WO2018160690A1 - Rna pharmaceutical formulations for prophylactic and therapeutic treatment of zika virus infection - Google Patents

Abstract

The present invention provides prophylactic and therapeutic pharmaceutical compositions comprising either siRNA or mRNA molecules for prevention and treatment of Zika virus infection and provides methods for their use.

Description

RNA PHARMACEUTICAL FORMULATIONS FOR PROPHYLACTIC AND THERAPEUTIC TREATMENT OF ZIKA VIRUS INFECTION

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Patent

Application No. 62/465,096, filed February 28, 2017, which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

This invention presents novel prophylactic and therapeutic agents for prevention and treatment of Zika virus (ZIKV) infection and methods for their use. BACKGROUND

Zika virus infection: Biology and Pathology

Initially identified in 1947 in Uganda, the Zika virus (a member of the flaviviridae family including West Nile Virus, Dengue and Chikungunya viruses) has recently garnered great interest because of a link between infection in pregnant women and miscarriage or birth defects in their infants. Transmission is via the bite of an infected Aedes mosquito, and a rapid increase in transmission observed in Brazil since late 2014 preceded an increase in the number of cases of microcephaly in infants reported to the Brazil Ministry of Health in 2015 [1, 2]. As of November 2016, a total of more than 4, 100 suspected microcephaly cases had been reported, many of which occurred in infants born to women who lived in or had visited areas where Zika virus transmission was observed [3]. There is now evidence of an association between Zika virus infection during early pregnancy and the occurrence of microcephaly. Viral infection has typically only resulted in patients presenting symptoms of mild fever, rash, conjunctivitis and arthralgia [4]. In the outbreak in Brazil, Zika virus (ZIKV) was detected by reverse transcription-polymerase chain reaction from the sera of eight patients and the result was confirmed by DNA sequencing. Phylogenetic analysis suggested that the ZIKV identified in this region belonged to the Asian clade [4]. ZIKA virus infections have now been identified and validated in the mainland US in Florida and gulf coast states, and the need for suitable prophylactic or therapeutic options is significant.

ZIKV is a single-strand positive RNA virus, belonging to family Flaviviridae; genus Flavivirus. The genome of ZIKV consists of about 10K nucleotides, which code at least three structural proteins (capsid (C), membrane (prM; processed to M) and envelope (E)), and seven non-structural proteins (NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5). In ZIKV, the single ORF is flanked by 5'- and 3'- untranslated regions (UTRs), respectively (Figure 1). Both the 5' and the 3' UTRs form several additional RNA elements that also play important roles in regulation of mRNA translation and are required for viral RNA replication, as well as in interactions with viral and cellular proteins. RNA elements of the 3' UTR greatly enhance viral RNA synthesis. ZIKV infection initiates from envelope (E) glycoproteins on the surface of the ZIKV virion binding to a host cell receptor, such as AXL and broadly neutralizing antibodies have been demonstrated against this E protein region [5]. ZIKV enters the host cell by endocytosis, and then its genome escapes from the endosome into the cytoplasm after viral-host membrane fusion driven by the fusion loop. In the cytoplasm, the viral genome is first translated into a large polyprotein, and then cleaved by host signal peptidases and a viral serine protease (during and after translation) to generate the viral proteins. Also in the cytoplasm, the positive strand RNA genome produces a negative strand RNA template to generate new copies of the genome.

AXL, a member of the receptor tyrosine kinase subfamily, has been identified as the major receptor for ZIKV binding. The study showed that blocking or silencing AXL dramatically reduced ZIKV replication in fibroblasts and alveolarepithelial cells [6]. The function of AXL. includes transducing signals from the extracellular matrix into the cytoplasm by binding growth factors, such as vitamin K-dependent protein growth-arrest-specific gene 6 (GAS6), as well as being involved in the stimulation of cell proliferation. AXL is broadly distributed in different cell types in humans.

Zika strains and similarities For a prophylactic or therapeutic to have viability it is important to ensure that it is able to function against a wide array (if not all) strains of a virus. We used bioinformatics to identify regions of sequence identity in the regions to be used as epitopes for vaccine or targets for therapeutic siRNA development across an extensive number of strains. ZIKV has circulated in both Africa and Asia since at least the 1950s. Haddow et al determined the nucleotide sequences of the open reading frames of five isolates from

Cambodia, Malaysia, Nigeria, Uganda, and Senegal collected between 1947 and 2010. Using phylogenetic analyses of these (and previously published) ZIKV sequences, the authors identified the existence of two main virus lineages (African and Asian) [7]. In 2013, Baronti et al were able to obtain the complete coding sequence of a Zika virus strain belonging to the Asian lineage, isolated from an infected patient returning from French Polynesia. Based on nucleotide and amino acid sequence composition, the African and Asian strains were most divergent while strains within a geographical area were least divergent [8]. More than 38 strains have now been sequenced and can be assigned to the African or Asian lineages. Advantages of mRNA vaccines

As a carrier for genetic information that specifies the amino acid sequence of the encoded protein, mRNA is a nontoxic molecule that allows transient protein expression of a desired protein product in nearly all transducable cell types. Compared with traditional plasmid and viral-based approaches, this approach allows design of patient personalized mRNAs that also benefit by not needing to pass through the nuclear membrane (as DNA does) and thus carries little to no risk of genomic integration. mRNA vaccines have also been demonstrated to promote immunogenicity in both very young and very old mice. If this translates to humans, then this would be beneficial since these demographics typically do not mount a robust immunological reaction to vaccines. Recently, self-amplifying mRNA vaccines have been demonstrated to be safe and efficacious against other viruses (e.g. Influenza). Generation of a robust immune response in infants and the elderly has always been an issue for influenza vaccines. However, mRNA vaccines may benefit in that they have been demonstrated to induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice. Vaccines based on mRNA or RNA replicons have also been shown to be immunogenic in a variety of animal models, including nonhuman primates [10]. Target selection for mRNA vaccine against ZIKV

Amongst all ZIKV proteins, E protein is responsible for the host cell receptor binding and virus entry into the cells. Therefore, E protein represents a major target for development of a neutralizing antibody. The ZIKV E protein contains three distinct domains (shown in Figure 1C): a central β-barrel domain (Domain I; aa. 1-51, 132-192 and 280-295), an extended dimerization domain (Domain II; aa. 52-131 and 193-279), and an immunoglobulin-like domain (Domain III; aa. 297-406) (Figure 1C.) [5]. The fusion loop, a hydrophobic sequence located in Domain II (aa. 98-109), is responsible for the pH-dependent conformational changes during viral-host membrane fusion. In several immunology studies, the results suggested that Domain III of E protein contains the determinants that can be recognized by a number of flavivirus type-specific neutralizing monoclonal antibodies (mAb) [5]. Cross- neutralizing mAbs targeting fusion loops (in Domain II) exhibit lower affinity (Table 1). The protective efficiency of those type-specific mAb has been proven in the animal model. However, ZIKV and dengue virus (DENV) share the same properties by which antibodies against select regions of the virus (e.g. the fusion loop of Envelope protein Domain II) may stimulate Antibody Dependent Enhancement (ADE) of immune response (when non-neutralizing antiviral proteins facilitate virus entry into host cells and increase infectivity in these cells resulting in higher viremia and more severe symptoms). Some people suffered a more severe form of DENV because of the cross-serotypic immune response, or ADE. When such a person was infected with stains of different DENV serotypes (totally 4 identified serotypes), the antibodies to the old strain interfered with the immune response to the new strain and resulted in more virus uptake [11]. siRNA: a flexible molecular platform for prophylactic and therapeutic efficacy against ZIKA virus RNA interference (RNAi) is a naturally occurring, highly specific mode of gene regulation that has broad potential applications in both research and therapeutic settings. The mechanics of RNAi are both exquisite and highly discriminating. At the onset, short (19-21bp) double stranded RNA sequences (referred to as short interfering RNAs, siRNAs) associate with the cytoplasmically localized RNA Interference Silencing Complex (RISC). The resultant ribonucleoprotein complex then searches the resident population of messenger RNAs (mRNAs) for complementary sequences, eventually degrading (and/or attenuating translation of) these transcripts and effectively down-regulating the expression of the targeted gene.

