shmiR comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 114; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence.

In one example, VEGFa_shmiR comprises an effector sequence set forth in SEQ ID NO: 115 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 and capable of forming a duplex therewith. For example, VEGFa_shmiR may comprise an effector sequence set forth in SEQ ID NO: 115 and an effector complement sequence set forth in SEQ ID NO: 114.

In one example, the sequence of VEGFa_shmiR may comprise, in a 5' to 3' direction:

a 5' flanking sequence of the pri-miRNA backbone;

the effector complement sequence;

the stemloop sequence;

the effector sequence; and a 3' flanking sequence of the pri-miRNA backbone.

In another example, the sequence of VEGFa_shmiR may comprise, in a 5' to 3' direction::

a 5' flanking sequence of the pri-miRNA backbone;

the effector sequence;

the stemloop sequence;

the effector complement sequence; and

a 3' flanking sequence of the pri-miRNA backbone.

Any suitable loop sequence known in the art may be included in VEGFa_shmiR. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.

Likewise, the pri-miRNA backbone included in VEGFa_shmiR may be selected from those known in the art, such as those already described herein. Preferably, however, the pri-miRNA backbone is a pri-miR-30a backbone. In accordance with an example in which the pri-miRNA backbone is a pri-miR-30a backbone, the 5' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 68 and the 3 ' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 69.

In one example, VEGFa_shmiR comprises or consists of the sequence set forth in SEQ ID NO: 116. In accordance with this example, the nucleic acid coding for

VEGFa_shmiR comprises a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117.

A plurality of nucleic acids in accordance with the present disclosure may comprise up to 10 nucleic acids, each encoding a shmiR or shRNA as described herein, such as two nucleic acids or three nucleic acids or four nucleic acids or five nucleic acids or six nucleic acids or seven nucleic acids or eight nucleic acids or nine nucleic acids or ten nucleic acids. In one example, the plurality of nucleic acids comprises two nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In another example, the plurality of nucleic acids comprises three nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises four nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises five nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises six nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises seven nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises eight nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises nine nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein. In one example, the plurality of nucleic acids comprises ten nucleic acids of the disclosure, each encoding a shmiR or shRNA as described herein.

In accordance with an example in which a plurality of nucleic acids is provided, two or more of the nucleic acids may form separate parts of the same polynucleotide. In another example, two or more of the nucleic acids in the plurality form parts of different

polynucleotides, respectively.

The or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, one or more transcriptional terminator sequences. For example, the or each nucleic acid may comprise a transcriptional terminator sequence at the 3 ' terminus of the sequence encoding the shmiR or shRNA. Such sequences will depend on the choice of promoter and will be known to a person of skill in the art. For example, where a nucleic acid of the disclosure is in operable linkage with a RNA pol III promoter, a transcriptional terminator sequence may include 'χχχχχ' _or ' XXXXXX'.

Alternatively, or in addition, the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, a transcription initiator sequence. Suitable transcription initiator sequences will be known to a skilled person.

Alternatively, or in addition, the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors. For example, the nucleic acids described herein may include a restriction site upstream and/or downstream of the DNA sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art.

In one example, the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting PGF. Exemplary nucleic acids encoding shmiRs targeting VEGFb and PGF respectively are described herein and shall be taken to apply mutatis mutandis to this example. For example, the shmiR targeting VEGFb may be selected from VEGFb_shmiR-l, VEGFb_shmiR-4 and VEGFb_shmiR-10 and the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l and a nucleic acid encoding PGF_shmiR-3.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding

VEGFb_shmiR-l and a nucleic acid encoding PGF_shmiR-7.

In another example, the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting VEGFa. Exemplary nucleic acids encoding shmiRs targeting VEGFb and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example. For example, the shmiR targeting VEGFb may be selected from VEGFb_shmiR- 1, VEGFb_shmiR-4 and VEGFb_shmiR-10 and the shmiR targeting VEGFa is

VEGFa_shmiR.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l and a nucleic acid encoding VEGFa_shmiR.

In another example, the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting PGF and at least one nucleic acid encoding a shmiR targeting VEGFa. Exemplary nucleic acids encoding shmiRs targeting PGF and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example. For example, the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10 and the shmiR targeting VEGFa is VEGFa_shmiR.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding PGF_shmiR-3 and a nucleic acid encoding VEGFa_shmiR.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding PGF_shmiR-7 and a nucleic acid encoding VEGFa_shmiR.

In another example, the plurality of nucleic acids comprises at least one nucleic acid encoding a shmiR targeting VEGFb, at least one nucleic acid encoding a shmiR targeting VEGFa, and at least one nucleic acid encoding a shmiR targeting PGF. Exemplary nucleic acids encoding shmiRs targeting VEGFb, PGF and VEGFa respectively are described herein and shall be taken to apply mutatis mutandis to this example. For example, the shmiR targeting VEGFb may be selected from VEGFb_shmiR-l, VEGFb_shmiR-4 and VEGFb_shmiR-10, the shmiR targeting VEGFa is VEGFa_shmiR, and the shmiR targeting PGF may be selected from PGF_shmiR-3, PGF_shmiR-7 and PGF_shmiR-10.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l, a nucleic acid encoding VEGFa_shmiR, and a nucleic acid encoding PGF_shmiR-3.

In one example, the plurality of nucleic acids comprises a nucleic acid encoding VEGFb_shmiR-l, a nucleic acid encoding VEGFa_shmiR, and a nucleic acid encoding PGF_shmiR-7.

A nucleic acid in accordance with the present disclosure may also be provided in the form of, or be comprised in, a DNA-directed RNA interference (ddRNAi) construct which is capable of expressing one or more shmiRs or shRNAs which is/are encoded by the nucleic acid(s) of the present disclosure. In this regard, one or more ddRNAi constructs comprising a nucleic acid of the disclosure is also provided.

In another example, a plurality of ddRNAi constructs, each comprising a nucleic acid encoding a shmiR or shRNA as described herein is provided, wherein:

(a) at least one of the plurality of ddRNAi constructs comprises a first nucleic acid of the plurality of nucleic acids as described herein; and

(b) at least one of the plurality of ddRNAi constructs comprises a second nucleic acid of the plurality of nucleic acids described herein; and

wherein the first and second nucleic acids encode shmiRs or shRNAs comprising effector sequences that are different to one another. Preferably, the effector sequences of the respective shmiRs or shRNAs target different gene transcripts.

The plurality of ddRNAi constructs described herein may comprise up to 10 ddRNAi constructs, each comprising one or more nucleic acids encoding a shmiR or shRNA as described herein, such as two ddRNAi constructs or three ddRNAi constructs or four ddRNAi constructs or five ddRNAi constructs or six ddRNAi constructs or seven ddRNAi constructs or eight ddRNAi constructs or nine ddRNAi constructs or ten ddRNAi constructs of the disclosure.

In yet another example, a ddRNAi construct of the disclosure comprises a plurality of nucleic acids as described herein, and thus encodes a plurality of shmiRs or shRNA targeting AMD-associated genes, wherein each of the shmiRs or shRNA are different to one another. Preferably, the effector sequences of the respective shmiRs or shRNAs target different gene transcripts.

In one example, the ddRNAi construct comprises at least two nucleic acids of the disclosure, wherein each of the nucleic acids encode different shmiRs.

One exemplary ddRNAi construct comprising a plurality of nucleic acids of the disclosure comprises, preferably in a 5' to 3' direction:

(a) a nucleic acid encoding VEGFb_shmiR- 1 ;

(b) a nucleic acid encoding VEGFa_shmiR; and

(c) a nucleic acid encoding PGF_shmiR-3.

In one example, the ddRNAi construct comprises, preferably in a 5' to 3' direction:

(a) a nucleic acid encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70:

(b) a nucleic acid encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116; and

(c) a nucleic acid encoding a PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82.

In one example, the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92: (b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104.

Another exemplary ddRNAi construct comprising a plurality of nucleic acids of the disclosure comprises, preferably in a 5' to 3' direction:

(a) a nucleic acid encoding VEGFb_shmiR- 1 ;

(b) a nucleic acid encoding VEGFa_shmiR; and

(c) a nucleic acid encoding PGF_shmiR-7.

(b) a nucleic acid encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116; and (c) a nucleic acid encoding a PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86.

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108.

In one example, a ddRNAi construct as described herein comprises a single promoter which is operably-linked to the or each nucleic acid encoding a shmiR or shRNA of the disclosure. However, it is preferred that each nucleic acid encoding a shmiR or shRNA of the disclosure is operably-linked to a separate promoter.

For example, the promoter(s) is(are) positioned upstream of the respective nucleic acid(s) encoding the shmiR(s) or shRNA(s). In a ddRNAi construct comprising multiple promoters, the promoters may be the same or different. Exemplary promoters are RNA pol

III promoters, such as for example, the U6 and HI promoters. Exemplary U6 promoters are

U6-1, U6-8 and U6-9 promoters.

(a) U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR- 1 ;

(b) U6-1 promoter upstream of a nucleic acid encoding VEGFa_shmiR; and

(c) U6-8 promoter upstream of a nucleic acid encoding PGF_shmiR-3.

For example, the ddRNAi construct may comprise, preferably in a 5' to 3' direction:

(a) U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70:

(b) U6-1 promoter upstream of a nucleic acid encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116; and

(c) U6-8 promoter upstream of a nucleic acid encoding a PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO: 82.

(a) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92: (b) U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104.

In another example, the ddRNAi construct of the disclosure comprises, preferably in a

5' to 3' direction:

(a) U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR- 1 ;

(b) U6-1 promoter upstream of a nucleic acid encoding VEGFa_shmiR; and

(c) U6-8 promoter upstream of a nucleic acid encoding PGF_shmiR-7.

(c) U6-8 promoter upstream of a nucleic acid encoding a PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO: 86.

For example, the ddRNAi construct of the disclosure may comprise, preferably in a 5' to 3' direction:

(a) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:

(b) U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108.

In any of the foregoing example, one or more of the promoters may be operably-linked to a proximal sequence element 7 (PSE7) i.e., rather than the proximal sequence element corresponding to the respective promoter.

Accordingly, in one example, the ddRNAi construct of the disclosure comprises, preferably in a 5' to 3' direction:

(a) PSE7 and a U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR-l;

(b) PSE7 and a U6-1 promoter upstream of a nucleic acid encoding VEGFa_shmiR; and

(c) PSE7 and a U6-8 promoter upstream of a nucleic acid encoding PGF_shmiR-3. For example, the ddRNAi construct may comprise, preferably in a 5' to 3' direction:

(a) PSE7 and a U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR-l comprising or consisting of the sequence set forth in SEQ ID NO:70:

(b) PSE7 and a U6-1 promoter upstream of a nucleic acid encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116; and

(c) PSE7 and a U6-8 promoter upstream of a nucleic acid encoding a PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO: 82.

(a) PSE7 and a U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:

(b) PSE7 and a U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) PSE7 and a U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104.

5' to 3' direction:

(a) PSE7 and a U6-9 promoter upstream of a nucleic acid encoding VEGFb_shmiR-l;

(c) PSE7 and a U6-8 promoter upstream of a nucleic acid encoding PGF_shmiR-7.

(c) PSE7 and a U6-8 promoter upstream of a nucleic acid encoding a PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO: 86.

(b) PSE7 and a U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and (c) PSE7 and a U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108.

The present disclosure also provides an expression vector, comprising a ddRNAi construct of the disclosure.

The present disclosure also provides plurality of expression vectors each of which comprises a ddRNAi construct of the disclosure. For example, one or more of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein. In another example, each of the plurality of expression vectors comprises a plurality of ddRNAi constructs as disclosed herein. In a further example, each of the plurality of expression vectors comprises a single ddRNAi construct as described herein. In any of the foregoing ways in this paragraph, the plurality of expression vectors may collectively express a plurality of shmiRs or shRNAs in accordance with the present disclosure.

In one example, the or each expression vector is a plasmid or a minicircle.

In one example, the plasmid or minicircle or expression vector or ddRNAi construct is complexed with a cationic DNA binding polymer.

In another example, the or each expression vector is a viral vector. For example, the viral vector is selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral vector (AdV) and a lentiviral (LV) vector.

The present disclosure also provides a composition comprising a ddRNAi construct and/or a plurality of ddRNAi constructs and/or expression vector and/or a plurality of expression vectors as described herein. In one example, the composition may also comprise one or more pharmaceutically acceptable carriers and/or diluents.

The present disclosure also provides a method of treating or preventing an ocular disease or disorder characterised by, or associated with, undesired neovascularization, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject.

In one example, the ocular disease or disorder to be treated or prevented is selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy,

Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, Purtscher's retinopathy and Retinal Vein Occlusion (RVO).

In one example, the disclosure provides a method of treating age-related macular degeneration (AMD) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject. In one example, the AMD is wet AMD. In one example, the AMD is dry AMD. In accordance with an example in which the AMD is dry AMD, treatment may comprise arresting or slowing progression of dry AMD to wet AMD.

In one example, the disclosure provides a method of treating diabetic retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating Diabetic Macular Edema (DME) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of reducing or inhibiting corneal neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

The present disclosure also provides a method of reducing or inhibiting choroidal neovascularisation (CNV) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein to the subject. In one example, the subject who is in need of reduction or inhibition of CNV is suffering from AMD, developing AMD or predisposed to AMD. For example, the AMD may be wet AMD.

In one example, the disclosure provides a method of treating cyclitis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating Hippel-Lindau Disease in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating retinopathy of prematurity in a subject, said method comprising administering to the subject a

therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating pterygium in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating histoplasmosis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of reducing or inhibiting iris neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating macular edema in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating glaucoma-associated neovascularization in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating Purtscher's retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating or preventing retinal vein occlusion (RVO) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In accordance with any method described herein, the administration to the subject is by intravitreal injection or subretinal injection.

In accordance with any method described herein, administration of the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, the plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition described herein to the subject is performed in conjunction with one or more other treatments known to be suitable for treatment of the ocular disease or condition associated with or

characterised by neovascularisation i.e., as an adjunctive therapy. For example, the administration may occur in combination with administration of one or more other agents known for treatment of AMD e.g., ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.

In one example, a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure is provided in the form of a kit. For example, a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure may be packaged together with one or more other therapeutic agents known for treating the ocular disease or condition associated with, or characterised by, neovascularisation as described herein. Such other therapeutic agents will be known to a person of skill in the art.

Alternatively, or in addition, a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition of the present disclosure may be packaged with instruction for use in a method of the disclosure.

The present disclosure also provides use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing an ocular disease or disorder characterised by, or associated with, undesired neovascularization. In one example, the ocular disease or disorder to be treated or prevented is selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated

neovascularization, Purtscher's retinopathy and Retinal Vein Occlusion (RVO).

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing AMD. In one example, the AMD is wet AMD. In another example, the AMD is dry AMD. In accordance with an example in which the AMD is dry AMD, treatment or prevention may comprise arresting or slowing progression of dry AMD to wet AMD. In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing diabetic retinopathy in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Diabetic Macular Edema (DME) in a subject in need thereof.

In one example, the disclosure also provides use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for reducing or inhibiting choroidal neovascularisation (CNV) in a subject in need thereof. . For example, a subject who is in need of reduction or inhibition of CNV may be suffering from AMD, developing AMD or predisposed to AMD. For example, the AMD may be wet AMD.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing cyclitis in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Hippel-Lindau Disease in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing retinopathy of prematurity in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing pterygium in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing histoplasmosis in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing iris neovascularization in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing macular edema in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing glaucoma-associated neovascularization in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing Purtscher's retinopathy in a subject in need thereof.

In one example, the disclosure provides for use of a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or a composition described herein in the preparation of a medicament for treating or preventing retinal vein occlusion (RVO) in a subject in need thereof.

The present disclosure also provides a nucleic acid, a plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors and/or composition described herein for use in therapy of an ocular disease or condition associated with, or characterised by, neovascularisation. For example, the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in treating or preventing an ocular disease or disorder selected from the group consisting of AMD e.g., wet AMD or dry AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, Purtscher's retinopathy and Retinal Vein Occlusion (RVO).

In one example, the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in treating or preventing AMD in a subject e.g., wet AMD.

In one example, the nucleic acid, the plurality of nucleic acids, the ddRNAi construct, a plurality of ddRNAi constructs, the expression vector, the plurality of expression vectors and/or the composition may be for use in reducing or inhibiting CNV in a subject, such as in a subject suffering from AMD, who is developing AMD or who is predisposed to AMD. For example, wet AMD.

