WO2009071679A1

WO2009071679A1 - Novel aav vector and uses thereof

Info

Publication number: WO2009071679A1
Application number: PCT/EP2008/066919
Authority: WO
Inventors: Thierry Vandendriessche; Marinee Chuah
Original assignee: Vib Vzw; Life Sciences Research Partners Vzw
Priority date: 2007-12-05
Filing date: 2008-12-05
Publication date: 2009-06-11

Abstract

The present invention relates to the field of gene therapy more specifically to the field of adeno-associated viral gene vectors. The present invention provides a novel recombinant AAV vector comprising a chimeric capsid protein fused to a Gly-Ala repeat domain.

Description

Novel AAV vector and uses thereof

Field of the invention

The present invention relates to the field of gene therapy more specifically to the field of adeno-associated viral gene vectors. The present invention provides a novel recombinant AAV vector comprising a chimeric capsid protein fused to a GIy-AIa repeat domain.

Background to the invention

Adeno-associated virus (AAV) is a parvovirus belonging to the genus Dependovirus. The virus is presently being evaluated as an attractive gene therapy vector because it possesses several promising features not found in other viruses. Indeed, AAV can infect a wide range of host cells, including non-dividing cells and it infects also cells from different species. More importantly, AAV has not been associated with any human or animal disease and does not appear to alter the physiological properties of the host cell upon integration. Furthermore, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.

Being a single-stranded DNA virus, AAV includes two large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, respectively: replication and capsid gene products allow for the replication, assembly, and packaging of a complete AAV virion. More specifically, a family of at least four viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1 , VP2, and VP3. Said rep and capsid genes are flanked by 165-bp inverted terminal repeats (ITRs). By replacement of the endogenous sequences between the ITRs, recombinant AAV (rAAV) creates a genetic cassette that can be packaged into intact virions and delivered preferentially to specific cell types. Following virus entry, nuclear trafficking, and uncoating, the released genome becomes double stranded and via intra- or intermolecular recombination of the ITRs persists as circular monomers or concatemers.

In nature, AAV is a helper virus-dependent virus, i.e., it requires co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but functional virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the functional virion. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. To construct functional recombinant AAV (rAAV) containing a nucleic acid, a suitable host cell line is transfected with an AAV vector containing a nucleic acid. AAV helper functions and accessory functions are then expressed in the host cell. In gene therapeutic uses - because the patient's cells lack the rep and cap genes, as well as the adenovirus accessory function genes - the rAAV are replication defective; i.e. they cannot further replicate and package their genomes. There are currently a number of AAV serotypes described which are able to infect humans. AAV-2 is the best characterized and has been used to successfully deliver transgenes to several cell lines, tissue types, and organs in a variety of in vitro and in vivo assays. Notwithstanding the promises of recombinant AAV vectors as a gene therapy vector there exists a severe limitation because of the immune response directed against vector and/or transgene product which significantly impedes the persistent expression of the transgenes encoding neoantigens. Long-term humoral immunity against several viral-vector systems prevails in a large part of the population, or may be induced upon the first vector administration. This may frustrate re-administration of the vector and lead to elimination of the transduced cells. Indeed, it is observed that after an initial treatment with a given AAV serotype, anti-AAV capsid neutralizing antibodies are often made which prevent subsequent treatments by the same serotype. Although the AAV virion induces a strong humoral immune response, it cannot transduce dendritic cells (DCs) efficiently and is thought to be less immunogenic for cytotoxic T-lymphocyte (CTL) induction. However, recently the ability of rAAV to induce a CTL response resulting in the eradication of transduced hepatocytes was suggested as lying at the basis for the failure of a recent rAAV clinical trial for factor IX gene addition therapy (Mingozzi F et al (2007 Nature Medicine 13, 4, 419). In the latter trial therapeutic levels of FIX in the blood were detected from 2 to 6 weeks postinjection of rAAV2/FIX and then decreased to baseline. It should be said that the scientific data on the CTL response against rAAV transduced cells are conflicting because in a later study Li C et al (2007) Journal of Virology 81 (4), 7540 did not demonstrate in vivo elimination of rAAV2- transduced liver cells or AAV2-transduced muscle cells by AAV2 capsid-specific cytotoxic T lymphocytes. Still other studies suggest elimination of rAAV-transduced cells by the activation of NK cells (Li H et al (2007) MoI. Ther. 15:792). It is clear that the precise mechanisms of the loss of stable expression of therapeutic proteins in AAV gene therapy clinical trials are not understood. To circumvent liver toxicity and CD8+ T-cell responses (while maintaining efficacy) the use of transient immunosuppression has been proposed in future gene therapy trials. However, the use of transient immunosuppression is not without risks and therefore there exists a clear need for the development of more robust vectors as a viable alternative. Summary of the invention

In the present invention we have developed a novel recombinant AAV vector which comprises a capsid protein that is coupled to a GIy-AIa repeat domain (GAR-domain). Said GAR-domain has been used to inhibit the T-cell mediated immune rejection of heterologous fusion proteins produced by adenoviral-transduced cells (Ossevoort M et al (2003) Gene Therapy 10, 2020).