Scientists have co-opted the endogenous RNAi machinery to advance a wide range of studies involving gene function analysis, pathway mapping, drug target validation, and host-pathogen interactions. As siRNAs can be designed to target virtually any gene and can be introduced into cells by a variety of methods, RNAi represents a highly flexible platform by which researchers and clinicians can control diseases, including infectious diseases.

RNA interference has previously been employed to target a range of human pathogenic viruses, including HIV, hepatitis, respiratory syncytial virus, polio virus, SARS coronavirus, Marburg, dengue, foot-and-mouth disease and Influenza. The ability to rapidly predict siRNA sequences with potency against a virus and the fact that siRNAs of the same length show similar molecular weight, charge and hydrophobicity, combined with highly efficient nanoparticle delivery systems that are agnostic for carrying these siRNAs independent of the sequence being targeted, makes RNAi a valuable tool in the arsenal against emerging viral threats and can act as an intermediate option while other therapeutic strategies, such as vaccines and small molecule therapeutics, are developed and validated. Additionally, RNAi has the ability to (1) efficiently limit viral replication without reliance on host immune functions, and (2) target multiple genes and/or sequences simultaneously, making this an ideal therapeutic approach for viruses like Zika (where a large number of strains may need to be targeted and no other options currently exist) or against other viruses (like Influenza), which have rapidly evolving genomes. Target selection for therapeutic siRNA against ZIKV

By performing recursive analyses of siRNAs against 28 sequenced Zika strains (including both African and Asian lineages), we identified siRNAs that were able to cover an extensive number of strains (Table 2). Furthermore, careful selection of multiple siRNAs can be seen to provide complete coverage against all strains used in our predictions (Table 3).

Combining 2 siRNA sequences can provide 100% coverage as seen by combining siRNA #62 and siRNA #62A (Table 3). However, in our Influenza studies, we have identified that siRNAs targeting different regions of the viral genome can produce synergistic silencing and antiviral efficacy compared with multiple siRNAs targeting the same gene. Therefore, while we can see complete coverage with certain combinations, we may see higher potency against the selected strains using a combination (such as 13 & 62) targeting distinct regions of the virus. By comparing combinations of the siRNAs as shown in Table 1, we can determine the relative potencies of each combination and compare this with the combination of 62 and 62A or 62A and 63 (targeting all strains). If we see a marked increase in potency, combining 2 siRNA sequences can provide 100% coverage as seen by combining siRNA #62 and siRNA #62a. However, in our Influenza studies, we have identified that siRNAs targeting different regions of the viral genome can produce synergistic silencing and antiviral efficacy compared with multiple siRNAs targeting the same gene. Therefore, while we can see complete coverage with certain combinations, we may see higher potency against the selected strains using a combination (such as 13 & 62) targeting distinct regions of the virus. By comparing combinations of the siRNAs as shown in Table 1, we can determine the relative potencies of each combination and compare this with the combination of 62 and 62A or 62A and 63 (targeting all strains, Table 3).

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The molecular basis and strategy of development of prophylactic and therapeutic RNA against zika virus infection. ZIKV is a single-strand positive RNA virus, belonging to family Flaviviridae; genus Flavivirus. The genome of ZIKV consists of about 10K nucleotides, which code at least three structural proteins (capsid (C), membrane (prM; processed to M) and envelope (E)), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). In ZIKV, the single ORF is flanked by 5'- and 3'- untranslated regions (UTRs), respectively. Amongst all ZIKV proteins, E protein is responsible for the host cell receptor binding and virus entry into the cells. Therefore, E protein represents a major target for development of a neutralizing antibody. The ZIKV E protein contains three distinct domains (shown in Figure 1): a central β-barrel domain (Domain I; aa. 1-51, 132-192 and 280-295), an extended dimerization domain (Domain II; aa. 52-131 and 193-279), and an

immunoglobulin-like domain (Domain III; aa. 297-406). Figure 2. Histidine-Lysine co-polymer enhances topical and subcutaneous siRNA deliveries in vivo. (A) The HKP/siRNA can self-assemble into nanoparticles (average 150nm in diameter). (B) The nanoparticles can be dissolved in aqueous solution, can be lyophilized into dry powder, and can be redissovled and mixed with methylcellulose, or with RNAse free water. (C) HKP/siRNA nanoparticle delivery to mouse respiratory track: upper airway, bronchi, alveoli.

Figure 3. Comparison of target knockdown of lung endogenous gene among HKP, DOTAP and D5W after oral tracheal deliveries of siRNA with three different dosing regimens. HKP demonstrated the efficient siRNA-mediated knockdown of the target gene at the 20μg dose.

Figure 4. Intraperitoneal delivery of PAA-siRNA formulation demonstrated a therapeutic efficacy against H1N1 in the viral challenged mice (n = 15). The viral challenges through intranasal administrations of lx LD50 H1N1 (A/California/04/2009) were conducted on day 1 (red arrow) for the virus only, Tamiflu and siRNA treatment groups. The H1N1 challenged mice were treated with various dosages of PAA-siRNA combination

(siRNA89-siRNA103 with a 1 : 1 ratio), 1 mg/kg, 5 mg/kg and 10 mg/kg, through

intraperitoneal administration daily, from day 2 to day 6 (black arrows). Adapting the same route and dosing regimen, 25 mg/kg Tamiflu was also administrated daily on the H1N1 infected mice. The therapeutic efficacy of 10 mg/kg/day of PAA-siRNA combination resulted in almost equal anti-influenza activity to that of 25 mg/kg/day of Tamiflu treatment.

Figure 5. The structures of Spermine-Lipid Conjugates (SLiC) species. The synthesis route for each of the five molecules are listed with the specific liposome chain, such as, Ri, R₂, R₃, R₄ and R₅, conjugated at the location of RiH, R₂H, R₃H, R₄H and R₅H respectively. The structures of the five SLiC species are illustrated with a spermine head and two lipid legs.

Figure 6. Target Gene Silencing by SLiC Liposome-Mediated siRNA Delivery In Vivo. TM4-packaged siRNA specific to cyclophilin-B was selected for being tested in a Balb/c mouse model through a respiratory route of delivery. In addition to Blank control and empty TM4 control, a HKP package cyclophilin-B siRNA was used as a positive control. Three different dosage: 25, 40 and 50 μg were tested. Both 40 and 50 μg siRNA dosages achieved significant target gene silencing (N=3, *P<0.05).

Figure 7. Evaluation of the cytokine response in the mouse lung after HKP-siRNA nanoparticles delivery. HKP-siRNA at different dosages were oraltracheally administrated in the mouse lungs. The total lung tissue was harvested for protein isolation and cytokine measurements by ELIS A assay.

Figure 8. A. Standard curve to measure IFN-a concentration was prepared according to in-house SOP (Lowry Method); B. IFN-a concentration in each sample was determined and normalized to total protein.