Brief description of the figures

Figure 1 shows (A) a map of the of pSilencer 2.1-U6 hygro vector, (B) an insert of VEGFb shmiR-1 in such construct, and (C) a predicted RNA folding structure of VEGFb shmiR-1.

Figure 2 illustrates: (A) strand preference activities of VEGFb shmiRs in a lucif erase reporter assay; and (B) strand preference activities of PGF shmiRs in a luciferase reporter assay. shmiRs showing higher anti-sense strand activities over sense strand activities were identified as preferred drug candidates.

Figure 3 illustrates: (A) hyperfunctional activities of selective VEGFb shmiRs; and (B) hyperfunctional activities of selective PGF shmiRs. VEGFb and PGF shmiRs inhibited luciferase protein expression in a dose dependent manner. Figure 4 shows down-regulation of endogenous VEGFb and PGF target expression by selected single shmiR constructs: (A) VEGFb shmiR knockdown activities in ARPE-19 cells; and (B) PGF shmiR knockdown activities in BeWo cells. Precent inhibition was calculated as relative activities to pSilencer negative control. Figures 5 provides an schematic of four ddRNAi constructs expressing three different shmiRs under control of three separate U6 promoters for simultaneously down regulation of VEGFa, VEGFb and PGF expression.

Figure 6 illustrates that when expressed by a triple construct, VEGFa shmiR-8 was capable of inhibiting VEGFa target expression in a dose dependent manner in ARPE-19 cells: (A) VEGFa mRNA inhibition levels determined using RT-qPCR analysis; (B) VEGFa protein inhibition levels analyzed using ELISA assay; and (C) VEGFa shmiR-8 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.

Figure 7 illustrates that when expressed by a triple construct, VEGFb shmiR- 1 efficiently inhibited VEGFb target expression in ARPE-19 cells; (A) VEGFb mRNA inhibition levels determined using RT-qPCR analysis and (b) VEGFb shmiR- 1 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.

Figure 8 illustrates that when expressed by a triple construct, PGF shmiR-3 and shmiR-7 could both independently down-regulate expression of endogenous PGF in JEG-3 cells: (A) PGF mRNA inhibition levels determined using RT-qPCR analysis; and (B, C) PGF shmiR-3 and PGF shmiR-7 expression levels analyzed using RT-qPCR assay. Precent inhibition was calculated as relative activities to TT034 negative control.

Figure 9 illustrates that there is high level expression of all three shmiRs (VEGFa- shmiR-8, VEGFb-shmiR-1 and PGF-shmiR-7) in retina layer of the eye following delivery of the AAV-based ddRNAi construct, CapVarl-BB201, to retina tissues. This figure also shows that expression of the shmiRs was detectable in RPE/Choroid layer. Key to the Sequence Listing

SEQ ID NO: 1 Target region 1 w thin RNA transcript of VEGFb (VEGFb- 1). SEQ ID NO: 2 Target region 2 w thin RNA transcript of VEGFb (VEGFb-2). SEQ ID NO: 3 Target region 3 w thin RNA transcript of VEGFb (VEGFb-3). SEQ ID NO: 4 Target region 4 w thin RNA transcript of VEGFb (VEGFb-4). SEQ ID NO: 5 Target region 5 w thin RNA transcript of VEGFb (VEGFb-5). SEQ ID NO: 6 Target region 6 w thin RNA transcript of VEGFb (VEGFb-6). SEQ ID NO: 7 Target region 7 w thin RNA transcript of VEGFb (VEGFb-7). SEQ ID NO: 8 Target region 8 w thin RNA transcript of VEGFb (VEGFb-8). SEQ ID NO: 9 Target region 9 w thin RNA transcript of VEGFb (VEGFb-9). SEQ ID NO: 10 Target region 10 within RNA transcript of VEGFb (VEGFb-10). SEQ ID NO: 11 Target region 1 w thin RNA transcript of PGF (PGF-1).

SEQ ID NO: 12 Target region 2 w thin RNA transcript of PGF (PGF-2).

SEQ ID NO: 13 Target region 3 w thin RNA transcript of PGF (PGF-3).

SEQ ID NO: 14 Target region 4 w thin RNA transcript of PGF (PGF-4).

SEQ ID NO: 15 Target region 5 w thin RNA transcript of PGF (PGF-5).

SEQ ID NO: 16 Target region 6 w thin RNA transcript of PGF (PGF-6).

SEQ ID NO: 17 Target region 7 w thin RNA transcript of PGF (PGF-7).

SEQ ID NO: 18 Target region 8 w thin RNA transcript of PGF (PGF-8).

SEQ ID NO: 19 Target region 9 w ithin RNA transcript of PGF (PGF-9).

SEQ ID NO: 20 Target region 10 within RNA transcript of PGF (PGF- 10).

SEQ ID NO: 21 Target region 11 within RNA transcript of PGF (PGF-11).

SEQ ID NO: 22 Target region 12 within RNA transcript of PGF (PGF- 12).

SEQ ID NO: 23 RNA effector complement sequence for shmiR designated

VEGFb_shmiR-l.

SEQ ID NO: 24: RNA effector sequence for shmiR designated VEGFb_shmiR- 1. SEQ ID NO: 25: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-2.

SEQ ID NO: 26: RNA effector sequence for shmiR designated VEGFb_shmiR-2. SEQ ID NO: 27: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-3.

SEQ ID NO: 28: RNA effector sequence for shmiR designated VEGFb_shmiR-3. SEQ ID NO: 29: RNA effector complement sequence for shmiR designated VEGFb_shmiR-4.

SEQ ID NO: 30: RNA effector sequence for shmiR designated VEGFb_shmiR-4.

SEQ ID NO: 31 : RNA effector complement sequence for shmiR designated

VEGFb_shmiR-5.

SEQ ID NO: 32: RNA effector sequence for shmiR designated VEGFb_shmiR-5.

SEQ ID NO: 33: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-6.

SEQ ID NO: 34: RNA effector sequence for shmiR designated VEGFb_shmiR-6.

SEQ ID NO: 35: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-7.

SEQ ID NO: 36: RNA effector sequence for shmiR designated VEGFb_shmiR-7.

SEQ ID NO: 37: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-8.

SEQ ID NO: 38: RNA effector sequence for shmiR designated VEGFb_shmiR-8.

SEQ ID NO: 39: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-9.

SEQ ID NO: 40: RNA effector sequence for shmiR designated VEGFb_shmiR-9.

SEQ ID NO: 41: RNA effector complement sequence for shmiR designated

VEGFb_shmiR-10.

SEQ ID NO: 42: RNA effector sequence for shmiR designated VEGFb_shmiR- 10.

SEQ ID NO: 43: RNA effector complement sequence for shmiR designated PGF_shmiR- 1.

SEQ ID NO: 44: RNA effector sequence for shmiR designated PGF_shmiR-l.

SEQ ID NO: 45: RNA effector complement sequence for shmiR designated PGF_shmiR-

2.

SEQ ID NO: 46: RNA effector sequence for shmiR designated PGF_shmiR-2.

SEQ ID NO: 47: RNA effector complement sequence for shmiR designated PGF_shmiR- 3.

SEQ ID NO: 48: RNA effector sequence for shmiR designated PGF_shmiR-3.

SEQ ID NO: 49: RNA effector complement sequence for shmiR designated PGF_shmiR- 4. SEQ ID NO: 50: RNA effector sequence for shmiR designated PGF_shmiR-4.

SEQ ID NO: 51: RNA effector complement sequence for shmiR designated PGF_shmiR-

5.

SEQ ID NO: 52: RNA effector sequence for shmiR designated PGF_shmiR-5.

SEQ ID NO: 53: RNA effector complement sequence for shmiR designated PGF_shmiR-

6.

SEQ ID NO: 54: RNA effector sequence for shmiR designated PGF_shmiR-6.

SEQ ID NO: 55: RNA effector complement sequence for shmiR designated PGF_shmiR-

7.

SEQ ID NO: 56: RNA effector sequence for shmiR designated PGF_shmiR-7.

SEQ ID NO: 57: RNA effector complement sequence for shmiR designated PGF_shmiR-

8.

SEQ ID NO: 58: RNA effector sequence for shmiR designated PGF_shmiR-8.

SEQ ID NO: 59: RNA effector complement sequence for shmiR designated PGF_shmiR-

9.

SEQ ID NO: 60: RNA effector sequence for shmiR designated PGF_shmiR-9.

SEQ ID NO: 61: RNA effector complement sequence for shmiR designated PGF_shmiR-

10.

SEQ ID NO: 62: RNA effector sequence for shmiR designated PGF_shmiR-10.

SEQ ID NO: 63: RNA effector complement sequence for shmiR designated PGF_shmiR-

11.

SEQ ID NO: 64: RNA effector sequence for shmiR designated PGF_shmiR-l 1.

SEQ ID NO: 65: RNA effector complement sequence for shmiR designated PGF_shmiR-

12.

SEQ ID NO: 66: RNA effector sequence for shmiR designated PGF_shmiR-12.

SEQ ID NO: 67: stemloop RNA sequence for shmiRs

SEQ ID NO: 68: 5' flanking sequence of pri-miR-30a backbone.

SEQ ID NO: 69: 3' flanking sequence of pri-miR-30a backbone.

SEQ ID NO: 70: RNA sequence for shmiR designated VEGFb_shmiR-l.

SEQ ID NO: 71: RNA sequence for shmiR designated VEGFb_ shmiR-2.

SEQ ID NO: 72: RNA sequence for shmiR designated VEGFb_shmiR-3.

SEQ ID NO: 73: RNA sequence for shmiR designated VEGFb_shmiR-4. SEQ ID NO: 74: RNA sequence for shmiR designated VEGFb_shmiR-5.

SEQ ID NO: 75: RNA sequence for shmiR designated VEGFb_shmiR-6.

SEQ ID NO: 76: RNA sequence for shmiR designated VEGFb_shmiR-7.

SEQ ID NO: 77: RNA sequence for shmiR designated VEGFb_shmiR-8.

SEQ ID NO: 78: RNA sequence for shmiR designated VEGFb_shmiR-9.

SEQ ID NO: 79: RNA sequence for shmiR designated VEGFb_shmiR-10.

SEQ ID NO: 80: RNA sequence for shmiR designated PGF_shmiR-l.

SEQ ID NO: 81: RNA sequence for shmiR designated PGF_shmiR-2.

SEQ ID NO: 82: RNA sequence for shmiR designated PGF_shmiR-3.

SEQ ID NO: 83: RNA sequence for shmiR designated PGF_shmiR-4.

SEQ ID NO: 84: RNA sequence for shmiR designated PGF_shmiR-5.

SEQ ID NO: 85: RNA sequence for shmiR designated PGF_shmiR-6.

SEQ ID NO: 86: RNA sequence for shmiR designated PGF_shmiR-7.

SEQ ID NO: 87: RNA sequence for shmiR designated PGF_shmiR-8.

SEQ ID NO: 88: RNA sequence for shmiR designated PGF_shmiR-9.

SEQ ID NO: 89: RNA sequence for shmiR designated PGF_shmiR-10.

SEQ ID NO: 90: RNA sequence for shmiR designated PGF_shmiR-l 1.

SEQ ID NO: 91: RNA sequence for shmiR designated PGF_shmiR-12.

SEQ ID NO: 92: DNA sequence coding for shmiR designated VEGFb_shmiR- 1.

SEQ ID NO: 93: DNA sequence coding for shmiR designated VEGFb_shmiR- 2.

SEQ ID NO: 94: DNA sequence coding for shmiR designated VEGFb_shmiR- 3.

SEQ ID NO: 95: DNA sequence coding for shmiR designated VEGFb_shmiR- 4.

SEQ ID NO: 96: DNA sequence coding for shmiR designated VEGFb_shmiR- 5.

SEQ ID NO: 97: DNA sequence coding for shmiR designated VEGFb_shmiR- 6.

SEQ ID NO: 98: DNA sequence coding for shmiR designated VEGFb_shmiR- 7.

SEQ ID NO: 99: DNA sequence coding for shmiR designated VEGFb_shmiR- 8.

SEQ ID NO: 100: DNA sequence coding for shmiR designated VEGFb_shmiR- 9.

SEQ ID NO: 101: DNA sequence coding for shmiR designated VEGFb_shmiR- 10.

SEQ ID NO: 102: DNA sequence coding for shmiR designated PGF_shmiR-l.

SEQ ID NO: 103: DNA sequence coding for shmiR designated PGF_shmiR-2.

SEQ ID NO: 104: DNA sequence coding for shmiR designated PGF_shmiR-3.

SEQ ID NO: 105: DNA sequence coding for shmiR designated PGF_shmiR-4. SEQ ID NO: 106: DNA sequence coding for shmiR designated PGF_shmiR-5.

SEQ ID NO: 107: DNA sequence coding for shmiR designated PGF_shmiR-6.

SEQ ID NO: 108: DNA sequence coding for shmiR designated PGF_shmiR-7.

SEQ ID NO: 109: DNA sequence coding for shmiR designated PGF_shmiR-8.

SEQ ID NO: 110: DNA sequence coding for shmiR designated PGF_shmiR-9.

SEQ ID NO: 111: DNA sequence coding for shmiR designated PGF_shmiR-10.

SEQ ID NO: 112: DNA sequence coding for shmiR designated PGF_shmiR-l l.

SEQ ID NO: 113: DNA sequence coding for shmiR designated PGF_shmiR-12.

SEQ ID NO: 114: RNA effector complement sequence for shmiR designated

VEGFa_shmiR.

SEQ ID NO: 115: RNA effector sequence for shmiR designated VEGFa_shmiR.

SEQ ID NO: 116: RNA sequence for shmiR designated VEGFa_shmiR.

SEQ ID NO: 117: DNA sequence coding for shmiR designated VEGFa_shmiR.

SEQ ID NO: 118: DNA sequence for primer designated VEGFb-shmiRl_fwd primer seq.

SEQ ID NO: 119: DNA sequence for primer designated VEGFa-shmiR8_fwd primer seq.

SEQ ID NO: 120: DNA sequence for primer designated PGF-shmiR7_fwd primer seq.

SEQ ID NO: 121: DNA sequence for primer designated PGF-shmiR3_fwd primer seq.

SEQ ID NO: 122: DNA sequence for primer designated VEGFb-shmiRl_RNA standard oligo seq.

SEQ ID NO: 123: DNA sequence for primer designated VEGFa-shmIR8_RNA standard oligo seq.

SEQ ID NO: 124: DNA sequence for primer designated PGF-shmiR7_RNA standard olig seq.

SEQ ID NO: 125: DNA sequence for primer designated PGF-shmiR3_RNA standard olig seq.

Detailed Description

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology,

immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

Throughout this specification, unless the context requires otherwise, the word

"comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Selected Definitions

By "RNA" is meant a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a β-D-ribo- furanose moiety. The terms include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly- produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the instant disclosure can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

The term "RNA interference" or "RNAi" refers generally to RNA-dependent silencing of gene expression initiated by double stranded RNA (dsRNA) molecules in a cell's cytoplasm. The dsRNA molecule reduces or inhibits transcription products of a target nucleic acid sequence, thereby silencing the gene or reducing expression of that gene.

As used herein, the term "double stranded RNA" or "dsRNA" refers to a RNA molecule having a duplex structure and comprising an effector sequence and an effector complement sequence which are of similar length to one another. The effector sequence and the effector complement sequence can be in a single RNA strand or in separate RNA strands. The "effector sequence" (often referred to as a "guide strand") is substantially complementary to a region of a target sequence, which in the present case, is a transcript of VEGFb, PGF or VEGFa. The "effector sequence" can also be referred to as the "antisense sequence". The "effector complement sequence" will be of sufficient complementary to the effector sequence such that it can anneal to the effector sequence to form a duplex. In this regard, the effector complement sequence will be substantially homologous to a region of target sequence. As will be apparent to the skilled person, the term "effector complement sequence" can also be referred to as the "complement of the effector sequence" or the sense sequence.

As used herein, the term "duplex" refers to regions in two complementary or substantially complementary nucleic acids (e.g., RNAs), or in two complementary or substantially complementary regions of a single- stranded nucleic acid (e.g., RNA), that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a stabilized duplex between the nucleotide sequences that are complementary or substantially complementary. It will be understood by the skilled person that within a duplex region, 100% complementarity is not required; substantial complementarity is allowable. Substantial complementarity includes may include 69% or greater

complementarity. For example, a single mismatch in a duplex region consisting of 19 base pairs (i.e., 18 base pairs and one mismatch) results in 94.7% complementarity, rendering the duplex region substantially complementary. In another example, two mismatches in a duplex region consisting of 19 base pairs (i.e., 17 base pairs and two mismatches) results in 89,5% complementarity, rendering the duplex region substantially complementary. In yet another example, three mismatches in a duplex region consisting of 19 base pairs (i.e., 16 base pairs and three mismatches) results in 84.2% complementarity, rendering the duplex region substantially complementary, and so on.

The dsRNA may be provided as a hairpin or stem loop structure, with a duplex region comprised of an effector sequence and effector complement sequence linked by at least 2 nucleotide sequence which is termed a stem loop. When a dsRNA is provided as a hairpin or stem loop structure it can be referred to as a "hairpin RNA" or "short hairpin RNAi agent" or "shRNA".

Other dsRNA molecules provided in, or which give rise to, a hairpin or stem loop structure include primary miRNA transcripts (pri-miRNA) and precursor microRNA (pre- miRNA). Pre-miRNA shRNAs can be naturally produced from pri-miRNA by the action of the enzymes Drosha and Pasha which recognize and release regions of the primary miRNA transcript which form a stem-loop structure. Alternatively, the pri-miRNA transcript can be engineered to replace the natural stem-loop structure with an artificial/recombinant stem- loop structure. That is, an artificial/recombinant stem-loop structure may be inserted or cloned into a pri-miRNA backbone sequence which lacks its natural stem-loop structure. In the case of stemloop sequences engineered to be expressed as part of a pri-miRNA molecule, Drosha and Pasha recognize and release the artificial shRNA. dsRNA molecules produced using this approach are known as "shmiRNAs", "shmiRs" or "microRNA framework shRNAs".

As used herein, the term "complementary" with regard to a sequence refers to a complement of the sequence by Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with either uracil (U) or thymine (T). A sequence may be complementary to the entire length of another sequence, or it may be complementary to a specified portion or length of another sequence. One of skill in the art will recognize that U may be present in RNA, and that T may be present in DNA. Therefore, an A within either of a RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence.

As used herein, the term "substantially complementary" is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between nucleic acid sequences e.g., between the effector sequence and the effector complement sequence or between the effector sequence and the target sequence. It is understood that the sequence of a nucleic acid need not be 100% complementary to that of its target or complement. The term encompasses a sequence complementary to another sequence with the exception of an overhang. In some cases, the sequence is complementary to the other sequence with the exception of 1-2 mismatches. In some cases, the sequences are complementary except for 1 mismatch. In some cases, the sequences are complementary except for 2 mismatches. In other cases, the sequences are complementary except for 3 mismatches. In yet other cases, the sequences are complementary except for 4 mismatches.

The term "encoded", as used in the context of a shRNA or shmiR of the disclosure, shall be understood to mean a shRNA or shmiR which is capable of being transcribed from a DNA template. Accordingly, a nucleic acid that encodes a shRNA or shmiR of the disclosure will comprise a DNA sequence which serves as a template for transcription of the respective shRNA or shmiR.

The term "DNA-directed RNAi construct" or "ddRNAi construct" refers to a nucleic acid comprising DNA sequence which, when transcribed produces a shRNA or shmiR molecule which elicits RNAi. The ddRNAi construct may comprise a nucleic acid which is transcribed as a single RNA that is capable of self-annealing into a hairpin structure with a duplex region linked by a stem loop of at least 2 nucleotides i.e., shRNA or shmiR, or as a single RNA with multiple shRNAs or shmiRs, or as multiple RNA transcripts each capable of folding as a single shRNA or shmiR respectively. The ddRNAi construct may be within an expression vector i.e., "ddRNAi expression construct", e.g., operably-linked to a promoter.

As used herein, the term "operably-linked" or "operable linkage" (or similar) means that a. coding nucleic acid sequence is linked to, or in association with, a regulatory sequence, e.g., a promoter, in a manner which facilitates expression of the coding sequence. Regulatory sequences include promoters, enhancers, and other expression control elements that are art-recognized and are selected to direct expression of the coding sequence.

A "vector" will be understood to mean a vehicle for introducing a nucleic acid into a cell. Vectors include, but are not limited to, p!asmids, phagemids, viruses, bacteria, and vehicles derived from viral or bacterial sources. A "plasmid" is a circular, double-stranded DNA molecule. A useful type of vector for use in accordance with the present disclosure is a viral vector, wherein heterologous DN A sequences are inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof. Certain vectors are capable of autonomous replication in a host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be stably integrated into the genome of a host cell, and are thereby replicated along with the host genome. As used herein, the term "expression vector" will be understood to mean a vector capable of expressing a RNA molecule of the disclosure.

As used herein, the terms "treating", "treat" or "treatment" and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.

A "therapeutically effective amount" is at least the minimum concentration or amount required to effect a measurable improvement of a particular disease or disorder (e.g., an ocular disease or disorder characterised by, or associated with, neovascularisation, such as AMD). A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the shmiR, nucleic acid encoding same, ddRNAi or expression construct to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the shRNA or shmiR, nucleic acid encoding same, ddRNAi or expression construct are outweighed by the therapeutically beneficial effects.

As used herein, the "subject" or "patient" can be a human or non-human animal suffering from or predisposed to an ocular disease or disorder characterised by, or associated with, neovascularisation, such as AMD. The "non-human animal" may be a primate, livestock (e.g. sheep, horses, cattle, pigs, donkeys), companion animal (e.g. pets such as dogs and cats), laboratory test animal (e.g. mice, rabbits, rats, guinea pigs), performance animal (e.g. racehorses, camels, greyhounds) or captive wild animal. In one example, the subject or patient is a mammal. In one example, the subject or patient is a primate. In one example, the subject or patient is a human.

The terms "reduced expression", "reduction in expression" or similar, refer to the absence or an observable decrease in the level of protein and/or mRNA product from the target gene(s) e.g., VEGFb, PGF and/or VEGFa. The decrease does not have to be absolute, but may be a partial decrease sufficient for there to a detectable or observable change as a result of the RNAi effected by the shmiR encoded by the nucleic acid of the disclosure. The decrease can be measured by determining a decrease in the level of mRNA and/or protein product from a target nucleic acid relative to a cell lacking the shmiR or shRNA, nucleic acid encoding same, ddRNAi construct or expression construct, and may be as little as 1 %, 5% or 10%, or may be absolute i.e., 100% inhibition. The effects of the decrease may be determined by examination of the outward properties i.e., quantitative and/or qualitative phenotype of the cell or organism, and may also include an assessment of vision or impairment thereof, rate of growth of abnormal blood vessels under the macula and/or fluid leakage from those blood vessels, following administration of a ddRNAi construct of the disclosure or a composition comprising same.

Agents for RNAi

In one example, the present disclosure provides a nucleic acid comprising a DNA sequence which encodes a short hairpin micro-RNA (shmiR), said shmiR comprising: an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone; wherein the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22. Preferably, the effector sequence will be less than 30 nucleotides in length. For example, a suitable effector sequence may be in the range of 17-29 nucleotides in length. In a particularly preferred example, the effector sequence will be 21 nucleotides in length. More preferably, the effector sequence will be 21 nucleotides in length and the effector complement sequence will be 20 nucleotides in length.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 1 A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-l". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 1. In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 2. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-2". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 2.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 3. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-3". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

3 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 3.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 4. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-4". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

4 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 4.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 5. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-5". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 5.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 6. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-6". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

6 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 6.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 7. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-7". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

7 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 7.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 8. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-8". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 8.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 9. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-9". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

9 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially

complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 9.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 10. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-10". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

10 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

10 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 10.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 11. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

11 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 11.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 12. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-2". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript of VEGFb comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 12.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 13. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-3". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 13 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

13 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: l 3.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 14. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-4". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

14 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 14.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 15. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-5". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 15.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 16. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-6". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

16 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 16.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 17. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-7". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 17.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 18. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-8". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

18 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

18 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 18.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 19. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-9". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

19 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 19. In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 20. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-10". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 20.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 21. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l l". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

21 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 21.

In one example, the shmiR comprises an effector sequence which is substantially complementary to a region of equivalent length within an RNA transcript of PGF comprising or consisting of the sequence set forth in SEQ ID NO: 22. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-12". For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO:

22 and contain 6 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 5 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 4 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 3 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 2 mismatch bases relative thereto. For example, the effector sequence may be substantially complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22 and contain 1 mismatch base relative thereto. For example, the effector sequence may be 100% complementary to a region of equivalent length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 22.

In accordance with an example in which the effector sequence of a shmiR of the disclosure is substantially complementary to a RNA transcript of VEGFb or PGF as described herein and contains 1, 2, 3, 4, 5 or 6 mismatch base(s) relative thereto, it is preferred that the mismatch(es) are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:23 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:23; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR- 1 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:24 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:24 may be the sequence set forth in SEQ ID NO:23. A shmiR in accordance with this example is hereinafter designated "VEFGb shmiR- '.

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:25 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:25; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-2 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:26 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:26 may be the sequence set forth in SEQ ID NO:25. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-2".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:27 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:27; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-3 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:28 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:28 may be the sequence set forth in SEQ ID NO:27. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-3".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:29 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:29; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-4 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:30 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:30 may be the sequence set forth in SEQ ID NO:29. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-4".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:31 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:31 ; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-5 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:32 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:32 may be the sequence set forth in SEQ ID NO:31. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-5".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:33 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:33; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-6 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:34 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:34 may be the sequence set forth in SEQ ID NO:33. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-6".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:35 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:35; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-7 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:36 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:36 may be the sequence set forth in SEQ ID NO:35. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-7".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:37 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:37; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-8 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:38 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:38 may be the sequence set forth in SEQ ID NO:37. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-8".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:39 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:39; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-9 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:40 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:40 may be the sequence set forth in SEQ ID NO:39. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-9".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding VEGFb_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:41 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:41; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, VEGFb_shmiR-10 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:42 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:42 may be the sequence set forth in SEQ ID NO:41. A shmiR in accordance with this example is hereinafter designated "VEGFb_shmiR-10".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:43 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:43; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-l encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:44 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:44 may be the sequence set forth in SEQ ID NO:41. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-l".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-2 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:45 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:45; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-2 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:46 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:46 may be the sequence set forth in SEQ ID NO:45. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-2".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-3 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:47 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:47; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-3 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:48 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:48 may be the sequence set forth in SEQ ID NO:47. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-3".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-4 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:49 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:49; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-4 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:50 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:50 may be the sequence set forth in SEQ ID NO:49. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-4".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-5 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:51 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:51; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-5 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:52 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:52 may be the sequence set forth in SEQ ID NO:51. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-5".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-6 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:53 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:53; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-6 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:54 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:54 may be the sequence set forth in SEQ ID NO:53. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-6".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-7 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:55 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:55; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-7 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:56 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:56 may be the sequence set forth in SEQ ID NO:55. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-7".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-8 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:57 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:57; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-8 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:58 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:58 may be the sequence set forth in SEQ ID NO:57. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-8".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-9 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:59 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:59; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-9 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:60 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:60 may be the sequence set forth in SEQ ID NO:59. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-9".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-10 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:61 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:61 ; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-10 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:62 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:62 may be the sequence set forth in SEQ ID NO:61. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-10".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-l l comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:63 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:63; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-l l encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:64 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:64 may be the sequence set forth in SEQ ID NO:63. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-H".

In one example, the nucleic acid as described herein comprises a DNA sequence encoding PGF_shmiR-12 comprising: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO:65 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO:65; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, PGF_shmiR-12 encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:66 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO:66 may be the sequence set forth in SEQ ID NO:65. A shmiR in accordance with this example is hereinafter designated "PGF_shmiR-12".

In any of the examples described herein, the shmiR encoded by the nucleic acid of the disclosure may comprise, in a 5' to 3' direction:

a 5' flanking sequence of the pri-miRNA backbone;

the effector complement sequence;

the stemloop sequence;

the effector sequence; and

a 3' flanking sequence of the pri-miRNA backbone.

a 5' flanking sequence of the pri-miRNA backbone;

the effector sequence;

the stemloop sequence;

the effector complement sequence; and

a 3' flanking sequence of the pri-miRNA backbone. Suitable loop sequences may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.

Suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in a nucleic acid of the disclosure may be selected from those known in the art. For example, the pri- miRNA backbone may be selected from a pri-miR-30a backbone, a pri-miR-155 backbone, a pri-miR-21 backbone and a pri-miR-136 backbone. Preferably, however, the pri-miRNA backbone is a pri-miR-30a backbone. In accordance with an example in which the pri- miRNA backbone is a pri-miR-30a backbone, the 5' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 68 and the 3' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 69. Thus, the nucleic acid encoding the shmiRs of the disclosure may comprise DNA sequence encoding the sequence set forth in SEQ ID NO: 68 and DNA sequence encoding the sequence set forth in SEQ ID NO: 69.

In one example, the nucleic acid described herein may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-113.

According to an example in which a nucleic acid described herein encodes a shmiR targeting VEGFb, the nucleic acid may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 92-101.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 92 and encodes a shmiR (VEGFb_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 70.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 93 and encodes a shmiR (VEGFb_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 71.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 94 and encodes a shmiR (VEGFb_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 72.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 95 and encodes a shmiR (VEGFb_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 73.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 96 and encodes a shmiR (VEGFb_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 74. In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 97 and encodes a shmiR (VEGFb_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 75.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 98 and encodes a shmiR (VEGFb_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 76.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 99 and encodes a shmiR (VEGFb_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 77.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 100 and encodes a shmiR (VEGFb_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 78.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 101 and encodes a shmiR (VEGFb_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 79.

Exemplary nucleic acids targeting VEGFb encode a shmiR selected from

VEGFb_shmiR-l, VEGFb_shmiR-4, and VEGFb_shmiR-10 as described herein.

According to an example in which the nucleic acid described herein encodes a shmiR targeting PGF, the nucleic acid may comprise a DNA sequence selected from the sequence set forth in any one of SEQ ID NOs: 102-113.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 102 and encodes a shmiR (PGF_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 80.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 103 and encodes a shmiR (PGF_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 81.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 104 and encodes a shmiR (PGF_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 82.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 105 and encodes a shmiR (PGF_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 83. In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 106 and encodes a shmiR (PGF_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 84.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 107 and encodes a shmiR (PGF_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 85.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 108 and encodes a shmiR (PGF_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 86.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 109 and encodes a shmiR (PGF_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 87.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 110 and encodes a shmiR (PGF_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 88.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 111 and encodes a shmiR (PGF_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 89.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 112 and encodes a shmiR (PGF_shmiR-l 1) comprising or consisting of the sequence set forth in SEQ ID NO: 90.

In one example, the nucleic acid described herein comprises or consists of a DNA sequence set forth in SEQ ID NO: 113 and encodes a shmiR (PGF_shmiR-12) comprising or consisting of the sequence set forth in SEQ ID NO: 91.

Exemplary nucleic acids targeting PGF encode a shmiR selected from PGF_shmiR-

3, PGF_shmiR-7, and PGF_shmiR-10 as described herein.

It will be understood by a person of skill in the art that effector/effector complement sequence combinations encoded by the nucleic acids described herein may also be expressed and thus provided in the form of a short hairpin RNA (shRNA). Accordingly, the present disclosure also provides a nucleic acid comprising a DNA sequence encoding a shRNA comprising:

an effector sequence of at least 17 nucleotides in length; a stemloop sequence; and

an effector complement sequence;

wherein the effector sequence is substantially complementary to an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22. Preferably, the effector sequence will be less than 30 nucleotides in length. For example, a suitable effector sequence may be in the range of 17-29 nucleotides in length.

the effector complement sequence;

the stemloop sequence; and

the effector sequence;

the stemloop sequence; and

the effector complement sequence.

Suitable loop sequences are described herein in the context of shmiRs and shall be taken to apply mutatis mutandis to each example in which a shRNA is described. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67.