Figure 9. GFP mRNA delivery to HeLa or HepG2 cells with DDS or Lipofectamine

MessengerMAX reagent (ThermoFisher). Cells were plated into wells of a 24-well plates at a density of 5xl0⁴cells per well in DMEM supplemented with 10% FBS. On the next day transfection complexes were prepared as follows: GFP mRNA (0.5ug) was diluted with 25ul of OPTI-MEM and added to DDS (2ug) or LipofectamineMessengerMAX reagent diluted with 25ul OPTI-MEM. Mixtures were incubated for 30min (for mGFP /DDS) or lOmin (mGFP/LipofectamineMessengerMAX) and then added to the cells. Fluorescent images were taken 24h after transfection using a Zeiss Axiovert inverted fluorescence microscope. DESCRIPTION OF THE INVENTION

The present invention provides RN A pharmaceutical formulations comprising either siRNA or mRNA sequences for the prevention and treatment of Zika virus infection. One formulation comprises siRNA sequences targeting the viral genome RNA and mRNA for knocking down the viral activity, and a carrier, such as Histidine-Lysine Co-polymers (HKP) or Spermine-liposome Conjugates (SLiC). Another formulation comprises mRNA sequences coding viral proteins critical for Zika viral infection and replication, and a carrier serving as an adjuvant for amplifying an immune response against the Zika virus within the host. The invention also provides methods of use for the pharmaceutical formulations for the prevention and therapeutic treatment of Zika virus Infection. siRNA Molecules

As used herein, an "siRNA molecule" or an "siRNA duplex" is a duplex

oligonucleotide, that is a short, double-stranded polynucleotide, that interferes with the expression of a gene in a cell, after the molecule is introduced into the cell, or interferes with the expression of a viral gene. For example, it targets and binds to a complementary nucleotide sequence in a single stranded (ss) target RNA molecule. siRNA molecules are chemically synthesized or otherwise constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5, 898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties. By convention in the field, when an siRNA molecule is identified by a particular nucleotide sequence, the sequence refers to the sense strand of the duplex molecule.

One or more of the ribonucleotides comprising the molecule can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide can be modified.

Additional modifications include the use of small molecules (e.g. sugar molecules), amino acids, peptides, cholesterol, and other large molecules for conjugation onto the siRNA molecule. The siRNA molecules of the invention target a conserved region of the genome of a ZIKV. As used herein, "target" or "targets" means that the molecule binds to a

complementary nucleotide sequence in a ZIKV gene, which is an RNA molecule, or it binds to mRNA produced by the gene. This inhibits or silences the expression of the viral gene and/or its replication. As used herein, a "conserved region" of a ZIKV gene is a nucleotide sequence that is found in more than one strain of the virus, is identical among the strains, rarely mutates, and is critical for viral infection and/or replication and/or release from the infected cell.

In one embodiment, the targeted conserved region of the genome comprises sequences coding for envelope protein or nonstructural proteins NS1, NS3, NS4B, or NS5. Thus, the siRNA molecule can target envelope gene expression, NS1 gene expression, NS3 gene expression, NS4B gene expression, or NS5 gene expression.

In another embodiment, the targeted conserved region of the genome is the 3' untranslated region (3'-UTR) of the virus. Thus, the siRNA molecule can target the

3'-UTR.

In one embodiment, the siRNA molecule is a double-stranded oligonucleotide with a length of about 19 to about 29 base pairs. In one aspect of this embodiment, the molecule is a double-stranded oligonucleotide with a length of 19 to 21 base pairs. In another aspect of this embodiment, it is a double-stranded oligonucleotide with a length of 25 base pairs. In all of these aspects, the molecule may have blunt ends at both ends, or sticky ends with overhangs at both ends (unpaired bases extending beyond the main strand), or a blunt end at one end and a sticky end at the other. In one particular aspect, it has blunt ends at both ends. In another particular aspect, the molecule has a length of 25 base pairs (25 mer) and has blunt ends at both ends. In another particular aspect, the siRNA molecule is one of those identified in Table 4.

The siRNA molecules of the invention also include ones derived from those listed in Table 4 or otherwise disclosed herein. The derived molecules can have less than the 25 base pairs shown for each molecule, down to 17 base pairs, so long as the "core" contiguous base pairs remain. That is, once given the specific sequences shown herein, a person skilled in the art can synthesize molecules that, in effect, "remove" one or more base pairs from either or both ends in any order, leaving the remaining contiguous base pairs, creating shorter molecules that are 24, 23, 22, 21, 20, 19, 18, or 17 base pairs in length, if starting with the 25 base pair molecule. For example, the derived molecules of the 25 mer molecules disclosed in Table 4 includes: a) 24 contiguous base pairs of any one or more of the molecules; b) 23 contiguous base pairs of any one or more of the molecules; c) 22 contiguous base pairs of any one or more of the molecules; b) 21 contiguous base pairs of any one or more of the molecules; d) 20 contiguous base pairs of any one or more of the molecules; e) 19 contiguous base pairs of any one or more of the molecules; f) 18 contiguous base pairs of any one or more of the molecules; and g) 17 contiguous base pairs of any one or more of the molecules. It is not expected that molecules shorter than 17 base pairs would have sufficient activity or sufficiently low off-target effects to be pharmaceutically useful; however, if any such constructs did, they would be equivalents within the scope of this invention. Alternatively, the derived molecules can have more than the 25 base pairs shown for each molecule, so long as the initial 25 contiguous base pairs remain. That is, once given the specific sequences disclosed herein, a person skilled in the art can synthesize molecules that, in effect, "add" one or more base pairs to either or both ends in any order, creating molecules that are 26 or more base pairs in length and containing the original 25 contiguous base pairs.

Pharmaceutical Compositions Comprising One or More siRNA Molecules

The invention includes a pharmaceutical composition comprising an siRNA molecule that targets a conserved region of the genome of a ZIKV and a pharmaceutically acceptable carrier. In one embodiment, the carrier condenses the molecules to form a nanoparticle. Alternatively, the composition may be formulated into nanoparticles. The compositions may be lyophilized into a dry powder. In one particular embodiment, the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In one embodiment, the composition comprises at least two different siRNA molecules that target one or more conserved regions of the genome of a ZIKV and a pharmaceutically acceptable carrier. Such compositions are sometimes referred to herein as a "cocktail." In one aspect of this embodiment, the gene sequences in the conserved regions of the ZIKV are critical for the viral infection of a mammal. In another aspect of this embodiment, the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey). In still another aspect, the mammal is a human.

The composition can include one or more additional siRNA molecules that target still other conserved regions of the ZIKV genome. In one embodiment, the composition comprises siRNA molecules that target the

Envelope (E) gene of the ZIKV genome. In one aspect of this embodiment, the molecules are selected from the group consisting of:

ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU ,

ZIKV14: CCUUGACAAGCAAUCAGACACUCAA , and ZIKV17: CCGGAACUCCACACUGGAACAACAA .

In one embodiment, the composition comprises siRNA molecules that target the NSl gene of the ZIKV genome. In one aspect of this embodiment, the molecule comprises ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA.

In one embodiment, the composition comprises siRNA molecules that target the NS3 gene of the ZIKV genome. In one aspect of this embodiment, the molecules are selected from the group consisting of:

ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA ,

ZIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU ,

ZIKV62A: CCUAUCAAGUAGCAUCUGCCGGAAU , and ZIKV62B: GCCUAUCAAGUAGCAUCUGCCGGAA . In one embodiment, the composition comprises siRNA molecules that target the NS4B gene of the ZIKV genome. In one aspect of this embodiment, the molecule comprises ZIKV74: CCACUUCAUACAACAACUACUCCUU.