It will be understood by a person of skill in the art that a nucleic acid in accordance with the present disclosure may be combined or used in conjunction with other therapeutic agents for treating AMD or inhibiting CNV. Accordingly, the present disclosure provides a nucleic acid comprising a DNA sequence encoding a shmiR as described herein (e.g., one or shmiRs designated VEGFb_shmiRl to VEGFb_shmiR-10 or PGF_shmiRl to PGF_shmiR- 12 described herein) in combination with one or more other agents for treating AMD or inhibiting CNV. In one example, a plurality of nucleic acids are provided comprising: (a) at least one nucleic acid as described herein; and

(b) at least one further nucleic acid selected from:

(i) a nucleic acid comprising a DNA sequence encoding a shmiR as described herein; or

(ii) a nucleic acid comprising a DNA sequence encoding a shmiR targeting VEGFa (designated herein as "VEGFa_shmiR") comprising:

an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence is substantially complementary to a region of corresponding length in the RNA transcript set forth in SEQ ID NO: 114; and

Preferably, the effector sequence of VEGFa_shmiR will be less than 30 nucleotides in length. For example, a suitable effector sequence of VEGFa_shmiR may be in the range of 17-29 nucleotides in length. In a particularly preferred example, the effector sequence of VEGFa_shmiR will be 21 nucleotides in length. More preferably, the effector sequence of VEGFa_shmiR will be 21 nucleotides in length and the corresponding effector complement sequence will be 20 nucleotides in length.

As described herein, VEGFa_shmiR will comprise an effector sequence which is substantially complementary to a region of corresponding length within an RNA transcript of VEGFa comprising or consisting of the sequence set forth in SEQ ID NO: 114. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 6 mismatch bases relative thereto. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 5 mismatch bases relative thereto. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 4 mismatch bases relative thereto. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 3 mismatch bases relative thereto. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 2 mismatch bases relative thereto. For example, the effector sequence of VEGFa_shmiR may be substantially complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114 and contain 1 mismatch base relative thereto. For example, the effector sequence of VEGFa_shmiR may be 100% complementary to a region of corresponding length within an RNA transcript comprising or consisting of the sequence set forth in SEQ ID NO: 114.

In accordance with an example in which the effector sequence of VEGFa_shmiR is substantially complementary to SEQ ID NO: 114 and contains 1, 2, 3, 4, 5 or 6 mismatch base(s) relative thereto, it is preferred that the mismatch(es) are not located within the region corresponding to the seed region of the shmiR i.e., nucleotides 2-8 of the effector sequence.

In one example, VEGFa_shmiR as described herein comprises: (i) an effector sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 114 with the exception of 1, 2, 3, 4, 5 or 6 base mismatches, provided that the effector sequence is capable of forming a duplex with a sequence set forth in SEQ ID NO: 114; and (ii) an effector complement sequence comprising a sequence which is substantially complementary to the effector sequence. For example, the shmiR encoded by the nucleic acid may comprise an effector sequence set forth in SEQ ID NO:l 15 and an effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 and capable of forming a duplex therewith. The effector complement sequence which is substantially complementary to the sequence set forth in SEQ ID NO: 115 may be the sequence set forth in SEQ ID NO: 115.

A suitable loop sequence for VEGFa_shmiR may be selected from those known in the art. However, an exemplary stemloop sequence is set forth in SEQ ID NO: 67. Similarly, suitable primary micro RNA (pri-miRNA or pri-R) backbones for use in VEGFa_shmiR may be selected from those known in the art, such as those already described herein. For example, the pri-miRNA backbone may be a pri-miR-30a backbone, in which the 5' flanking sequence is set forth in SEQ ID NO: 68 and the 3 ' flanking sequence is set forth in SEQ ID NO: 69. Thus, a nucleic acid encoding the VEGFa_shmiR may comprise DNA sequence encoding the sequence set forth in SEQ ID NO: 68 and DNA sequence encoding the sequence set forth in SEQ ID NO: 69.

In one example, the nucleic acid encoding VEGFa_shmiR comprises or consists of a DNA sequence set forth in SEQ ID NO: 117 and encodes a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116.

A plurality of nucleic acids as described herein may comprise two or more nucleic acids encoding shmiRs as described herein, such as two, or three, or four, or five, or six, or seven, or eight, or nine, or ten nucleic acids encoding shmiRs as described herein.

In one example, the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting VEGFb as described herein (i.e., VEGFb_shmiR-l to VEGFb_shmiR- 10) and at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR- l to PGF_shmiR- 12). For example, a plurality of nucleic acids of the disclosure may comprise:

a nucleic acid comprising a DNA sequence encoding one of VEGFb_shmiR-l, VEGFb_shmiR-4 or VEGFb_shmiR- 10 as described herein; and

a nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,

PGF_shmiR-7 or PGF_shmiR- 10 as described herein.

In another example, the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting VEGFb as described herein (i.e.,

VEGFb_shmiR-l to VEGFb_shmiR-10) and at least one nucleic acid encoding a shmiR targeting VEGFa (i.e., VEGFa_shmiR). For example, a plurality of nucleic acids of the disclosure may comprise:

a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR as described herein. In another example, the plurality of nucleic acids described herein comprises at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR-l to PGF_shmiR-12) and at least one nucleic acid encoding a shmiR targeting VEGFa (i.e., VEGFa_shmiR). For example, a plurality of nucleic acids of the disclosure may comprise:

a nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,

PGF_shmiR-7 or PGF_shmiR-10 as described herein; and

a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR as described herein.

VEGFb_shmiR-l to VEGFb_shmiR-10), at least one nucleic acid encoding a shmiR targeting PGF (i.e., PGF_shmiR-l to PGF_shmiR-12) and at least one nucleic acid encoding a shmiR targeting VEGFa (i.e., VEGFa_shmiR). For example, a plurality of nucleic acids of the disclosure may comprise:

a nucleic acid comprising a DNA sequence encoding one of VEGFb_shmiR-l,

VEGFb_shmiR-4 or VEGFb_shmiR-10 as described herein;

a nucleic acid comprising a DNA sequence encoding one of PGF_shmiR-3,

PGF_shmiR-7 or PGF_shmiR-10 as described herein; and

In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 ; and

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR.

In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 ;

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR; and

(iii) a nucleic acid comprising a DNA sequence encoding PGF-shmiR-3.

In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 ; and

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR; and

(iii) a nucleic acid comprising a DNA sequence encoding PGF-shmiR-7. Exemplary nucleic acids of the disclosure encoding shmiRs designated VEGFb_shmiR-l, VEGFb_shmiR-4, VEGFb_shmiR-10, PGF_shmiR-3, PGF_shmiR-7, PGF_shmiR-10 and VEGFa_shmiR are described herein and shall be taken to apply mutatis mutandis to each example in which a plurality of nucleic acids of the disclosure is described In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 24 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 23; and

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114.

In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence encoding VEGFb_shmiR- 1 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 24 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 23;

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114; and

(iii) a nucleic acid comprising a DNA sequence encoding a PGF_shmiR-3 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 48 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO:47.

In one example, the plurality of nucleic acids of the disclosure comprises:

(ii) a nucleic acid comprising a DNA sequence encoding VEGFa_shmiR comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 115 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 114; and (iii) a nucleic acid comprising a DNA sequence encoding a PGF_shmiR-7 comprising an effector sequence consisting of the sequence set forth in SEQ ID NO: 56 and an effector complement sequence consisting of the sequence set forth in SEQ ID NO: 55.

In one example, the plurality of nucleic acids of the disclosure comprises:

(i) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 70); and

(ii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116).

In one example, the plurality of nucleic acids of the disclosure comprises:

(ii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116); and

(iii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 82).

In one example, the plurality of nucleic acids of the disclosure comprises:

(iii) a nucleic acid comprising a DNA sequence comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 86).

polynucleotides, respectively. In another example, the plurality of nucleic acids described herein are provided as multiple components e.g., multiple compositions. For example, each of the nucleic acids of the plurality may be provided separately. Alternatively, in an example where at least three nucleic acids of the disclosure are provided, at least one of the nucleic acids may be provided separately and two or more of the plurality provided together.

In some examples, the or each nucleic acid in accordance with the present disclosure may comprise, or be in operable linkage with, additional elements e.g., to facilitate transcription of the RNA. For example, the or each nucleic acid may comprise a promoter operably-linked to the sequence encoding a shmiR described herein. Other regulatory elements e.g., transcriptional terminators and initiators, are known in the art and/or described herein.

Alternatively, or in addition, the or each nucleic acid in accordance with the present disclosure may comprise one or more restriction sites e.g., to facilitate cloning of the nucleic acid(s) into cloning or expression vectors. For example, the nucleic acids described herein may include a restriction site upstream and/or downstream of the sequence encoding a shmiR or shRNA of the disclosure. Suitable restriction enzyme recognition sequences will be known to a person of skill in the art. However, in one example, the nucleic acid(s) of the disclosure may include a BamHl restriction site (GGATCC) at the 5' terminus i.e., upstream of the sequence encoding the shmiR, and a Hindlll restriction site (AAGCTT) at the 3 ' terminus i.e., downstream of the sequence encoding the shmiR. ddRNAi

In one example, the or each nucleic acid of the disclosure is provided in the form of, or is comprised in, a DNA-directed RNAi (ddRNAi) construct. Accordingly, in one example, the present disclosure provides a ddRNAi construct comprising a nucleic acid as described herein. In another example, the present disclosure provides a ddRNAi construct comprising a plurality of nucleic acids described herein. Exemplary nucleic acids encoding shmiRs targeting VEGFb and PGF, provided alone or in combination with a nucleic acid encoding a shmiR targeting VEGFa, are described herein and shall be taken to apply mutatis mutandis to this example of the disclosure. In one example, the ddRNAi construct comprises a nucleic acid of the disclosure operably-linked to a promoter.

In accordance with an example in which the ddRNAi construct comprises a plurality of the nucleic acids described herein , each of the nucleic acids may be operably-linked to a promoter. In one example, the nucleic acids in the ddRNAi construct may be operably- linked to the same promoter. In one example, the nucleic acids in the ddRNAi construct may be operably-linked to different promoters.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 92 and encodes a shmiR

(VEGFb_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 70. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 93 and encodes a shmiR

(VEGFb_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 71. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 94 and encodes a shmiR

(VEGFb_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 72. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 95 and encodes a shmiR

(VEGFb_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 73. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 96 and encodes a shmiR

(VEGFb_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 74. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 97 and encodes a shmiR

(VEGFb_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 75. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs:

102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 98 and encodes a shmiR

(VEGFb_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 76. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 99 and encodes a shmiR

(VEGFb_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 77. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117. In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 100 and encodes a shmiR

(VEGFb_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 78. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 101 and encodes a shmiR

(VEGFb_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 79. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 102-113 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 104, 108, 111 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 102 and encodes a shmiR

(PGF_shmiR-l) comprising or consisting of the sequence set forth in SEQ ID NO: 80. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 103 and encodes a shmiR

(PGF_shmiR-2) comprising or consisting of the sequence set forth in SEQ ID NO: 81. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 104 and encodes a shmiR

(PGF_shmiR-3) comprising or consisting of the sequence set forth in SEQ ID NO: 82. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 105 and encodes a shmiR

(PGF_shmiR-4) comprising or consisting of the sequence set forth in SEQ ID NO: 83. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 106 and encodes a shmiR

(PGF_shmiR-5) comprising or consisting of the sequence set forth in SEQ ID NO: 84. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 107 and encodes a shmiR

(PGF_shmiR-6) comprising or consisting of the sequence set forth in SEQ ID NO: 85. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 108 and encodes a shmiR

(PGF_shmiR-7) comprising or consisting of the sequence set forth in SEQ ID NO: 86. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117. In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 109 and encodes a shmiR

(PGF_shmiR-8) comprising or consisting of the sequence set forth in SEQ ID NO: 87. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 110 and encodes a shmiR

(PGF_shmiR-9) comprising or consisting of the sequence set forth in SEQ ID NO: 88. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 111 and encodes a shmiR

(PGF_shmiR-10) comprising or consisting of the sequence set forth in SEQ ID NO: 89. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 112 and encodes a shmiR

(PGF_shmiR-l l) comprising or consisting of the sequence set forth in SEQ ID NO: 90. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

In one example, a ddRNAi of the disclosure comprises a nucleic acid comprising or consisting of a DNA sequence set forth in SEQ ID NO: 113 and encodes a shmiR

(PGF_shmiR-12) comprising or consisting of the sequence set forth in SEQ ID NO: 91. The ddRNAi construct may comprise one or more further nucleic acids of the disclosure, such as a nucleic acid comprising a DNA sequence set forth in any one of SEQ ID NOs: 92-101 or 117. In a preferred example, the further nucleic acids comprise a DNA sequence selected from the sequences set forth in SEQ ID NOs: 92, 95, 101 and 117.

An exemplary ddRNAi construct comprising a two nucleic acids of the disclosure comprises:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70); and

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in

SEQ ID NO: 116).

The present disclosure also provides a ddRNAi construct comprising at least three nucleic acids described herein, each of which encode shmiRs targeting different genes.

In one example, the disclosure provides a ddRNAi construct comprising:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID

NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70);

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116); and

(c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82). In a preferred example, (a) to (c) are configured in a 5' to 3' direction relative to one another within the ddRNAi construct.

In one example, the disclosure provides a ddRNAi construct comprising:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70);

SEQ ID NO: 116); and (c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86). In a preferred example, (a) to (c) are configured in a 5' to 3' direction relative to one another within the ddRNAi construct.

In each of the foregoing examples describing a ddRNAi construct of the disclosure, the or each nucleic acid comprised therein may be operably-linked to a promoter. For example, the ddRNAi construct as described herein may comprise a single promoter which is operably-linked to the or each nucleic acid comprised therein e.g., to drive expression of one or more shmiRs from the ddRNAi construct. In another example, each shmiR-encoding nucleic acid comprised in the ddRNAi construct is operably-linked to a separate promoter.

According to an example in which multiple promoters are present, the promoters can be the same or different. For example, the construct may comprise multiple copies of the same promoter with each copy operably-linked to a different nucleic acid of the disclosure. In another example, each promoter operably-linked to a shmiR-encoding nucleic acid of the disclosure is different. For example, in a ddRNAi construct encoding three shmiRs, each nucleic acids encoding one of respective shmiRs is operably-linked to a different promoter.

According to a further example in which a ddRNAi construct encodes three or more shmiRs, two (or more) of the nucleic acids encoding the shmiRs are linked to the same promoter and one (or more) of the nucleic acids encoding the other shmiR(s) is/are linked to a different promoter(s).

In one example, the or each promoter is a constitutive promoter. The term

"constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably-linked nucleic acid sequence in the absence of a specific stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a coding sequence in substantially any cell and any tissue. The promoters used to transcribe shmiRs from the nucleic acid(s) of the disclosure include promoters for ubiquitin, CMV, β-actin, histone H4, EF-la or pgk genes controlled by RNA polymerase II, or promoter elements controlled by RNA polymerase I.

In one example, a Pol II promoter such as CMV, SV40, Ul, β-actin or a hybrid Pol II promoter is employed.

In another example, a promoter controlled by RNA polymerase III is used, such as a U6 promoter (U6-1, U6-8, U6-9), HI promoter, 7SL promoter, a human Y promoter (hYl, hY3, hY4 (see Maraia, et al, Nucleic Acids Res 22(15):3045-52(1994)) and hY5 (see Maraia, et al, Nucleic Acids Res 24(18):3552-59(1994)), a human MRP-7-2 promoter, an Adenovirus VA1 promoter, a human tRNA promoter, or a 5s ribosomal RNA promoter.

Suitable promoters for use in a ddRNAi construct of the disclosure are described in US Patent No. 8,008,468 and US Patent No. 8, 129,510.

In one example, the promoter is a RNA pol III promoter. For example, the promoter is a U6 promoter (e.g., a U6-1, U6-8 or U6-9 promoter). In another example, the promoter is a HI promoter.

In the case of a ddRNAi construct of the disclosure encoding a plurality of shmiRs described herein, each of the nucleic acids in the ddRNAi construct is operably-linked to a U6 promoter e.g., each operably-linked to a separate U6 promoter.

In one example, the ddRNAi construct comprises a U6- 1 promoter.

In one example, the ddRNAi construct comprises a U6-8 promoter.

In one example, the ddRNAi construct comprises a U6-9 promoter.