In one embodiment, the composition comprises siRNA molecules that target the NS5 gene of the ZIKV genome. In one aspect of this embodiment, the molecule comprises ZIKV103 : GGUGCGCAGGAUCAUAGGUGAUGAA.

In one embodiment, the composition comprises siRNA molecules that target the 3'-UTR region of the ZIKV genome. In one aspect of this embodiment, the molecule comprises: ZIKV105: C C GAG A AC GC C AUGGC AC GG A AG A A .

In another embodiment, the composition comprises a cocktail, MSTZIKV13, wherein a first siRNA molecule comprises ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU and a second siRNA molecule comprises ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA.

In another embodiment, the composition comprises a cocktail, MSTZIKV62, wherein a first siRNA molecule comprises ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA and a second siRNA molecule comprises ZIKV74: CCACUUCAUACAACAACUACUCCUU.

In another embodiment, the composition comprises a cocktail, MSTZIKV62B, wherein a first siRNA molecule comprises ZIKV62B:

GC CU AUC A AGU AGC AUCUGCC GGA A and a second siRNA molecule comprises ZIKV17: CCGGAACUCCACACUGGAACAACAA.

In another embodiment, the composition comprises a cocktail, MSTZIKV103, wherein a first siRNA molecule comprises ZIKV103 :

GGUGCGCAGGAUCAUAGGUGAUGAA, a second siRNA molecule comprises KIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU, and a third siRNA molecule comprises ZIKVl 05 : CCGAGAACGCCAUGGC ACGGAAGAA.

In one aspect of all of these embodiments, a pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle. In one aspect of all of these embodiments, the siRNA molecules comprise 25 mer blunt-end siRNA molecules and the carrier comprises a Histidine-Lysine copolymer or Spermine-Lipid Conjugate (SLiC) and cholesterol.

Pharmaceutically Acceptable Carriers for the siRNA Molecules Pharmaceutically acceptable carriers include saline, sugars, polypeptides, polymers, lipids, creams, gels, micelle materials, and metal nanoparticles. In one embodiment, the carrier comprises at least one of the following: a glucose solution, a polycationic binding agent, a cationic lipid, a cationic micelle, a cationic polypeptide, a hydrophilic polymer grafted polymer, a non-natural cationic polymer, a cationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligand functionalized cationic polymer, a ligand

functionalized-hydrophilic polymer grafted polymer, and a ligand functionalized liposome. In another embodiment, the polymers comprise a biodegradable histidine-lysine polymer, a biodegradable polyester, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer, a cationic lipid, or a PEGylated PEL Cationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.

In one embodiment, the carrier is a polymer. In one aspect of this embodiment, the polymer comprises a histidine-lysine copolymer (HKP). Such copolymers are described in U.S. Pat. Nos. 7,070,807 B2, 7,163,695 B2, and 7,772,201 B2, which are incorporated herein by reference in their entireties. In one aspect of this embodiment, the HKP forms a nanoparticle with the siRNA molecule, wherein the diameter of the nanoparticle is about lOOnm to about 400 nm. In another aspect of this embodiment, the HKP comprises the structure (R)K(R)-K(R)-(R)K(X), where R =KHHHKHHHKHHHKHHHK, K = lysine, and H = histidine. In another aspect, the HKP and the siRNA molecules self-assemble into nanoparticles or can be formulated into nanoparticles.

In another embodiment, the carrier is a liposome. In one aspect of this embodiment, the liposome comprises a cationic lipid conjugated with cholesterol. In a further aspect, the cationic lipid comprises a spermine head and one or two oleyl alcoholic tails. In a further aspect, the liposome comprises a Spermine-Liposome-Conjugate (SLiC) and cholesterol. Examples of such molecules are disclosed in Figure 5. In another aspect, the liposome and the siRNA molecules self-assemble into nanoparticles or can be formulated into nanoparticles. Methods of Using the Pharmaceutical Compositions

The invention also includes methods of using the siRNA molecules and

pharmaceutical compositions containing them to prevent or treat ZIKV disease. As used herein "treat" or "treatment" refers to reducing the severity of or curing ZIKV disease. A therapeutically effective amount of the composition of the invention is administered to a mammal. In one embodiment, the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey). In one aspect of this embodiment, the mammal is a laboratory animal, such as a rodent. In another aspect of this embodiment, the mammal is a non-human primate, such as a monkey. In still another aspect of this embodiment, the mammal is a human. As used herein, a "therapeutically effective amount" is an amount that prevents, reduces the severity of, or cures ZIKV disease. Such amounts are determinable by persons skilled in the art, given the teachings contained herein. In one embodiment, a therapeutically effective amount of the pharmaceutical composition administered to a human comprises about 1 mg of the siRNA molecules per kilogram of body weight of the human to about 5 mg of the siRNA molecules per kilogram of body weight of the human.

Routes of administration are also determinable by persons skilled in the art, given the teachings contained herein. Such routes include intranasal administration and airway instillation, such as through use of an airway nebulizer. Such routes also include injection instillation and intraperitoneal, intravenous, intradermal, intravaginal, and subcutaneous administration. mRNA Vaccines The invention provides a vaccine comprising an mRNA molecule that codes for an amino acid sequence encoded by a conserved region of the genome of a ZIKV and a pharmaceutically acceptable carrier comprising a polymer or a liposome. As used herein, a "conserved region" of a ZIKV gene is a nucleotide sequence that is found in more than one strain of the virus, is identical among the strains, rarely mutates, and is critical for viral infection and/or replication and/or release from the infected cell. In one embodiment, the gene sequence in the conserved region of the ZIKV genome is critical for the viral infection of a mammal. In another embodiment, the conserved region of the genome comprises gene sequences coding for the Envelope protein of ZIKV. In one aspect of this embodiment, the gene sequences code for amino acid sequences within Domain III of the Envelope protein.

In one embodiment, the polymer comprises a Histidine-Lysine co-polymer (HKP). In one aspect of this embodiment, the HKP and the mRNA molecules self-assemble into nanoparticles. In another aspect of this embodiment, the HKP and mRNA molecules are formulated into nanoparticles. In one embodiment, the liposome comprises a Spermine-Lipid Conjugate (SLiC) and cholesterol. In one aspect of this embodiment, the SLiC and cholesterol and the siRNA self-assemble into nanoparticles. In another aspect of this embodiment, the SLiC and cholesterol and the mRNA molecules are formulated into nanoparticles. The SLiC and cholesterol also acts as an adjuvant for amplifying the immune response. Use of the mRNA Vaccines

The mRNA vaccines of the invention are used to prevent a ZIKV infection or reduce its severity. Thus, the invention includes method of preventing or reducing the severity of a ZIKV infection in a mammal comprising administering to the mammal a therapeutically effective amount of the vaccine prior to infection. The vaccine is administered to the mammal through injection instillation or intradermal, intravenous, intraperitoneal, intravenous, intravaginal, or subcutaneous administration. In one embodiment, the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey). In one aspect of this embodiment, the mammal is a laboratory animal, such as a rodent. In another aspect of this embodiment, the mammal is a non-human primate, such as a monkey. In still another aspect of this embodiment, the mammal is a human.

The following examples illustrate certain aspects of the invention and should not be construed as limiting the scope thereof.

Example 1: Design mRNA targeting the envelope protein of ZIKV

Amongst all ZIKV proteins, E protein represents a major target for development of a neutralizing antibody. The ZIKV E protein contains three distinct domains (shown in Figure 1). We selected the sequence of Envelope Domain III of the virus to be the immunogenic regions of mRNA vaccine (Table 1). We used BLAST searches for identifying a region within

Domain III that should provide the necessary immune response without inducing Antibody Dependent Enhancement (ADE) in response to other flaviviruses (e.g. DENV; Supplement 1). To avoid ADE, we gave up Envelope Domain II as this region shares high homology with the regions in common with DENV (Supplement 1). Example 2: mRNA transfection into cells and measurement of mRNA expression in vitro.

The mRNA constructs containing Envelope Domain II of ZIKV will be transfected into human cells in vitro using a variety of commercially available transfection agent. Cells to be used for these studies included HEK293T, VERO cells, A549 cells and others. We will also examine electroporation (using MaxCyte technology) as an option for delivery. The various delivery processes are aimed at determining which will give good uptake into a variety of cells and to see subsequent expression of the construct. The aim is to determine protein production by each construct and also to determine whether the product is secreted from the cells. This process is not necessarily identified a clinically viable delivery process. mRNA will be detected in live cells using SmartFlare probes (Millipore) or through use of QRTPCR. mRNA of GFP was taken as the positive control (Figure 9). Example 3: Detection of mRNA uptaking into cells using SmartFlare technology

These smart flares are beads that have a sequence attached that, when recognizing the RNA sequence in the cell, produce an increase in fluorescence. Smartflares will be designed against several regions along the constructs in case steric hindrance reduces signal from one region.

Example 4: Detection of the protein expression in the culture media

The protein expressed by the mRNA construct will be identified and quantitated by RPHPLC using an analytical C18 column (250mm x 2.1mm; Phenomenex). Protein detection will use a dual wavelength detector. A gradient of 0.1%TFA/Acetonitrile will be adjusted over time to allow analytical separation of protein peaks. In initial experiments, fractions will be collected and submitted for Mass Spectrometry to determine the presence of the expected sequence. The secreted product and the product manufactured within the cells will be compared using protein sequencing. To mitigate enzyme degradation of the sample, we will use enzyme inhibitors in the media and concentrated media from multiple wells in order to detect the product on HPLC. Example 5: Examination of siRNAs against Zika in a CPE assay

Specific siRNAs for testing will be provided to Immuquest (Frederick, MD) for analysis in their Zika CPE assay. We will first optimize siRNA delivery into the cells to be used for this study (Vero cells) by comparing multiple commercial and proprietary delivery agents (including lipofectamine, Oligofectamine, Spermine and PEG-PEI). These experiments will use siRNAs against housekeeping genes combined with qRTPCR measurements to show degree of silencing with each formulation. The optimal formulation will then be used to examine efficacy of the designed siRNAs against Zika in a CPE assay. In this experiment, control and test siRNAs will be administered at select intervals ahead of the virus and the degree of effect monitored by examining the change in the CPE value. We will provide test and control siRNAs for delivery at varying doses in the same delivery vehicle. These materials will be supplied as blinded samples.

Example 6: Determine best nanoparticle for delivery We have developed several different varieties of nanoparticle for mRNA/siRNA delivery. These range from branched polypeptides (e.g. Histidine Lysine Ploymer or HKP) and various derivatives that, like the HKP, have been modified with targeting ligands to allow tissue specific delivery or PEGylated varieties that should assist in uptake across the mucosa of the vagina. We have demonstrated delivery topically to open wounds, and systemically to various tumors as well as specific targeted delivery to the lung and liver (avoiding uptake by Kupffer cells) using these formulations. We also have developed a spermine/spermidine co-polymer carrier and a liposomal delivery agent that, like the HKP, protect the siRNA and provide high efficiency delivery to tissues. Evaluate mRNA/siRNA-nanoparticle biological activity in cell culture. The various peptide/lipid formulations will be evaluated for their ability to form nanoparticles with single siRNAs or combinations of siRNAs against different targets. Binding with the nucleic acid will be evaluated by gel electrophoresis. Nanoparticle formation will be studied by particle size measurements using DLS (Dynamic Light Scattering) and the charge/size distribution measured using a nanoparticle size/charge instrument (Malvern instruments D9000). Morphology/size distribution of the nanoparticles will be confirmed by electron microscopy (TEM). The biological activity of these molecules will be evaluated in cell culture prior to in vivo studies. Particles formed at different N/P (Nitrogen/Phosphate) ratios will be tested in 2 different cell lines for their ability to deliver fluorescently labeled siRNA.

We will examine the ability of HKP and the other nanoparticles to deliver fluorescently labeled mRNA or siRNAs into the tissues of AG129 mice. We will then demonstrate efficacy by silencing housekeeping genes such as GAPDH in these local cells. These experiments will determine depth of penetration of the mRNA or siRNAs into the surrounding tissues. Studies to characterize the delivery vehicle will include 1) biophysical properties; 2) in vitro efficiency of mRNA and siRNA delivery; 3) delivery efficiency and toxicity in mice; and 4) the efficacy of siRNA-mediated gene silencing in Zika infected mice.

Example 7: SLiC/mRNA/siR A nanoparticle SLiC Liposome Preparation Regular methods will be tried at first to prepare liposomes with newly synthesized SLiC molecules, such as thin film method, solvent injection and so on without much success. Norbert Maurer et al reported a method of liposome preparation in which siRNA or oligonucleotide solution will be slowly added under vortexing to the 50% ethanol solution (v/v) of liposome and ethanol was later removed by dialysis. The nanoparticles thus derived will be small in size and homogeneous. In this method mRNA and siRNA will be directly wrapped by cationic lipids during formation of liposome, while in most other methods mRNA or siRNA are loaded (or entrapped) into preformed liposome, such as Lipofectamine 2000. Lipids dissolved in ethanol are in so-called metastable state in which liposomes are not very stable and tend to aggregate. We will then prepare un-loaded or pre-formed liposomes using modified Norbert Maurer's method. (Maurer, N., A., Mori, L., Palmer, M. A., Monck, K. W. C, Mok, B., Mui, Q. F., Akhong, and P. R., Cullis. 1999. Lipid-based systems for the intracellular delivery of genetic drugs. Mol. Membr. Biol. 16, 129-140, incorporated herein by reference in its entirety.) We found that stable liposome solution could be made by simply diluting ethanol to the final concentration of 12.5% (v/v).

Liposomes were prepared by addition of lipids (cationic SLiC /cholesterol, 50:50, mol %) dissolved in ethanol to sterile dd-LbO. The ethanolic lipid solution need to be added slowly under rapid mixing. Slow addition of ethanol and rapid mixing were critical for the success in making

SLiC liposomes, as the process allows formation of small and more homogeneous liposomes. Unlike conventional methods in which mRNA or siRNAs are loaded during the process of liposome formulation and ethanol or other solvent is removed at end of manufacturing, our SLiC liposomes were formulated with remaining ethanol still in the solution so that liposomes were thought to be still in metastable state. When mRNA or siRNA solution was mixed/loaded with liposome solution cationic groups lipids will interact with anionic siRNA and condense to form core. SLiC liposomes' metastable state helped or facilitated liposome structure transformation to entrap mRNA or siRNA more effectively. Because of the entrapment of mRNA/siRNA, SLiC liposomes become more compact and homogeneous.

Physiochemical Characterization of SLiC Liposome

After the liposome formation, we have developed an array of assays to characterize the physicochemical properties of SLiC liposome, including particle size, surface potential, morphology study, mRNA or siRNA loading efficiency and biological activity, etc. The particle size and zeta-potentials of SLiC liposomes were measured with Nano ZS Zeta Sizer (Malvern Instruments, UK). Each new SLiC liposome was tested for particle size and zeta-potential when ethanol contents changed from 50% to 25% and to 12.5%. Data were derived from formulations of different ethanol contents. All SLiC liposomes were prepared at lmg/ml in concentration and loaded with siRNA (2: 1, w/w). Each of SLiC Liposomes was composed of cationic SLiC and cholesterol dissolved in ethanol at 12.5%, e.g. TM2 (12.5). The average particle sizes of three sequential measurements and the average zeta-potentials of three sequential measurements were illustrated in Table 5.