In some examples, promoters of variable strength are employed. For example, use of two or more strong promoters (such as a Pol Ill-type promoter) may tax the cell, by, e.g., depleting the pool of available nucleotides or other cellular components needed for transcription. In addition, or alternatively, use of several strong promoters may cause a toxic level of expression of RNAi agents e.g., shmiRs, in the cell. Thus, in some examples one or more of the promoters in the multiple -promoter ddRNAi construct is weaker than other promoters in the construct, or all promoters in the construct may express the shmiRs at less than a maximum rate. Promoters may also be modified using various molecular techniques, or otherwise, e.g., through modification of various regulatory elements, to attain weaker levels or stronger levels of transcription. One means of achieving reduced transcription is to modify sequence elements within promoters known to control promoter activity. For example, the Proximal Sequence Element (PSE) is known to affect the activity of human U6 promoters (see Domitrovich, et al., Nucleic Acids Res 31: 2344-2352 (2003). Replacing the PSE elements present in strong promoters, such as the human U6-1, U6-8 or U6-9 promoters, with the element from a weak promoter, such as the human U6-7 promoter (i.e., PSE7), reduces the activity of the hybrid U6-1, U6-8 or U6-9 promoters. This approach has been used in the examples described in this application, but other means to achieve this outcome are known in the art. Promoters useful in some examples of the present disclosure can be tissue-specific or cell- specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective transcription of a nucleic acid of interest to a specific type of tissue (e.g., ocular tissues) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., liver). The term "cell-specific" as applied to a promoter refers to a promoter which is capable of directing selective

transcription of a nucleic acid of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue.

In one example, a ddRNAi construct of the disclosure may additionally comprise one or more enhancers to increase expression of the shmiRs encoded by the nucleic acids described herein. Enhancers appropriate for use in examples of the present disclosure will be known to those skilled in the art.

In a further example, a ddRNAi construct of the disclosure may comprise a

transcriptional terminator linked to a nucleic acid encoding a shmiR of the disclosure. In the case of a ddRNAi construct comprising a plurality of nucleic acids described herein i.e., encoding multiple shmiRs, the terminators linked to each nucleic acid can be the same or different. According to an example in which a RNA pol III promoter is employed, the terminator may be a contiguous stretch of 4 or more or 5 or more or 6 or more T residues.

In some examples, where different promoters are used, the terminators can be different and are matched to the promoter from the gene from which the terminator is derived. Such terminators include the SV40 poly A, the AdV VAl gene, the 5S ribosomal RNA gene, and the terminators for human t-RNAs. In addition, promoters and terminators may be mixed and matched, as is commonly done with RNA pol II promoters and terminators.

In one example, the promoter and terminator combinations used for each nucleic acid in a ddRNAi construct comprising a plurality of nucleic acids is different to decrease the likelihood of DNA recombination events between components.

One exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding

VEGFb_shmiR-l comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter. Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding

PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO: 82) operably-linked to a U6 promoter e.g., a U6-8 promoter.

Another exemplary ddRNAi construct of the disclosure comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding

PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO: 86) operably-linked to a U6 promoter e.g., a U6-8 promoter.

According to one example in which the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein, the ddRNAi construct may comprise:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and

SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter.

According to another example in which the ddRNAi construct of the disclosure comprises a plurality of nucleic acids described herein, the ddRNAi construct may comprise:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in

SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID

NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter. According to one example in which the ddRNAi construct of the disclosure comprises at least three nucleic acids described herein, the ddRNAi construct may comprise:

(a) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;

(b) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter;

(c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.

According to another example in which the ddRNAi construct of the disclosure comprises at least three nucleic acids described herein, the ddRNAi construct may comprise:

SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;

(c) a nucleic acid comprising or consisting of the sequence set forth in SEQ ID

NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter.

An exemplary ddRNAi construct of the disclosure comprises, in a 5' to 3' direction:

Another exemplary ddRNAi construct of the disclosure comprises, in a 5' to 3' direction: (a) U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:

The present disclosure also provides a plurality of ddRNAi constructs comprising two or more ddRNAi constructs, each comprising nucleic acid encoding a shmiR of the disclosure operably-linked to a suitable promoter as described herein.

In one example, the plurality of ddRNAi constructs comprises:

(a) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and

(b) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter.

In another example, the plurality of ddRNAi constructs comprises:

(b) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.

In another example, the plurality of ddRNAi constructs comprises:

(a) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter; and (b) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter.

According to an example in which the plurality of ddRNAi constructs comprises three ddRNAi constructs, the plurality of ddRNAi constructs comprises:

(a) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92 (encoding VEGFb_shmiR- 1 comprising or consisting of the sequence set forth in SEQ ID NO:70) operably-linked to a U6 promoter e.g., a U6-9 promoter;

(b) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117 (encoding VEGFa_shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116) operably-linked to a U6 promoter e.g., a U6-1 promoter; and

(c) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104 (encoding PGF_shmiR-3 comprising or consisting of the sequence set forth in SEQ ID NO:82) operably-linked to a U6 promoter e.g., a U6-8 promoter.

According to another example in which the plurality of ddRNAi constructs comprises three ddRNAi constructs, the plurality of ddRNAi constructs comprises:

(c) a ddRNAi construct which comprises a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108 (encoding PGF_shmiR-7 comprising or consisting of the sequence set forth in SEQ ID NO:86) operably-linked to a U6 promoter e.g., a U6-8 promoter. In addition, the or each ddRNAi construct can comprise one or more multiple cloning sites and/or unique restriction sites that are located strategically, such that the promoter, nucleic acid encoding the shmiR and/or other regulator elements are easily removed or replaced. The or each ddRNAi construct can be assembled from smaller oligonucleotide components using strategically located restriction sites and/or complementary sticky ends. The base vector for one approach according to the present disclosure comprises plasmids with a multilinker in which all sites are unique (though this is not an absolute requirement). Sequentially, each promoter is inserted between its designated unique sites resulting in a base cassette with one or more promoters, all of which can have variable orientation.

Sequentially, again, annealed primer pairs are inserted into the unique sites downstream of each of the individual promoters, resulting in a single-, double- or multiple-expression cassette construct. The insert can be moved into, e.g. an AdV backbone or an AAV backbone using two unique restriction enzyme sites (the same or different ones) that flank the single-, double- or multiple-expression cassette insert.

Generation of the or each construct can be accomplished using any suitable genetic engineering techniques known in the art, including without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing. If the or each construct is a viral construct, the construct comprises, for example, sequences necessary to package the ddRNAi construct into viral particles and/or sequences that allow integration of the ddRNAi construct into the target cell genome. In some examples, the or each viral construct additionally contains genes that allow for replication and propagation of virus, however such genes will be supplied in trans. Additionally, the or each viral construct cam contain genes or genetic sequences from the genome of any known organism incorporated in native form or modified. For example, a viral construct may comprise sequences useful for replication of the construct in bacteria.

The or each construct also may contain additional genetic elements. The types of elements that may be included in the construct are not limited in any way and may be chosen by one with skill in the art. For example, additional genetic elements may include a reporter gene, such as one or more genes for a fluorescent marker protein such as GFP or RFP; an easily assayed enzyme such as beta-galactosidase, luciferase, beta-glucuronidase, chloramphenical acetyl transferase or secreted embryonic alkaline phosphatase; or proteins for which immunoassays are readily available such as hormones or cytokines.

Other genetic elements that may find use in embodiments of the present disclosure include those coding for proteins which confer a selective growth advantage on cells such as adenosine deaminase, aminoglycodic phosphotransferase, dihydrofolate reductase, hygromycin-B-phosphotransferase, drug resistance, or those genes coding for proteins that provide a biosynthetic capability missing from an auxotroph. If a reporter gene is included along with the or each construct, an internal ribosomal entry site (IRES) sequence can be included. In one example, the additional genetic elements are operably linked with and controlled by an independent promoter/enhancer. In addition a suitable origin of replication for propagation of the construct in bacteria may be employed. The sequence of the origin of replication generally is separated from the ddRNAi construct and other genetic sequences. Such origins of replication are known in the art and include the pUC, ColEl, 2-micron or SV40 origins of replication.

Expression vectors

In one example, a ddRNAi construct of the disclosure is included within an expression vector. In accordance with an example in which a plurality of ddRNAi constructs is provided, each ddRNAi construct may be included within its own expression vector.

In one example, the or each expression vector is a plasmid, e.g., as is known in the art.

In one example, a suitable plasmid expression vector is a Psh vector e.g., with a U6 promoter and proximal sequence element 7 (PSE7).

In one example, the expression vector is mini-circle DNA. Mini-circle DNA is described in U.S. Patent Publication No. 2004/0214329. Mini-circle DNA are useful for persistently high levels of nucleic acid transcription. The circular vectors are characterized by being devoid of expression-silencing bacterial sequences. For example, mini-circle vectors differ from bacterial plasmid vectors in that they lack an origin of replication, and lack drug selection markers commonly found in bacterial plasmids, e.g. β-lactamase, tet, and the like. Consequently, minicircle DNA becomes smaller in size, allowing more efficient delivery.

In one example, the expression vector is a viral vector. A viral vector based on any appropriate virus may be used to deliver a nucleic acid or ddRNAi construct of the disclosure. In addition, hybrid viral systems may be of use. The choice of viral delivery system will depend on various parameters, such as the tissue targeted for delivery, transduction efficiency of the system, pathogenicity, immunological and toxicity concerns, and the like.

Commonly used classes of viral systems used in gene therapy can be categorized into two groups according to whether their genomes integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as extrachromosomal episomes (adeno-associated virus, adenoviruses and herpesviruses). In one example, a viral vector of the disclosure integrates into a host cell's chromatin. In another example, a viral vector of the disclosure persists in a host cell's nucleus as an extrachomosomal episome.

In one example, a viral vector is an adenoviral (AdV) vector. Adenoviruses are medium-sized double-stranded, non-enveloped DNA viruses with linear genomes that is between 26-48 Kbp. Adenoviruses gain entry to a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells.

Adenoviruses are heavily reliant on the host cell for survival and replication and are able to replicate in the nucleus of vertebrate cells using the host's replication machinery.

In one example, a viral vector is from the Parvoviridae family. The Parvoviridae is a family of small single-stranded, non-enveloped DNA viruses with genomes approximately 5000 nucleotides long. Included among the family members is adeno-associated virus (AAV). In one example, a viral vector of the disclosure is an AAV. AAV is a dependent parvovirus that generally requires co-infection with another virus (typically an adenovirus or herpesvirus) to initiate and sustain a productive infectious cycle. In the absence of such a helper virus, AAV is still competent to infect or transduce a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells. Because progeny virus is not produced from AAV infection in the absence of helper virus, the extent of transduction is restricted only to the initial cells that are infected with the virus. It is this feature which makes AAV a desirable vector for the present disclosure.

Furthermore, unlike retrovirus, adenovirus, and herpes simplex virus, AAV appears to lack human pathogenicity and toxicity (Kay, et ah, Nature. 424: 251 (2003)). Since the genome normally encodes only two genes it is not surprising that, as a delivery vehicle, AAV is limited by a packaging capacity of 4.5 single stranded kilobases (kb). However, although this size restriction may limit the genes that can be delivered for replacement gene therapies, it does not adversely affect the packaging and expression of shorter sequences such as shmiRs and shRNAs.

Another viral delivery system useful with the ddRNAi constructs of the disclosure is a system based on viruses from the family Retroviridae. Retroviruses comprise single- stranded RNA animal viruses that are characterized by two unique features. First, the genome of a retrovirus is diploid, consisting of two copies of the RNA. Second, this RNA is transcribed by the virion-associated enzyme reverse transcriptase into double- stranded DNA. This double- stranded DNA or provirus can then integrate into the host genome and be passed from parent cell to progeny cells as a stably-integrated component of the host genome.

In some examples, a viral vector is a lentivirus. Lentivirus vectors are often pseudotyped with vesicular steatites virus glycoprotein (VSV-G), and have been derived from the human immunodeficiency virus (HIV); visan-maedi, which causes encephalitis (visna) or pneumonia in sheep; equine infectious anemia virus (EIAV), which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immunodeficiency virus (BIV) which causes lymphadenopathy and lymphocytosis in cattle; and simian immunodeficiency virus (SIV), which causes immune deficiency and encephalopathy in non-human primates. Vectors that are based on HIV generally retain <5% of the parental genome, and <25% of the genome is incorporated into packaging constructs, which minimizes the possibility of the generation of reverting replication-competent HIV. Biosafety has been further increased by the development of self-inactivating vectors that contain deletions of the regulatory elements in the downstream long-terminal-repeat sequence, eliminating transcription of the packaging signal that is required for vector mobilization. One of the main advantages to the use of lentiviral vectors is that gene transfer is persistent in most tissues or cell types, even following cell division of the transduced cell.

A lentiviral-based construct used to express shmiRs and/or shRNAs from the nucleic acids and ddRNAi constructs of the disclosure comprises sequences from the 5' and 3' long terminal repeats (LTRs) of a lentivirus. In one example, the viral construct comprises an inactivated or self-inactivating 3' LTR from a lentivirus. The 3' LTR may be made self- inactivating by any method known in the art. For example, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, e.g., the TATA box, Spl and NF-kappa B sites. As a result of the self-inactivating 3' LTR, the provirus that is integrated into the host genome will comprise an inactivated 5' LTR. The LTR sequences may be LTR sequences from any lentivirus from any species. The lentiviral-based construct also may incorporate sequences for MMLV or MSCV, RSV or mammalian genes. In addition, the U3 sequence from the lentiviral 5' LTR may be replaced with a promoter sequence in the viral construct. This may increase the titer of virus recovered from the packaging cell line. An enhancer sequence may also be included.

Other viral or non-viral systems known to those skilled in the art may be used to deliver the ddRNAi or nucleic acid of the present invention to cells of interest, including but not limited to gene-deleted adenovirus-transposon vectors (see Yant, et al., Nature Biotech. 20:999-1004 (2002)); systems derived from Sindbis virus or Semliki forest virus (see Perri, et al, J. Virol. 74(20):9802-07 (2002)); systems derived from Newcastle disease virus or Sendai virus.

Testing nucleic acids and ddRNAi constructs of the disclosure Cell Culture Models

Exemplary cell culture -based methods which are useful for determining the ability of nucleic acids, ddRNAi constructs, expression constructs or compositions of the disclosure to reduce or inhibit expression of AMD-associated genes are described in Examples 2-8 herein.

Animal Models

There are a number animal models available to study wet AMD, including rodent, pig and non-human primate models established by laser or direct mechanical/surgical injury to the RPE/Bruch's membrane complex, alteration of the RPE and surrounding environment by external interventions (such as exogenous compounds injected in the subretinal space), VEGF-eluting scleral pellets, or internally (such as genetic knock-out models). A number of these rodent models are reviewed in Pennesi et al., (2012) Mol. Aspects Med., 33(4): 487- 509 and Grossniklaus et al., (2010) Prog. Retin. Eye Res., 26(6): 500-519, Lukason et al., (2011) Molecular Therapy, 19(2):260-265, and Lai et al., (2002) Gene Therapy, 9:804-813, the contents of each of which is incorporated by reference herein and the methods described therein contemplated for use in determining efficacy of nucleic acids, ddRNAi constructs, expression constructs or compositions of the disclosure for treating wet AMD or reducing CNV. An exemplary animal model-based method for determining efficacy of nucleic acids, ddRNAi constructs, expression constructs or compositions of the disclosure for treating wet AMD or reducing CNV is described in Example 9 hereof.

Carriers

In some examples, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are provided in a composition with a carrier.

In some examples, the carrier is a lipid-based carrier, cationic lipid, or liposome nucleic acid complex, a liposome, a micelle, a virosome, a lipid nanoparticle or a mixture thereof.

In some examples, the carrier is a polymer-based carrier such as a cationic polymer- nucleic acid complex.

In a further example, the carrier is a cyclodextrin -based carrier such as a cyclodextrin polymer- nucleic acid complex.

In a further example, the carrier is a protein-based carrier such as a cationic peptide- nucleic acid complex.

In another example, the carrier is a lipid nanoparticle. Exemplary nanoparticles are described, for example, in US7514099.

In some examples, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with a lipid nanoparticle composition comprising a cationic lipid/Cholesterol/PEG-C-DMA/DSPC (e.g., in a 40/48/2/10 ratio), a cationic lipid/Cholesterol/PEG-DMG/DSPC (e.g., in a 40/48/2/10 ratio), or a cationic lipid/Cholesterol/PEG-DMG (e.g., in a 60/38/2 ratio). In some examples, the cationic lipid is Octyl CL in DMA, DL in DMA, L-278, DLinKC2DMA, or MC3.

In another example, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with any of the cationic lipid formulations described in WO 2010/021865; WO 2010/080724; WO 2010/042877; WO 2010/105209 or WO 2011/022460.