Further analysis of the physiochemical perimeters of the SLiC liposome suggested that ethanol concentrations were positively proportional to particle sizes (the lower of ethanol concentration, the smaller of particle sizes), but negatively proportional to zeta-potential (the lower of ethanol concentration, the higher of zeta-potential at the same time). The higher surface potential will render particles more stable in solution. In addition to stability in solution, data shown later also indicated that toxicity was lower with lower ethanol concentration, too. Therefore, to put all factors together, ethanol concentration of 12.5% (v/v) was selected as solvent to suspend cholesterol as well as SLiC into the master working stock solution before they were used to make liposome formulations.

In contrast to bare SLiC liposome formulation, liposomes particle sizes became much smaller when they were loaded with mRNA or siRNA at 2: 1 (w/w) resulting in particle sizes in the range of 110 to 190nm in diameter and much lower PDI values. Conventional consideration of liposomal structure dictates that mRNA or siRNA is loaded or interacted with cationic lipids through electrostatic forces and liposomes wraps mRNA or siRNA to form spherical particles in shape in order to reduce surface tension. As the result, the liposomes particle sizes became much smaller after loaded with mRNA or siRNA. Liposomes formulated with mRNA or siRNA also have lower surface charge, which could be explained by neutralizing effect from loaded mRNA or siRNA. Example 8: Mouse model study for mRNA vaccine

To investigate the protective efficiency of mRNA vaccine against ZIKV infection in vivo, AG129 mice will be used as the animal model. We will perform all mouse studies in Biosafety level-2 conditions.

All AG129 mice will be five-week old at the beginning of this study. 20 mice of prophylactic group will be intravenously injected with mRNA combination encapsidated with HKP-SLiC nanoparticle system from the tail vein. The other 20 mice of control group will be injected with PBS. 14 days later, the serum of all mice will be collected, and the mice from both prophylactic group and control group will be intravenously injected with ZIKV.

All mice will be weighed and the survival of each group will be counted daily. The serum of infected mice will be taken at 1, 3, 5, 7, 9 and 14-day post-infection. The tumor necrosis factor alpha (TNF-a) will be detected using enzyme-linked immunosorbent assay (ELISA). The tissues including testes, spleen, liver, heart, brain and kidney will be collected at 24 and 72 h post-infection. The total RNA from the tissues will be extracted, one-step quantitative real-time PCR and 5'-RACE assay will be performed as described in the above in vitro study part. The viral titers in the sera and tissues will be detected in Vero cells with serially dilution. The results from vaccination group will be compared with the control groups to evaluate the protective efficiency of the mRNA vaccine candidates.

Example 9: Design of siRNAs against ZIKV infection

By performing recursive analyses of siRNAs against 28 sequenced Zika strains (including both African and Asian lineages) we identified siRNAs that were able to cover an extensive number of strains (Table 2). Furthermore, careful selection of multiple siRNAs can be seen to provide complete coverage against all strains used in our predictions (Table 3). Further sequence analysis showed that the 11 elected anti-ZIKV siRNA target the highly homologous regions of Envelope (E) protein, non-structure protein NS1, NS3, NS4B, NS5 and 3'-UTR in all ZIKV strains (Table 4 and Figure 1). If the mRNA translation and genome replication of ZIKV mimic DENV and take place in the cytoplasm, the siRNA candidates should work efficiently to degrade both positive and negative strand of the viral RNAs in ZIKV.

Example 10: Cell Culture Based Screening for Potent Anti-ZIKV siRNA Oligos siRNAs will be tested for their silencing activities using a reporter assay where the regions of the ZIKV genome to be silenced are incorporated into an expression vector. An appropriate ZIKV gene segment will be cloned into psiCheck™-2 (Promega). The cells will be co-transfected with reporter plasmids and siRNAs and the expression of renilla luciferase will be normalized to firefly luciferase expression (transfection efficiency control). Using RTPCR we will examine the ability of the siRNAs (both individually and in combinations) to silence their respective sequences of interest by transfecting in the plasmid containing the reporter construct, administering the siRNA and monitoring the reporter output at 24, 48 and 72h post transfection. Specifically, Vero cells will be transfected with single siRNA at 20 nM and infected (5 hours post transfection) with a strain of Zika. At T= 24 or 48hrs post infection, global expression profiling of the viral genome and viral titering will be performed using standard protocols (RT-PCR). The siRNAs with high activity will be further validated by performing a dose response of the siRNAs.

Example 11: Identify potent combinations of siRNAs to improve strain coverage.

Using similar experimental conditions to those used in Example 10, we will supply siRNA mixtures for evaluation in the CPE assay. We will selecte the siRNAs to include in the mixtures based on their efficacy as well as the degree of overlap between Zika strains.

Essentially, we believe that testing the combinations shown in Table 1 will allow identification of suitable combinations for subsequent in vivo tests based on potency in the CPE assay and/or strain coverage (for equipotent combinations strain coverage will be a selection criteria). We will identify either single siRNAs or combinations that demonstrate the greatest efficacy in silencing and inhibiting the viral genes and which produce the greatest effect on ZIKV in the CPE assay.

Example 12: Develop and characterize nanoparticles for in vivo delivery of siRNAs. The most critical and challenging aspect of developing an siRNA-based therapeutic is the delivery of the siRNA into the cells where ZIKV infection can be transmitted. Since ZIKV has been shown to be transmitted by sexual intercourse we have elected to pursue intravaginal siRNA delivery as a means of validating this as a preliminary approach to therapy. Once more is understood about the etiology of the disease we may be able to adjust the delivery approach to treat therapeutically. The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. However, there are many biological barriers and factors that protect the vagina from foreign particles, such as a thick and elastic mucus layer that may bind and inhibit access of these agents.

One of the biggest barriers to successful siRNA delivery is the mucus layer [12]. Cross-linked mucin fibers limit the size and hydrophobicity of reagents that can penetrate it. In regards to optimal nanoparticle size for siRNA delivery the upper limit for effective access and uptake is 0.5um and the lower limit appears to be 0.2um [12, 13]. However, despite these reported limitations, others have successfully demonstrated siRNA delivery intravaginally [14, 15] using other carriers. We have several nanoparticles that are below this size - including the branched peptide Histidine Lysine copolymer (HKP) that has shown excellent delivery characteristics to wounds and systemically. This will allow exploration of other modifications e.g. PLGA, PEG or PEG-PEI to this HKP nanoparticle or evaluation of a spermine/spermidine copolymer that exhibits suitable molecular characteristics for delivery including size, charge and hydrophobicity. Previous studies have shown significant success with PLGA, PEG and other modifications to existing nanoparticle formulations and we have demonstrated functionalization of our nanoparticles with these moieties while retaining this optimal size, ability to carry and protect the siRNAs while penetrating the mucus layer. Finally, others have shown that spray dried powders can enhance intravaginal siRNA delivery [16]. We have also demonstrated that we can lyophilize our HKP nanoparticle complexed with siRNA without adversely affecting its characteristic size or delivery capacity.