In another example, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are conjugated to or complexed with another compound, e.g., to facilitate delivery of the RNA or ddRNAi or expression construct. Non- limiting, examples of such conjugates are described in US 2008/0152661 and US

2004/0162260 (e.g., CDM-LBA, CDM-Pip-LBA, CDM-PEG, CDM-NAG, etc.).

In another example, polyethylene glycol (PEG) is covalently attached to a nucleic acid or ddRNAi construct or expression construct of the disclosure. The attached PEG can be any molecular weight, e.g.,. from about 100 to about 50,000 daltons (Da).

In yet other example, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are formulated with a carrier comprising surface- modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes), such as is disclosed in for example, WO 96/10391; WO 96/10390; or WO 96/10392.

In some examples, the nucleic acids or ddRNAi constructs or expression constructs of the disclosure can also be formulated or complexed with polyethyleneimine or a derivative thereof, such as polyethyleneimine -polyethyleneglycol-N-acetylgalactosamine (PEI-PEG- GAL) or polyethyleneimine -polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG- triGAL) derivatives. In one example, a RNA or ddRNAi or expression construct of the disclosure is formulated as described in U.S. Patent Application Publication No.

2003/0077829.

In other examples, one or more of the nucleic acids or ddRNAi constructs or expression vectors of the disclosure is/are complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 2001/0007666.

Other carriers include cyclodextrins (see for example, Gonzalez et al., 1999,

Bioconjugate Chem., 10, 1068-1074; or WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example US 2002130430).

Compositions and methods of treatment

One or more nucleic acids, ddRNAi constructs or expression vectors of the disclosure may be formulated in a pharmaceutical composition for preventing or treating a disease or disorder of the eye characterised by undesired neovascularization. The term "disease or disorder of the eye characterized by undesired neovascularization" refers to any disease or disorder in which neovascularization causes or contributes to damage to the eye or a particular structure of the eye (e.g., retina, macula, rods, cones, retinal pigment epithelium, Bruch's membrane, etc.) or causes or contributes to impairment of vision from the eye. Diseases and disorders of the eye which are contemplated by this term include, but are not limited to, wet AMD, diabetic retinopathy, Diabetic Macular Edema (DME), corneal neovascularization, choroidal neovascularization, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization, Purtscher's retinopathy, Retinal Vein Occlusion (RVO), and the like. Although dry AMD is not primarily characterized by neovascularization, the fact that patients who develop the wet form of AMD are believed to have had the dry form of AMD first, supports the conclusion that the treatments described herein will be beneficial in the treatment of dry AMD e.g., to arrest or slow its progress to wet AMD, and that dry AMD may be included in this disease category.

Other examples of ocular diseases in which cellular degeneration has been implicated and for which the composition of the disclosure may be useful include retinal detachment, chorioretinal degenerations, retinal degenerations, photoreceptor degenerations, RPE degenerations, mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies and cone degenerations, particularly when such diseases or conditions are associated with a disease or disorder of the eye characterized by undesired neovascularization as described herein.

Accordingly, in one example, the disclosure provides a method of treating AMD in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein. In one example, the AMD is wet AMD. In another example, the AMD is dry AMD. In accordance with an example in which the AMD is dry AMD, treatment may comprise arresting or slowing progression of dry AMD to wet AMD.

In one example, the disclosure provides a method of treating Diabetic Macular Edema

(DME) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of reducing or inhibiting choroidal neovascularisation (CNV) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating pterygium in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein. In one example, the disclosure provides a method of treating histoplasmosis in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

In one example, the disclosure provides a method of treating Purtscher's retinopathy in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.In one example, the disclosure provides a method of treating or preventing retinal vein occlusion (RVO) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid, plurality of nucleic acids, a ddRNAi construct, a plurality of ddRNAi constructs, an expression vector, a plurality of expression vectors or a composition as described herein.

The therapeutic compositions of the disclosure may be used alone or in combination with one or more agents or compositions known to be suitable for treatment of the disease or condition. For example, the therapeutic compositions of the disclosure may be used alone or in combination with one or more agents or compositions known to be suitable for treatment of AMD, such as, for example, ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.

Compositions will desirably include materials that increase the biological stability of the nucleic acids, ddRNAi constructs or expression vectors of the disclosure and/or materials that increase the ability of the compositions to penetrate the eye, in particular, cells in and around the macula. The therapeutic compositions of the disclosure may be administered in pharmaceutically acceptable carriers (e.g., physiological saline), which are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. One having ordinary skill in the art can readily formulate a pharmaceutical composition that comprises one or more nucleic acids, ddRNAi constructs or expression vectors of the disclosure. In some cases, an isotonic formulation is used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some examples, a vasoconstriction agent is added to the formulation. The compositions according to the present disclosure are provided sterile and pyrogen free. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in

pharmaceutical formulations, are described in Remington: The Science and Practice of Pharmacy (formerly Remington's Pharmaceutical Sciences), Mack Publishing Co., a standard reference text in this field, and in the USP/NF.

Routes of administration include, but are not limited to, intravitreal injection, periocular injection and/or subretinal injection. An exemplary and preferred route of administration is intravitreal injection. Targeted transfection of the eye in vivo for delivery of nucleic acids, ddRNAi constructs or expression vectors of the disclosure may be accomplished through intravitreal injection or subretinal injection with a composition comprising one or more nucleic acids, ddRNAi constructs or expression vectors as described herein complexed with a suitable carrier. Such compositions are useful for pharmaceutical applications and may readily be formulated in a suitable sterile, non-pyrogenic vehicle, e.g., buffered saline for injection, for intravitreal, periocular and/or subretinal injection.

The volume, concentration, and formulation of the pharmaceutical composition as well as the dosage regimen may be tailored specifically to maximize cellular delivery while minimizing toxicity such as an inflammatory response. For example, an injection volume of 0.05ml is commonly used for intravitreal injection of existing point of care drugs for AMD, but this may be varied between about 0.01ml to about 0.2ml to achieve the optimal volume, concentration, and formulation combination. Kits

The present disclosure also provides the nucleic acids, ddRNAi constructs, expression vectors and/or compositions of the disclosure in a kit form. The kit may comprise a container. The kit typically contains one or more nucleic acids, ddRNAi constructs, expression vectors or compositions of the disclosure with instructions for administration. In some examples, the kit contains more than one nucleic acids, ddRNAi constructs, expression vectors or composition of the disclosure. In some examples, the kit contains more than one nucleic acids, ddRNAi constructs, expression vectors or compositions of the disclosure packed together with one or more other compounds for treatment of AMD, such as, for example, ranibizumab, aflibercept, bevacizumab, pegaptanib sodium and/or verteporfin.

Examples

Example 1: Design and generation of ddRNAi constructs targeting VEGFb and PGF

Messenger RNA (mRNA) sequences corresponding to VEGFb and PGF from human and macaca species were aligned, and conserved regions identified from the sequence alignments. Potential target regions for design of ddRNAi constructs were identified from those regions which were conserved across the alignments. Ten target regions were identified for VEGFb (Table 1) and twelve target regions were identified for PGF (Table 2).

DNA-directed RNA interference (ddRNAi) constructs coding for short-hairpin microRNAs (shmiRs) (also referred to hereinafter as "shmiR constructs") were then designed based on the target regions identified for VEGFb and PGF. To produce the shmiR constructs, DNA sequences coding for the respective effector and effector complement sequence combinations corresponding to the target regions for VEGFb and PGF were generated (set forth in Tables 3 and 4 respectively) and incorporated into a pre-miRNA backbone. Each shmiR construct comprised a DNA sequence coding for: a 5' flanking region of the pri-miRNA backbone (SEQ ID NO: 68); an effector complement sequence (see column 2 of Tables 3 and 4); a stem/loop junction sequence (SEQ ID NO 67); an effector sequence (see column 4 of Tables 3 and 4); and a 3 ' flanking region of the pri-miRNA backbone (SEQ ID NO: 69). The shmiR constructs coding for shmiRs targeting VEGFb and PGF are set forth in Tables 5 and 6 respectively, and the shmiR encoded by those shmiR constructs are set forth in Tables 7 and 8, respectively. Each shmiR construct was cloned between the BamHI / Hindlll sites of pSilencer 2.1-U6 hygro vector (ThermoFisher), which contained a human U6-1 promoter to drive expression of the shmiR. A map of the vector and an insert for a representative shmiR construct (designated "VEGFb shmiR- 1") are shown in Figures 1A and IB respectively. The secondary structure of the expressed VEGFb shmiR-1 predicted using m-Fold program is shown in Figures 1C.

Table 1 - Targeted regions in VEGFb

Table 2 - Targeted regions in PGF

Table 3 - shmiR effector and effector complement sequences for VEGFb

shmiR ID KITcetor complement sequence (5^* - J') SEQ I D NO: Effeclor sequence (5^* - 3') SEQ ID NO:

VEGFb_shmiR-l AGGAAAGUGGUGUCAUGGAU SEQ ID NO: 23 AUCCAUGACACCACUUUCCUC _: SEQ ID NO: 24

VEGFb_shmiR-2 AUGGGCACCGUGGCCAAACA SEQ ID NO: 25 UGUUUGGCCACGGUGCCCAU⁽ - SEQ ID NO: 26

VEGFb_shmiR-3 GCACCAAGUCCGGAUGCAGA SEQ ID NO: 27 UCUGCAUCCGGACUUGGUGC1 _j SEQ ID NO: 28

VEGFb_shmiR-4 GGGAGAUGUCCCUGGAAGAA SEQ ID NO: 29 UUCUUCCAGGGACAUCUCCCC _: SEQ ID NO: 30

VEGFb_shmiR-5 GGCUUAGAGCUCAACCCAGA SEQ ID NO: 31 UCUGGGUUGAGCUCUAAGCC< SEQ ID NO: 32

VEGFb_shmiR-6 AACAAAGAGGAGCCUGGUAA SEQ ID NO: 33 UUACCAGGCUCCUCUUUGUUi 2 SEQ ID NO: 34

VEGFb_shmiR-7 AAGACCUCAGCCCAGGCAGA SEQ ID NO: 35 UCUGCCUGGGCUGAGGUCUU ₃ SEQ ID NO: 36

VEGFb_shmiR-8 AUCAUCAAACAGGACAGAGU SEQ ID NO: 37 ACUCUGUCCUGUUUGAUGAU Q SEQ ID NO: 38

VEGFb_shmiR-9 AGGACAGAGUUGGAAGAGGA SEQ ID NO: 39 UCCUCUUCCAACUCUGUCCUC j SEQ ID NO: 40

VEGFb_shmiR-10 GGAUUUGGGCUUUGGUACAA SEQ ID NO: 41 UUGUACCAAAGCCCAAAUCC( SEQ ID NO: 42

Table 4 - shmiR effector and effector complement sequences

Table 5 - VEGFb shmiR-encoding construct

slimiK II) \ K( i l- l) shmiK en oding cassettes (r> - 3 ) S Q I I) NO:

VEGFb_shmiR-l GGTATATTGCTGTTGACAGTGAGCGAAGGAAAGTGGTGTCATGGATAC TGTGAAGCAGATGG SEQ ID NO: 92

GTATCCATGACACCACTTTCCTCCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-2 GGTATATTGCTGTTGACAGTGAGCGAATGGGCACCGTGGCCAAACAAC TGTGAAGCAGATGG SEQ ID NO: 93

GTTGTTTGGCCACGGTGCCCATGCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-3 GGTATATTGCTGTTGACAGTGAGCGTGCACCAAGTCCGGATGCAGAAC^r rGTGAAGCAGATGG SEQ ID NO: 94

GTTCTGCATCCGGACTTGGTGCTCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-4 GGTATATTGCTGTTGACAGTGAGCGAGGGAGATGTCCCTGGAAGAAAC TGTGAAGCAGATGG SEQ ID NO: 95

GTTTCTTCCAGGGACATCTCCCCCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-5 GGTATATTGCTGTTGACAGTGAGCGAGGCTTAGAGCTCAACCCAGAAC^r rGTGAAGCAGATGG SEQ ID NO: 96

GTTCTGGGTTGAGCTCTAAGCCCCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-6 GGTATATTGCTGTTGACAGTGAGCGAAACAAAGAGGAGCCTGGTAAAC TGTGAAGCAGATG SEQ ID NO: 97

GGTTTACCAGGCTCCTCTTTGTTCCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-7 GGTATATTGCTGTTGACAGTGAGCGAAAGACCTCAGCCCAGGCAGAAC TGTGAAGCAGATGG SEQ ID NO: 98

GTTCTGCCTGGGCTGAGGTCTTGCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-8 GGTATATTGCTGTTGACAGTGAGCGAATCATCAAACAGGACAGAGTAC TGTGAAGCAGATGG SEQ ID NO: 99

GTACTCTGTCCTGTTTGATGATGCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-9 GGTATATTGCTGTTGACAGTGAGCGAAGGACAGAGTTGGAAGAGGAAC TGTGAAGCAGATG SEQ ID NO: 100

GGTTCCTCTTCCAACTCTGTCCTGCGCCTACTGCCTCGGACTTCAA

VEGFb_shmiR-10 GGTATATTGCTGTTGACAGTGAGCGAGGATTTGGGCTTTGGTACAAACl GTGAAGCAGATGG SEQ ID NO: 101

GTTTGTACCAAAGCCCAAATCCCCGCCTACTGCCTCGGACTTCAA

Table 6 - PGF shmiR-encoding construct

slimiK II) Pii l- shmiR sequences (r> - 3 ) S Q I I) NO:

PGF_shmiR-l GGTATATTGCTGTTGACAGTGAGCGATCAGAAGGGAGCTGCTGl ^"CTACTGTGAAGCAGATGG SEQ ID NO: 102

GTAGACAGCAGCTCCCTTCTGAGCGCCTACTGCCTCGGACTTCA A

PGF_shmiR-2 GGTATATTGCTGTTGACAGTGAGCGACCAGAAGATGCTCGAACC :ACACTGTGAAGCAGATGG SEQ ID NO: 103

GTGTGGTTCGAGCATCTTCTGGACGCCTACTGCCTCGGACTTCA^

PGF_shmiR-3 GGTATATTGCTGTTGACAGTGAGCGAGGTCATGAGGCTGTTCCC TTACTGTGAAGCAGATGG SEQ ID NO: 104

GTAAGGGAACAGCCTCATGACCGCGCCTACTGCCTCGGACTTCA A

PGF_shmiR-4 GGTATATTGCTGTTGACAGTGAGCGAACCATGCAGCTCCTAAAC rATACTGTGAAGCAGATGG SEQ ID NO: 105

GTATCTTTAGGAGCTGCATGGTGCGCCTACTGCCTCGGACTTCA.

PGF_shmiR-5 GGTATATTGCTGTTGACAGTGAGCGACTACGTGGAGCTGACGTT CTACTGTGAAGCAGATGG SEQ ID NO: 106

GTAGAACGTCAGCTCCACGTAGGCGCCTACTGCCTCGGACTTCA A

PGF_shmiR-6 GGTATATTGCTGTTGACAGTGAGCGAGCCCTCTATTTATTAGCO AACTGTGAAGCAGATGG SEQ ID NO: 107

GTTTGGCTAATAAATAGAGGGCACGCCTACTGCCTCGGACTTCA A

PGF_shmiR-7 GGTATATTGCTGTTGACAGTGAGCGACAGGAATTCAGTGCCTTC AAACTGTGAAGCAGATGG SEQ ID NO: 108

GTTTGAAGGCACTGAATTCCTGACGCCTACTGCCTCGGACTTCA.

PGF_shmiR-8 GGTATATTGCTGTTGACAGTGAGCGAAAAGAGAGAAGCCAGCC ACAACTGTGAAGCAGATG SEQ ID NO: 109

GGTTGTGGCTGGCTTCTCTCTTTCCGCCTACTGCCTCGGACTTCA A

PGF_shmiR-9 GGTATATTGCTGTTGACAGTGAGCGATGCTACCTGTTCTTGGGC( TACTGTGAAGCAGATGGG SEQ ID NO: 110

TAGGCCCAAGAACAGGTAGCAGCGCCTACTGCCTCGGACTTCA^ >L

PGF_shmiR-10 GGTATATTGCTGTTGACAGTGAGCGAAGAACATTCAGCTCTGG^ LGAACTGTGAAGCAGATGG SEQ ID NO: 111

GTTCTCCAGAGCTGAATGTTCTGCGCCTACTGCCTCGGACTTCA^

PGF_shmiR-l l GGTATATTGCTGTTGACAGTGAGCGATTGTACTGGGACATTGTTt TACTGTGAAGCAGATGGG SEQ ID NO: 112

TAGAACAATGTCCCAGTACAAGCGCCTACTGCCTCGGACTTCAA

PGF_shmiR-12 GGTATATTGCTGTTGACAGTGAGCGTCTGCCAAGCCAGATTCTC TTACTGTGAAGCAGATGGG SEQ ID NO: 113

TAAGAGAATCTGGCTTGGCAGTCGCCTACTGCCTCGGACTTCAA

Table 7 - VEGFb shmiR sequences

Table 8 - PGF shmiR sequences

slimiK II) PG F shmiK sequences (5^* - 3") SFQ I I) NO:

PGF_shmiR-l GGUAUAUUGCUGUUGACAGUGAGCGAUCAGAAGGGAGCUGCU GUCUACUGUGAAGCAGA SEQ ID NO: 80

UGGGUAGACAGCAGCUCCCUUCUGAGCGCCUACUGCCUCGGAC :UUCAA

PGF_shmiR-2 GGUAUAUUGCUGUUGACAGUGAGCGACCAGAAGAUGCUCGAA CCACACUGUGAAGCAGAU SEQ ID NO: 81

GGGUGUGGUUCGAGCAUCUUCUGGACGCCUACUGCCUCGGAC1 JUCAA

PGF_shmiR-3 GGUAUAUUGCUGUUGACAGUGAGCGAGGUCAUGAGGCUGUUC CCUUACUGUGAAGCAGAU SEQ ID NO: 82

GGGUAAGGGAACAGCCUCAUGACCGCGCCUACUGCCUCGGACl JUCAA

PGF_shmiR-4 GGUAUAUUGCUGUUGACAGUGAGCGAACCAUGCAGCUCCUAA AGAUACUGUGAAGCAGAU SEQ ID NO: 83

GGGUAUCUUUAGGAGCUGCAUGGUGCGCCUACUGCCUCGGACl UUCAA

PGF_shmiR-5 GGUAUAUUGCUGUUGACAGUGAGCGACUACGUGGAGCUGACG UUCUACUGUGAAGCAGAU SEQ ID NO: 84

GGGUAGAACGUCAGCUCCACGUAGGCGCCUACUGCCUCGGACl JUCAA

PGF_shmiR-6 GGUAUAUUGCUGUUGACAGUGAGCGAGCCCUCUAUUUAUUAG CCAAACUGUGAAGCAGAU SEQ ID NO: 85

GGGUUUGGCUAAUAAAUAGAGGGCACGCCUACUGCCUCGGAC UUCAA

PGF_shmiR-7 GGUAUAUUGCUGUUGACAGUGAGCGACAGGAAUUCAGUGCCU UCAAACUGUGAAGCAGAU SEQ ID NO: 86

GGGUUUGAAGGCACUGAAUUCCUGACGCCUACUGCCUCGGAC1 JUCAA

PGF_shmiR-8 GGUAUAUUGCUGUUGACAGUGAGCGAAAAGAGAGAAGCCAGC CACAACUGUGAAGCAGAU SEQ ID NO: 87

GGGUUGUGGCUGGCUUCUCUCUUUCCGCCUACUGCCUCGGACl JUCAA

PGF_shmiR-9 GGUAUAUUGCUGUUGACAGUGAGCGAUGCUACCUGUUCUUGG GCCUACUGUGAAGCAGAU SEQ ID NO: 88

GGGUAGGCCCAAGAACAGGUAGCAGCGCCUACUGCCUCGGAC1 JUCAA

PGF_shmiR-10 GGUAUAUUGCUGUUGACAGUGAGCGAAGAACAUUCAGCUCUG GAGAACUGUGAAGCAGA SEQ ID NO: 89

UGGGUUCUCCAGAGCUGAAUGUUCUGCGCCUACUGCCUCGGAi UUCAA

PGF_shmiR-l l GGUAUAUUGCUGUUGACAGUGAGCGAUUGUACUGGGACAUUC RUUCUACUGUGAAGCAGA SEQ ID NO: 90

UGGGUAGAACAAUGUCCCAGUACAAGCGCCUACUGCCUCGGAi UUCAA

PGF_shmiR-12 GGUAUAUUGCUGUUGACAGUGAGCGUCUGCCAAGCCAGAUUC UCUUACUGUGAAGCAGAU SEQ ID NO: 91

GGGUAAGAGAAUCUGGCUUGGCAGUCGCCUACUGCCUCGGACl UUCAA

Example 2: Activity and strand-specificity of ddRNAi constructs

Dual luciferase reporter assays were then performed to determine the activities of shmiRs when expressed from the shmiR constructs. For these experiments, reporter constructs containing regions of VEGF-b and PGF cDNA were cloned into the 3 ' UTR of a firefly luciferase expression vector, pGL3-control (Promega), using Xbal / Fsel restriction enzyme sites. Three separate regions of VEGF-b cDNA were designed to prepare reporter constructs that could be used to assay VEGFb shmiR 1-10. Similarly, four separate regions of PGF cDNA were designed for assaying PGF shmiR 1- 12. The regions of VEGFb and PGF included in the respective report constructs are shown in Table 9. In addition, these cDNA regions were cloned in both orientations, which allowed the strand preference of RISC loading to be determined. "Anti-sense" reporters were used to assay activity of effector strand while "Sense" reporter constructs assayed activity of passenger strand. shmiR constructs with strong effector activity and weak passenger activity were strongly favoured for potential therapeutic use because they are likely to produce less off target effects in vivo.

Table 9

VEGFb or PGF shmiR and reporter constructs were co-transfected into HEK293 cells using Lipofectamine 2000 reagents (Thermofisher) according to manufacturer's protocol. Briefly, HEK293 cell were seeded at a density of 2.5 x 10^A4 cells per well onto 96-well culture plate one day prior to transfection. For each well of transfection, 100 ng of shmiR construct, lOng of Luciferase reporter construct and lng of Renilla reporter construct (served as loading controls) was co-transfected using 0.3uL of Lipotectamine 2000. In a parallel transfection, pSilencer vector containing sequence coding for a non-active shmiR was used to serve as negative control (Thermofisher). 48 hour post-transfection, cell lysates were collected and analyzed using Dual Luciferase Reporter Assay System (Promega). Percent inhibition was calculated as relative luciferase activity to the pSilencer control.

Results of typical experiments are shown in Figure 2A for VEGFb shmiRs and Figure 2B for PGF shmiRs. These data showed that all ddRNAi constructs exhibited significant silencing of the antisense target, but differed significantly in activity against the sense target, reflecting differences in RISC loading of passenger strands between the different shmiR constructs.

Example 3: Hyperfuntional properties of ddRNAi constructs

Hyperfunctional properties of the shmiR constructs were then tested using the same dual luciferase reporter assay as described in Example 2. VEGFb or PGF shmiR "sense" and "anti-sense" reporter constructs were co-transfected into HEK293 cells using Lipofectamine 2000 reagents (Thermofisher) according to manufacturer's protocol. Briefly, HEK293 cell were seeded at a density of 2.5 x 10^A4 cells per well onto 96-well culture plate one day prior to transfection. For each well of transfection, a serial dilution of shmiR construct (lOOng, 50ng, 20ng, 5ng, 1.67ng, 0.56ng, 0.19ng, 0.06ng and 0.02ng), lOng of Luciferase reporter construct and lng of Renilla reporter construct (served as loading controls) was co- transfected using 0.3uL of Lipotectamine 2000. In a parallel transfection, pSilencer vector containing a sequence coding for a non-active shmiR was used to serve as negative control (Thermofisher). 48 hour post-transfection, cell lysates were collected and analyzed using Dual Luciferase Reporter Assay System (Promega). Percent inhibition was calculated as relative luciferase activity to the pSilencer control.

These data stratified the ability of each shmiR construct to inhibit expression of luciferase reporter carrying target sequences in a dose dependent manner (Figure 3A and 3B). Based on these data VEGFb shmiR- 1, VEGFb shmiR-4 and VEGFb shmiR-10, and PGF shmiR-3, PGF shmiR-7 and PGF shmiR-10, were chosen for further analyses. Example 4: Down-regulation of VEGFb and PGF expression by individual ddRNAi constructs

To confirm the activity of individual shmiR constructs against endogenously expressed VEGFb and PGF targets, VEGFb and PGF single shmiR constructs (5 ug) were transfected into ARPE-19 (human retinal pigment epithelial) and BeWo (human placental choriocarcinoma) cells (5.6 x 10^A5 cells) using Neon Electroporation system

(Thermofisher). After electroporation, ARPE-19 cells were plated onto 24-well plate at density of 7x 10^A4 cells per well. BeWo cells were plated onto 12-well plate at density of 2 xl0^A5 cells per well. At 48hr, 72hr and 96hr hour post-transfection, RNAs were extracted using miRNeasy mini Kit (Qiagen). VEGFb and PGF mRNA expression levels were detected by RT-qPCR.

RT-qPCR analysis: 100 ng of total RNA was used to synthesize cDNA using High Capacity cDNA Reverse Transcription Kit (Thermofisher). qPCR amplification of VEGFb, PGF and GAPDH (as loading control) were performed using TaqMan gene expression assays (VEGFb: Hs00173634_ml, PGF: Hs00182176_ml, GAPDH: Hs02758991_gl) and PCR Master Mix (Thermofisher). Standard real-time PCR conditions were used: initial denaturation at 95°C for lOmin followed by 40 cycles of 95°C for 15sec and 60°C for 1 min.

The expression level of each mRNA was normalized to GAPDH. Percent mRNA inhibition was calculated as relative mRNA levels to the pSilencer control (Figure 4A and 4B).

As shown in Figure 4, VEGFb shmiRs down-regulated VEGFb mRNA expression with percent inhibition ranging from 70% to 92% at 96 hr time point (Figure 4A), while PGF shmiR inhibited PGF mRNA expression ranging from 50% to 71% in BeWo cells (Figure 4B).

Example 5: Design of the triple shmiR constructs concomitantly expressing three shmiRs targeting VEGFa, VEGFb and PGF

ddRNAi constructs that can simultaneously express three shmiRs were generated by incorporating three shmiR constructs, each operably-linked to a U6 promoter, into a pUC57- mini-Kan vector (GenScript). Each triple shmiR construct was comprised of (i) a wildtype or modified U6-9 promoter upstream of a VEGFb shmiR, (ii) a wildtype or modified U6-1 promoter upstream of a VEGFa shmiR and (iii) a wildtype or modified U6-8 promoter upstream of a PGF shmiR. The triple shmiR constructs were flanked by Sphl and EcoRI restriction enzyme sites to facilitate subcloning into adenovirus or adeno- associated virus (AAV) based vectors. Four such constructs were generated and subcloned into adenovirus expression vector for further testing (Figure 5). These triple shmiR adenovirus vectors were as follows:

(1) wtU6-Psh7: wildtype promoters with VEGFb shmiR- 1, VEGFa shmiR- 8 and PGF shmiR-7;

(2) wtU6-Psh3: wildtype promoters with VEGFb shmiR- 1, VEGFa shmiR- 8 and PGF shmiR-3;

(3) PSE-Psh7: modified promoters with VEGFb shmiR- 1, VEGFa shmiR-8 and PGF shmiR- 7; and (4) PSE-Psh3: modified promoters with VEGFb shmiR- 1, VEGFa shmiR-8 and PGF shmiR- 3.

The VEGFa shmiR-8 construct was comprised of DNA sequences coding for: a 5' flanking region of the pri-miRNA backbone (SEQ ID NO: 68); an effector complement sequence (SEQ ID NO: 114); a stem/loop junction sequence (SEQ ID NO 67); an effector sequence (SEQ ID NO: 115); and a 3 ' flanking region of the pri-miRNA backbone (SEQ ID NO: 69). The DNA sequence of the VEGFa shmiR- 8 construct is set forth in SEQ ID NO: 117 and encodes the shmiR set forth in SEQ ID NO: 116.

Example 6: Down-regulation of VEGFa target expression by triple shmiR constructs.

ddRNAi activities of the four AAV-based triple shmiR constructs described

Example 5 were tested first by packaging the constructs into functional adenovirus. The adenoviruses were then used to infect relevant tissue culture cells expressing relevant targets. To evaluate VEGFa shmiR activities, ARPE-19 cells, a human retinal pigment epithelial cell line, were employed. Briefly, ARPE-19 cells were infected with wtU6-Psh7 or PSE-Psh7 adenovirus in suspension and then cultured on 12-well plates. In a parallel set of samples, ARPE-19 cells were infected with adenovirus expressing a clinical construct TT034 as negative control or a single VEGFa shmiR-8 as positive control. Each well contained a suspension of 1.2 x 10^A5 cells and adenovirus of the indicated MOIs (10, 30, 100, 175 and 300). 72hr and 144hr post-infection, RNA was extracted using miRNeasy mini kit (Qiagen). VEGFa mRNA and shmiR expression levels were detected using RT-qPCR analysis (Figure 6 A and 6C). Cell culture medium was collected and secreted VEGFa protein levels were measured using ELISA assay (Figure 6B).

Reverse transcription-quantitative real time PCR (RT-qPCR) of VEGFa, VEGFb and PGF mRNA: 100 ng of total RNA was used to synthesize cDNA using High Capacity cDNA Reverse Transcription Kit (Thermofisher). qPCR amplification of VEGFa, VEGFb, PGF and GAPDH (as loading control) were performed using TaqMan gene expression assays (VEGFa: Hs00900055_ml, VEGFb: Hs00173634_ml, PGF: Hs00182176_ml,

GAPDH: Hs02758991_gl) and PCR Master Mix (Thermofisher). Standard real-time PCR conditions were used: initial denaturation at 95°C for lOmin followed by 40 cycles of 95°C for 15sec and 60°C for 1 min. Percent inhibition of mRNA was calculated as relative expression levels to TT034 negative control. Reverse transcription-quantitative real time PCR (RT-qPCR) of VEGFa, VEGFb and

PGF shmiR: Production of shmiR from the respective constructs was measured using miScript PCR system (Qiagen). For each RT-qPCR analysis, 50ng of total RNA were converted into cDNA using Qiagen' s miScript II RT kit. Quantitative PCR of shmiR was carried out using miScript SYBR PCR kit with custom primers and RNA oligo standards listed as following: Primer name Primer sequence (5' to 3 ')

VEGFb-shmiRl_fwd primer seq ATCCATGACACCACTTTCCTC (SEQ ID NO: 118)

VEGFa-shmiR8_fwd primer seq TATGTGGGTGGGTGTGTCTAC (SEQ ID NO: 119)

PGF-shmiR7_fwd primer seq TTGAAGGCACTGAATTCCTGA (SEQ ID NO: 120)

PGF-shmiR3_fwd primer seq AAGGGAACAGCCTCATGACCG (SEQ ID NO: 121)

VEGFb-shmiRl_RNA standard olij *o seq AUCCAUGACACCACUUUCCUC (SEQ ID NO: 122)

VEGFa-shmIR8_RNA standard olij *o seq UAUGUGGGUGGGUGUGUCUAC (SEQ ID NO: 123)

PGF-shmiR7_RNA standard oligo seq UUGAAGGCACUGAAUUCCUGA (SEQ ID NO: 124)

PGF-shmiR3_RNA standard oligo seq AAGGGAACAGCCUCAUGACCG (SEQ ID NO: 125)

Real-time PCR conditions were as following: initial denaturation at 95 °C for 15min followed by 40 cycles of 94°C for 15sec, 55°C for 30sec and 70°C for 30sec. Copy number of shmiR expression levels was calculated based on RT-qPCR standards constituted of RNA oligo s. ELISA assay for detecting secreted VEGFa protein in cell culture medium: S ecreted

VEGFa protein was analyzed using Duoset ELISA development system (R&D systems) according to manufacturer instructions. Percent inhibition of protein levels was calculated as relative expression levels to TT034 negative control.

As shown in Figure 6, VEGFa shmiR-8 embedded in the triple shmiR constructs was able to inhibit VEGFa expression level in a dose dependent manner. VEGFa shmiR-8 controlled by the modified U6-1 promoter was expressed at much lower levels and thus it took longer time to accumulate enough shmiR to achieve inhibition levels similar to that of the wildtype U6-1 promoter.

Example 7: Down-regulation of VEGFb target expression by triple shmiR constructs.