Example 13: Mice model study for anti-ZIKV siRNA

To investigate the efficiency of siRNA combination against ZIKV virus infection in vivo, AG129 mice will be used as the animal model. We will perform all mouse studies in Biosafety level-2 conditions. All AG129 mice will be five-week old at the beginning of this study. 20 mice of treatment group will be intravenously injected with siRNA combination encapsidated with HKP-SLiC nanoparticle system from the tail vein. The other 20 mice of control group will be injected with PBS. 24 h later, mice from treatment group and control group will be infected intravenously with ZIKV, respectively. siRNA or PBS will be intravenously injected into the treatment or control group at 0, 24, 48 and 72 h post-infection.

All mice will be weighed and the survival of each group was counted daily. The serum of infected mice will be taken at 1, 3, 5, 7, 9 and 14-day post-infection. The tumor necrosis factor alpha (TNF-a) will be detected using enzyme-linked immunosorbent assay (ELISA). The studies are expected to demonstrate that the cell cultures infected with ZIKV exhibit up-regulated expression of tumor necrosis factor-a (TNF-a), and interleukin-ΐβ (IL-Ιβ). The tissues including testes, spleen, liver, heart, brain and kidney will be collected at 24, 28 and 72 h post-infection. The total RNA from the tissues were extracted, one-step quantitative real-time PCR and 5 '-RACE assay will be performed. The viral titers in the sera and tissues will be detected in Vero cells with serially dilution. The results from treatment groups will be compared with the control groups to evaluate the anti-viral efficiency of the therapeutic siRNA candidates.

Table 1. Characteristics of ZIKV mRNA vaccine candidate proteins.

Table 2. Table showing strains used for siRNA prediction and siRNA sequence overlap.

Table 3. Percentage of strain coverage by combining 2 siRNAs from table above

Table 4. The list of sequence and targets for anti-ZIKV siRNA candidates

Table 5 Characterization indexes of five SLiC species and five SLiC-siRNA nanoparticles, including particle sizes, poly-dispersity index (PDI) and Zeta-potential.

Supplement 1: The Envelope gene amino acid sequence alignment results of ZIKV with all four serotypes of DENV

High homology was indicated in red frame, while low homology was indicated in green frame.

Dengue virus serotype 1

Zika vs Dengue virus serotype 2

Zika vs Dengue virus serotype 3

Zika vs Dengue virus serotype 4

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Perera-Lecoin M, Surasombatpattana P, Talignani L, Thomas F, Cao-Lormeau VM, Choumet V, Briant L, Despres P, Amara A, Yssel H, Misse D. Biology of Zika Virus Infection in Human Skin Cells. Journal of Virology. 2015: 89(17):8880-96.

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Vasilakis N, Heang V, Weaver SC, Haddow A, Tesh RB, Sovann L, Palacios G. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. Cell Host Microbe. 2016: 19(5):696-704.

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Complete coding sequence of zika virus from a French polynesia outbreak in 2013. Genome Announc. 2014: 2(3). pii: e00500-14. doi: 10.1128/genomeA.00500-14.

9. Liang, H., Lee, M., and Jin, X. Guiding dengue vaccine development using

knowledge gained from the success of the yellow fever vaccine. Cellular & Molecular Immunology. 2015: 13, 36-46.

10. Petsch B, Schnee M, Vogel AB, Lange E, Hoffmann B, Voss D, Schlake T, Thess A, Kallen KJ, Stitz L, Kramps T. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat Biotechnol. 2012:

30(12): 1210-6.

11. Charles AS, Christofferson RC. Utility of a Dengue-Derived Monoclonal Antibody to Enhance Zika Infection In Vitro. PLoS Curr. 2016 Jul 5;8.

12. Yang,S., Chen, Y.,Ahmadie, R., Ho, EA. Advancements in the Field of

IntravaginalsiRNA Delivery. Journal of Controlled Release. 2013 : 167; 29-39

13. Lai SK, O'Hanlon DE, Harrold S, Man ST, Wang YY, Cone R, Hanes J.. Rapid

transport of large polymeric particles in fresh undiluted human mucus. Proc Nat AcdSci. 2007: 104: 1482-1487. 14. Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT, Knipe DM, Lieberman J. An SiRNA-based microbicide protects mice from lethal Herpes Simplex 2 virus infection. Nature. 2006: 439; 89-94.

15. Woodrow KA, Cu Y, Booth CJ, Saucier- Sawyer JK, Wood MJ, Saltzman WM.

Intravaginal gene silencing using biodegradabalepolyer nanoparticles densely loaded with small interfering RNA. Nat Mater. 2009: 8; 526-533.

16. Wu N, Zhang X, Li F, Zhang T, Gan Y, Li J. Spray-dried powders enhance vaginal siRNAdelivery by potentially modulating the mucus molecular sieve structure.

International Journal of Nanomedicine. 2015: 10 5383-5396. The disclosures of all publications identified herein, including issued patents and published patent applications, and all database entries identified herein by url addresses or accession numbers are incorporated herein by reference in their entirety.

Although this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising at least two different siRNA molecules that target one or more conserved regions of the genome of a Zika Virus (ZIKV) and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a polymer or a liposome and wherein the siRNA molecules and the carrier form a nanoparticle.

2. The composition of claim 1, wherein the gene sequences in the conserved regions of the ZIKV are critical for the viral infection of a mammal.

3. The composition of claim 2, wherein the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).

4. The composition of any one of claims 1-3, wherein the targeted conserved regions of the genome comprise the 3'-UTR (untranslated region) and gene sequences coding for ZIKV proteins selected from the group consisting of Envelope (E) protein and non-structure proteins NS1, NS3, NS4B, and NS5.

5. The composition of claim 4, wherein the siRNA molecules are selected from the group consisting of:

ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU , ZIKV14: CCUUGACAAGCAAUCAGACACUCAA , ZIKV17: CCGGAACUCCACACUGGAACAACAA , ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA, ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA , ZIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU , ZIKV62A: CCUAUCAAGUAGCAUCUGCCGGAAU , ZIKV62B: GCCUAUCAAGUAGCAUCUGCCGGAA , ZIKV74: CCACUUCAUACAACAACUACUCCUU ,

ZIKV103 : GGUGCGCAGGAUCAUAGGUGAUGAA , and

ZIKVl 05 : CCGAGAACGCC AUGGCACGGAAGAA

6. The composition of claim 4, wherein the siRNA molecules that target the Envelope (E) gene are selected from the group consisting of:

ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU ,

ZIKV14: CCUUGACAAGCAAUCAGACACUCAA , and

ZIKV17: CCGGAACUCCACACUGGAACAACAA .

7. The composition of claim 4, wherein the siRNA molecule that targets the non-structure protein NS1 gene comprises:

ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA .

8. The composition of claim 4, wherein the siRNA molecules that target the non-structure protein NS3 gene are selected from the group consisting of:

ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA , ZIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU , ZIKV62A: CCUAUCAAGUAGCAUCUGCCGGAAU , and ZIKV62B: GCCUAUCAAGUAGCAUCUGCCGGAA .

9. The composition of claim 4, wherein the siRNA molecule that targets the non-structure protein NS4B gene comprises:

ZIKV74: CCACUUCAUACAACAACUACUCCUU .

10. The composition of claim 4, wherein the siRNA molecule that targets the non-structure protein NS5 gene comprises:

ZIKV103 : GGUGCGCAGGAUCAUAGGUGAUGAA .

11. The composition of claim 4, wherein the siRNA molecule that targets the 3 ' -UTR

comprises:

ZIKV105: CCGAGAACGCCAUGGCACGGAAGAA .

12. The composition of claim 1, comprising a siRNA cocktail, MST^{zn v13}, wherein a first siRNA molecule comprises ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU and a second siRNA molecule comprises ZIKV30:

GCCAUGGCACAGUGAAGAGCUUGAA.