VEGFb shmiR activities in the triple shmiR constructs were examined by infecting ARPE-19 cells with wtU6-Psh7 or PSE-Psh7 adenovirus described in Example 6. At 72hr and 144hr post-infection, RNAs were extracted. VEGFb mRNA and shmiR expression levels were detected by RT-qPCR. Percent inhibition of mRNA was calculated as relative expression levels to TT034 negative control.

As shown in Figure 7, both wildtype and modified U6-9 promoters expressed VEGFb shmiR- 1 at very high levels (Figure 7B). This led to strong VEGFb mRNA inhibition, achieving 80% -90% inhibition at fairly low MOI of 10 in a short period of time (72hr post-infection, Figure 7A).

Example 8: Down-regulation of PGF target expression by triple shmiR constructs. To determine PGF shmiR activities in triple shmiR constructs, JEG-3 cells, and a human placental choriocarcinoma cell line that expressed high levels of PGF target were used.

JEG-3 cells were infected with wtU6-Psh7, wtU6-Psh3, PSE-Psh7, or PSE-Psh3 adenovirus in suspension and then cultured on 12-well plates. Each well contained a suspension of 8 x 10^A4 cells and adenovirus of the indicated MOIs (100, 175, 300 and 600). In a parallel set of samples, JEG-3 cells were infected with adenovirus expressing a clinical construct TT034 as negative control. At 72hr and 144hr post-infection, RNAs were extracted. PGF mRNA and shmiR expression levels were detected by RT-qPCR. Percent inhibition of mRNA was calculated as relative expression levels to TT034 negative control (Figure 8 A, 8B and 8C).

As shown in Figure 8, good expression levels of PGF shmiR-3 and shmiR-7 were already detected in JEG-3 cells at 72hr time point. Correspondingly, PGF mRNA expression was down regulated by either PGF shmiR-3 or shmiR-7 at levels ranging from 50% to 80%.

Example 9: Inhibition of Choroidal Neovascularization in a Rat Model

In vivo efficacy of triple ddRNAi construct is examined using a rat laser-induced choroidal neovascularisation (CNV) model. Briefly, wtU6-Psh7 - a triple shmiR ddRNAi construct described in Example 5 is subcloned into a single-strand AAV expression vector (named as BB-201). To ensure that BB-201 can be delivered into retina efficiently, AAV2 based capsid variant (CapVar7) is applied to package BB-201 into functional AAV virus using triple transfection methodology. The final drug substance is labeled as CapVar7-BB- 201. In parallel, a triple ddRNAi control construct ("TT035") is also packaged using the same capsid protein into a control substance, labelled as CapVar7-TT035.

CapVar7-BB-201 and TT035 are administrated into Rat eyes through a single intravitreal injection. Three dosing cohorts each with five animals (10 eyes) are used for both the drug substance BB-201 and the control substance TT035. The dosing levels are: 2 e8, 2 e9 and 2 elO vg per eye in 2.5 uL injection volume.

Four weeks after drug administration, CNV is induced using laser diode at 810 nm wavelength. Three to four spots of laser burns are generated in each eye. Two and three weeks after laser treatment, CNV lesion size are examined using Fundus Fluorescein Angiography.

Example 10: Delivery and expression of a triple shmiR construct in a non-human primate model

In vivo expression of triple ddRNAi construct was examined using a non-human primate model, St. Kitts African green monkeys (Chlorocebus sabaeus).

CapVarl-BB-201 (described in Example 9 above) was administrated into monkey eyes through a single intravitreal injection. The dosing levels were 1 el2 vg per eye in ΙΟΟμί injection volume. Eight weeks after drug administration, eye globes were harvested and dissected into five segments as macular region and temporal, nasal, superior and inferior petals. Retina and RPE/Choroid tissue layers were further separated from each segment. RNA were extracted from Retina and RPE/choroid tissue of macular and temporal region using miRNeasy mini kit (Qiagen). shmiR expression levels were determined using RT- qPCR analysis RNA as described in Example 6.

As shown in Figure 9, all three shmiRs were expressed at high levels in retina layer. Lower levels of shmiR expression was also detected at RPE/Choroid layer. This

demonstrates the potential of using triple shmiR construct BB-201 to deliver shmiRs to the appropriate tissue for treatment AMD disease.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

WE CLAIM:

1. A nucleic acid comprising a DNA sequence which encodes a short hairpin micro- RNA (shmiR), said shmiR comprising:

an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence is substantially complementary to a region of equivalent length in an RNA transcript of VEGFb set forth set forth in any one of SEQ ID NOs: 1-10 or a region of equivalent length in an RNA transcript of PGF set forth set forth in any one of SEQ ID NOs: 11-22.

2. The nucleic acid according to claim 1, wherein the shmiR is selected from the group consisting of:

VEGFb_shmiR- 1 comprising an effector complement sequence set forth in SEQ ID NO:23 and an effector sequence set forth in SEQ ID NO:24;

VEGFb_shmiR-2 comprising an effector complement sequence set forth in SEQ ID NO:25 and an effector sequence set forth in SEQ ID NO:26;

VEGFb_shmiR-3 comprising an effector complement sequence set forth in SEQ ID NO:27 and an effector sequence set forth in SEQ ID NO:28;

VEGFb_shmiR-4 comprising an effector complement sequence set forth in SEQ ID NO:29 and an effector sequence set forth in SEQ ID NO:30;

VEGFb_shmiR-5 comprising an effector complement sequence set forth in SEQ ID NO:31 and an effector sequence set forth in SEQ ID NO:32;

VEGFb_shmiR-6 comprising an effector complement sequence set forth in SEQ ID NO:33 and an effector sequence set forth in SEQ ID NO:34;

VEGFb_shmiR-7 comprising an effector complement sequence set forth in SEQ ID NO:35 and an effector sequence set forth in SEQ ID NO:36;

VEGFb_shmiR-8 comprising an effector complement sequence set forth in SEQ ID NO:37 and an effector sequence set forth in SEQ ID NO:38; VEGFb_shmiR-9 comprising an effector complement sequence set forth in SEQ ID NO:39 and an effector sequence set forth in SEQ ID NO:40;

VEGFb_shmiR-10 comprising an effector complement sequence set forth in SEQ ID NO:41 and an effector sequence set forth in SEQ ID NO:42;

PGF_shmiR-l comprising an effector complement sequence set forth in SEQ ID NO:43 and an effector sequence set forth in SEQ ID NO:44;

PGF_shmiR-2 comprising an effector complement sequence set forth in SEQ ID NO:45 and an effector sequence set forth in SEQ ID NO:46;

PGF_shmiR-3 comprising an effector complement sequence set forth in SEQ ID NO:47 and an effector sequence set forth in SEQ ID NO:48;

PGF_shmiR-4 comprising an effector complement sequence set forth in SEQ ID NO:49 and an effector sequence set forth in SEQ ID NO:50;

PGF_shmiR-5 comprising an effector complement sequence set forth in SEQ ID NO:51 and an effector sequence set forth in SEQ ID NO:52;

PGF_shmiR-6 comprising an effector complement sequence set forth in SEQ ID NO:53 and an effector sequence set forth in SEQ ID NO:54;

PGF_shmiR-7 comprising an effector complement sequence set forth in SEQ ID NO:55 and an effector sequence set forth in SEQ ID NO:56;

PGF_shmiR-8 comprising an effector complement sequence set forth in SEQ ID NO:57 and an effector sequence set forth in SEQ ID NO:58;

PGF_shmiR-9 comprising an effector complement sequence set forth in SEQ ID NO:59 and an effector sequence set forth in SEQ ID NO:60;

PGF_shmiR-10 comprising an effector complement sequence set forth in SEQ ID NO:61 and an effector sequence set forth in SEQ ID NO:62;

PGF_shmiR-l l comprising an effector complement sequence set forth in SEQ ID NO:63 and an effector sequence set forth in SEQ ID NO:64; and

PGF_shmiR-12 comprising an effector complement sequence set forth in SEQ ID NO:65 and an effector sequence set forth in SEQ ID NO:66.

3. The nucleic acid according to claim 1 or claim 2, wherein the shmiR comprises, in a 5' to 3' direction:

(i) a 5' flanking sequence of the pri-miRNA backbone; the effector complement sequence;

a stemloop sequence;

the effector sequence; and

a 3' flanking sequence of the pri-miRNA backbone; or

(ii) a 5' flanking sequence of the pri-miRNA backbone;

the effector sequence;

a stemloop sequence;

the effector complement sequence; and

a 3' flanking sequence of the pri-miRNA backbone.

4. The nucleic acid according to any one of claims 1 to 3, wherein the stemloop sequence is the sequence set forth in SEQ ID NO: 67.

5. The nucleic acid according to any one of claims 1 to 4, wherein the pri-miRNA backbone is a pri-miR-30a backbone.

6. The nucleic acid according to any one of claims 3 to 5, wherein the 5' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 68 and the 3' flanking sequence of the pri-miRNA backbone is set forth in SEQ ID NO: 69.

7. The nucleic acid according to any one of claims 3 to 5, wherein the shmiR comprises a sequence set forth in any one of SEQ ID NOs: 70-91.

8. The nucleic acid according to any one of claims 1 to 7, wherein the DNA sequence which encodes the shmiR is set forth in any one of SEQ ID NO: 92-113.

9. A plurality of nucleic acids, comprising:

(a) at least one nucleic acid according to any one of claims 1 to 8; and

(b) at least one further nucleic acid selected from:

(i) a nucleic acid according to any one of claims 1 to 8; or

(ii) a nucleic acid comprising a DNA sequence encoding a shmiR targeting VEGFa (VEGFa_shmiR) comprising: an effector sequence of at least 17 nucleotides in length;

an effector complement sequence;

a stemloop sequence; and

primary micro RNA (pri-miRNA) backbone;

wherein the effector sequence is substantially complementary to a region of equivalent length in the RNA transcript set forth in SEQ ID NO: 114;

10. The plurality of nucleic acids according to claim 9, wherein VEGFa_shmiR comprises an effector complement sequence set forth in SEQ ID NO: 114 and an effector sequence set forth in SEQ ID NO: 115.

11. The plurality of nucleic acids according to claim 9 or claim 10, wherein:

the stemloop sequence of VEGFa_shmiR is the sequence set forth in SEQ ID NO:

67;

the 5' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 68; and

the 3' flanking sequence of the pri-miRNA backbone of VEGFa_shmiR is set forth in SEQ ID NO: 69.

12. The plurality of nucleic acids according to any one of claims 9 to 11, wherein the VEGFa_shmiR comprises the sequence set forth in SEQ ID NO: 116.

13. The plurality of nucleic acids according to any one of claims 9 to 12, wherein the nucleic acid encoding VEGFa_shmiR comprises the DNA sequence set forth in SEQ ID NO: 117.

14. The plurality of nucleic acids according to any one of claims 9 to 13, comprising at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting PGF.

15. The plurality of nucleic acids according to claim 14, wherein the shmiR targeting VEGFb is VEGFb_shmiR-l and the shmiR targeting PGF is PGF_shmiR-3 or PGF_shmiR- 7.

16. The plurality of nucleic acids according to any one of claims 9 to 13, comprising at least one nucleic acid encoding a shmiR targeting VEGFb and at least one nucleic acid encoding a shmiR targeting VEGFa.

17. The plurality of nucleic acids according to claim 16, wherein the shmiR targeting VEGFb is VEGFb_shmiR-l and the shmiR targeting VEGFa is VEGFa_shmiR.

18. The plurality of nucleic acids according to any one of claims 9 to 13, comprising at least one nucleic acid encoding a shmiR targeting PGF and at least one nucleic acid encoding a shmiR targeting VEGFa.

19. The plurality of nucleic acids according to claim 18, wherein the shmiR targeting PGF is PGF_shmiR-3 or PGF_shmiR-7 and the shmiR targeting VEGFa is VEGFa_shmiR.

20. The plurality of nucleic acids according to any one of claims 9 to 19, comprising at least one nucleic acid encoding a shmiR targeting VEGFb, at least one nucleic acid encoding a shmiR targeting PGF and at least one nucleic acid encoding a shmiR targeting VEGFa.

21. The plurality of nucleic acids according to claim 20, wherein the shmiR targeting VEGFb is VEGFb_shmiR-l, the shmiR targeting PGF is PGF_shmiR-3 or PGF_shmiR-7, and the shmiR targeting VEGFa is VEGFa_shmiR.

22 A DNA-directed RNA interference (ddRNAi) construct comprising a nucleic acid according to any one of claims 1 to 8 or a plurality of nucleic acids according to any one of claims 9 to 21.

23. The ddRNAi construct according to claim 22, comprising at least two nucleic acids according to any one of claims 1 to 8, wherein each of the nucleic acids encode different shmiRs.

24. The ddRNAi construct according to claim 23, said ddRNAi construct comprising in a 5' to 3' direction:

(a) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:70:

(b) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO: 116; and

(c) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:82.

25. The ddRNAi construct according to claim 24, said ddRNAi construct comprising in a 5' to 3' direction:

26. The ddRNAi construct according to claim 23, said ddRNAi construct comprising in a 5' to 3' direction:

(c) a nucleic acid encoding a shmiR comprising or consisting of the sequence set forth in SEQ ID NO:86.

27. The ddRNAi construct according to claim 26, said ddRNAi construct comprising in a 5' to 3' direction:

28. The ddRNAi construct according to any one of claims 22 to 27, comprising a RNA pol III promoter upstream of the or each nucleic acid encoding a shmiR.

29. The ddRNAi construct of claim 22, wherein the or each RNA pol III promoter is selected from a U6 and a HI promoter.

30. The ddRNAi construct of claim 28 or claim 29, wherein the or each RNA pol III promoter is a U6 promoter selected from a U6-9 promoter, a U6-1 promoter and U6-8 promoter.

32. The ddRNAi construct according to claim 30, said ddRNAi construct comprising in a 5' to 3' direction:

33. The ddRNAi construct according to claim 24, said ddRNAi construct comprising in a 5' to 3' direction:

34. The ddRNAi construct according to any one of claims 28 to 33, wherein one or more of the promoters is operably-linked to a proximal sequence element 7 (PSE7).

35. The ddRNAi construct according to claim 34, said ddRNAi construct comprising in a 5' to 3' direction:

(a) PSE7 and U6-9 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO:92:

(b) PSE7 and U6-1 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 117; and

(c) PSE7 and U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 104.

36. The ddRNAi construct according to claim 34, said ddRNAi construct comprising in a 5' to 3' direction:

(c) PSE7 and U6-8 promoter upstream of a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 108.

37. An expression vector comprising the ddRNAi construct of any one of claims 22 to 36.

38. A plurality of expression vectors, each expression vector comprising a ddRNAi construct according to any one of claims 22 to 36 capable of expressing one or more shmiRs.

39. The expression vector of claim 37 or the plurality of expression vectors according to claim 38, wherein the or each expression vector is a plasmid or minicircle.

40. The expression vector of claim 37 or the plurality of expression vectors according to claim 38, wherein the or each expression vector is a viral vector selected from the group consisting of an adeno-associated viral (AAV) vector, a retroviral vector, an adenoviral (AdV) vector and a lentiviral (LV) vector.

41. A composition comprising a DNA Directed RNA interference (ddRNAi) construct according to any one of claims 22 to 36 or an expression vector according to any one of claims 37 or 39 to 40 or a plurality of expression vectors according to any one of claims 38 to 40.

42. The composition according to claim 41 further comprising one or more

pharmaceutically acceptable carriers.

43. A method of treating age-related macular degeneration (AMD) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 1 to 8 or a plurality of nucleic acids according to any one of claims 9 to 21 or a ddRNAi construct according to any one of claims 22 to 36 or an expression vector according to any one of claims 37 or 39 or 40 or a plurality of expression vectors according to any one of claims 38 to 40 or a composition according to claim 41 or 42.

44. The method according to claim 43, wherein the AMD is wet AMD.

45. A method of reducing or inhibiting choroidal neovascularisation (CNV) in a subject, said method comprising administering to the subject a therapeutically effective amount of a nucleic acid according to any one of claims 1 to 8 or a plurality of nucleic acids according to any one of claims 9 to 21 or a ddRNAi construct according to any one of claims 22 to 36 or an expression vector according to any one of claims 37 or 39 or 40 or a plurality of expression vectors according to any one of claims 38 to 40 or a composition according to claim 41 or 42.

46. The method according to any one of claims 43 to 45, wherein the administration to the subject is by intravitreal injection.