13. The composition of claim 1, comprising a siRNA cocktail, MST^{zn v62}, wherein a first siRNA molecule comprises ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA and a second siRNA molecule comprises ZIKV74:

CCACUUCAUACAACAACUACUCCUU.

14. The composition of claim 1, comprising a siRNA cocktail, MST^{zn v62B}, wherein a first siRNA molecule comprises ZIKV62B: GCCUAUCAAGUAGCAUCUGCCGGAA and a second siRNA molecule comprises ZIKV17:

CCGGAACUCCACACUGGAACAACAA.

15. The composition of claim 1, comprising a siRNA cocktail, MST^{zn v103}, wherein a first siRNA molecule comprises ZIKV103 : GGUGC GC AGG AUC AU AGGUG AUGA A, a second siRNA molecule comprises KTKV63 :

CCUAUCAGGUUGCAUCUGCCGGAAU, and a third siRNA molecule comprises ZIKVl 05 : CCGAGAACGCC AUGGCACGGAAGAA.

16. The composition of any one of claims 1-3, wherein the polymer comprises a

Histidine-Lysine co-polymer (HKP).

17. The composition of claim 4, wherein the polymer comprises a Histidine-Lysine

co-polymer (HKP).

18. The composition of any one of claims 5-15, wherein the polymer comprises a

Histidine-Lysine co-polymer (HKP).

19. The composition of any one of claims 16-18, wherein the HKP comprises the structure (R)K(R)-K(R)-(R)K(X), where R =KHHHKHHHKHHHKHHHK, K = lysine, and H = histidine.

20. The composition of any one of claims 1-3, wherein the liposome comprises a

Spermine-Lipid Conjugate (SLiC) and cholesterol.

21. The composition of claim 4, wherein the liposome comprises a Spermine-Lipid

Conjugate (SLiC) and cholesterol.

22. The composition of any one of claims 5-15, wherein the liposome comprises a

Spermine-Lipid Conjugate (SLiC) and cholesterol.

23. The composition of any one of claims 20-22, wherein the SLiC comprises one of the structures TM1, TM2, TM3, TM4, or TM5 shown in Figure 5.

24. The composition of claim 23, wherein the SLiC comprises the structure TM4 shown in Figure 5.

25. The composition of any one of claims 1-24, wherein the siRNA molecules comprise oligonucleotides with a length of 19-29 base pairs.

26. The composition of any one of claims 1-24, wherein the siRNA molecules comprise oligonucleotides with a length of 19-21 base pairs.

27. The composition of any one of claims 1-24, wherein the siRNA molecules comprise oligonucleotides with a length of 25 base pairs.

28. A method of treating a mammal with a ZIKV infection comprising administering to said mammal a therapeutically effective amount of the composition of claim 1.

29. A method of treating a mammal with a ZIKV infection comprising administering to said mammal a therapeutically effective amount of the composition of any one of claims 2-27.

30. The method of claims 28 or 29, wherein the composition is administered to the mammal through injection instillation.

31. The method of claims 28 or 29, wherein the composition is administered to the mammal through intranasal administration.

32. The method of claims 28 or 29, wherein the composition is administered to the mammal through intradermal administration.

33. The method of claims 28 or 29, wherein the composition is administered to the mammal through intravenous administration.

34. The method of claims 28 or 29, wherein the composition is administered to the mammal through subcutaneous administration.

35. The method of claims 28 or 29, wherein the composition is administered to the mammal through intravaginal administration.

36. The method of any one of claims 28-35, wherein the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).

37. The method of claim 36, wherein the mammal is a human.

38. An siRNA molecule that targets a conserved region of the genome of a ZIKV.

39. The siRNA molecule of claim 38, wherein the targeted conserved region of the genome comprises the 3'-UTR and gene sequences coding for ZIKV proteins selected from the group consisting of Envelope (E) protein, non-structure protein NS1, non-structure protein NS3, non-structure protein NS4B, and non-structure protein NS5.

40. The siRNA molecule of claim 38, wherein the wherein the siRNA molecules are

selected from the group consisting of:

ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU ,

ZIKV14: CCUUGACAAGCAAUCAGACACUCAA ,

ZIKV17: CCGGAACUCCACACUGGAACAACAA ,

ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA,

ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA ,

ZIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU ,

ZIKV62A: CCUAUCAAGUAGCAUCUGCCGGAAU ,

ZIKV62B: GCCUAUCAAGUAGCAUCUGCCGGAA ,

ZIKV74: CCACUUCAUACAACAACUACUCCUU ,

ZIKV103 : GGUGCGCAGGAUCAUAGGUGAUGAA , and

ZIKV105: CCGAGAACGCCAUGGCACGGAAGAA .

41. The siRNA molecule of any one of claims 38-40, wherein the siRNA molecule

comprises oligonucleotides with a length of 19-29 base pairs.

42. The siRNA molecule of any one of claims 38-40, wherein the siRNA molecule

comprises oligonucleotides with a length of 19-21 base pairs.

43. The siRNA molecule of any one of claims 38-40, wherein the siRNA molecule

comprises oligonucleotides with a length of 25 base pairs.

44. The siRNA molecule of claim 40, wherein the siRNA molecules comprise derivatives of the identified siRNA molecules, the derivatives having 17-24 contiguous base pairs of original 25 contiguous base pairs of the identified molecules or one or more base pairs in addition to the original 25 contiguous base pairs of the identified molecules.

45. A composition comprising the siRNA molecule of any one of claims 38-44 and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a polymer or a liposome and wherein the siRNA molecules and the carrier form a nanoparticle.

46. A method of treating a mammal with a ZIKV infection comprising administering to said mammal a therapeutically effective amount of the composition of claim 45.

47. The method of claim 46, wherein the mammal is a human.

48. A pharmaceutical composition comprising an mRNA molecule that codes for an amino acid sequence encoded by a conserved region of the genome of a Zika Virus (ZIKV) and a pharmaceutically acceptable carrier comprising a polymer or a liposome.

49. The composition of claim 48, wherein the gene sequence in the conserved region of the ZIKV is critical for the viral infection of a mammal.

50. The composition of claim 49, wherein the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).

51. The composition of claim 48, wherein the conserved region of the genome comprises gene sequences coding for the Envelope protein of ZIKV.

52. The composition of claim 51, wherein the gene sequences code for amino acid

sequences within Domain III of the Envelope protein.

53. The composition of any one of claims 48-52, wherein the polymer comprises a

Histidine-Lysine co-polymer (HKP).

54. The composition of any one of claims 48-52, wherein the liposome comprises a

Spermine-Lipid Conjugate (SLiC) and cholesterol.

55. The composition of any one of claims 48-53, wherein the HKP and mRNA molecules are formulated into nanoparticles.

56. The composition of any one of claims 48-51, wherein the SLiC and the mRNA

molecules are formulated into nanoparticles.

57. The composition of claim 53, wherein the HKP and the mRNA molecules self-assemble into nanoparticles.

58. The composition of claim 54, wherein the SLiC and cholesterol and the siRNA

self-assemble into nanoparticles.

59. A method of preventing or reducing the severity of a ZIKV infection in a mammal comprising administering to said mammal a therapeutically effective amount of the composition of claim 48 prior to infection.

60. A method of preventing or reducing the severity of a ZIKV infection in a mammal comprising administering to said mammal a therapeutically effective amount of the composition of any one of claim 49-58 prior to infection.

61. The method of claims 59 or 60, wherein the composition is administered to the mammal through injection instillation or intradermal, intravenous, intravaginal, or subcutaneous administration.

62. The method of any one of claims 59-61, wherein the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).

63. The method of claim 62, wherein the mammal is a human.