Our novel recombinant vector surprisingly leads to a sustained production of therapeutic proteins in an animal host, as it could not have been predicted that such a large sequence would not interfere with protein expression.

Brief description of the figures

Figure 1 shows a real-time qPCR on the AAV-EBNA vector particles. DNA was extracted from the particles and subjected to a real-time qPCR (TaqMan) with vector specific primers/probe (see Example 1 ). AAV particles that encapsidate the corresponding vector genomes should yield a signal, as shown here for the GFP (Fig. 1A) and FIX (Fig. 1 B) vectors.

Figure 2 represents a microscopic fluorescent image of different cell types transduced with the (stealth) AAV vector. It clearly shows that the EBNA-modified AAV vector can transduce different cell types in a dose-dependent manner consistent with increasing percentage of GFP- positive cells and increased GFP expression level/cell. This indicates that the EBNA-modified AAV vector is functional and capable of delivering genes (in casu GFP reporter gene) to different cell types.

Figure 3 shows a microscopic fluorescent image of hepatocytes. Left panel: negative control animals injected with PBS (phosphate buffer saline), right panel: hepatocytes transduced with the Gar-modified vector.

In Figure 4, values and standard curve of human FIX in mouse plasma are shown. The standard curve is based on serially diluted known amounts of human FIX spiked in mouse plasma (diamonds on graph). The actual OD values of the mouse plasma from the recipient mouse injected with the Gar-AAV-FIX vector was plotted (circles) onto this linear standard curve. Detailed description of the invention

The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, current edition, vol. I & Il (B. N. Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology, VoIs. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (current edition); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

Recombinant adeno-associated virus (rAAV) vectors are promising delivery gene delivery systems, based on the defective and non-pathogenic parvovirus adeno-associated virus. Most vectors are derived from a plasmid substrate which retains only the AAV inverted terminal repeats (ITRs) flanking the transgene cassette of choice. The deleted viral coding sequences are present on a separate template, referred to as an AAV helper of packaging plasmid. Generation of rAAV requires transfection of the vector and packaging constructs into adenovirus (Ad)-infected cells. Due to the lack of homology between vector and helper sequences, rAAV produced in this system is essentially free of wild-type AAV.

In a first embodiment the invention provides a recombinant adeno-associated vector (AAV) comprising a chimeric capsid protein which is fused to a domain of about 25-250 amino acids consisting of a repeat domain of (GIy-AIa). In a particular embodiment a domain of about 50- 200 amino acids is used. In another particular embodiment a domain of about 100-150 amino acids is used. In a particular embodiment said capsid protein is selected from the list consisting of VP1 and/or VP2 and/or VP3. Preferably said capsid protein is VP2. In a particular embodiment said recombinant AAV vector is derived from AAV-2.

In another particular embodiment said recombinant adeno-associated vector (AAV) comprises a chimeric capsid protein having an amino acid sequence depicted in SEQ ID NO: 2. In yet another particular embodiment said recombinant AAV vector comprises a heterologous transgene operably linked to regulatory sequences which direct its expression in a host cell. In a preferred embodiment said heterologous transgene encodes for a blood clotting factor. In yet another embodiment the invention provides a pharmaceutical composition comprising a recombinant AAV vector according to the before mentioned embodiments in addition to a physiologically compatible carrier. In yet another embodiment the invention provides an isolated chimeric VP2 gene comprising the nucleotide sequence as depicted in SEQ ID NO: 1 which encodes a chimeric VP2 protein depicted in SEQ ID NO: 2.

The fusion (or coupling which is an equivalent word) between the AAV capsid protein (such as for example VP2) and the GAr domain is in one embodiment a genetic fusion resulting in a chimeric capsid protein. Said genetic fusion can be an aminoterminal fusion with a capsid protein (such as VP2) or a carboxyterminal fusion with a capsid protein (such as VP2). In a particular embodiment said chimeric capsid protein is provided in trans by the AAV helper or AAV packaging construct during the recombinant AAV vector production process. GAr- domains are described in WO9746573 (The University of Virginia Patent Foundation) and in particular the example (pp. 8-11 ) described in the latter patent application is herein incorporated by reference.

Different strategies are commonly known in the art to incorporate peptides into the AAV capsid. The capsid is a tightly packaged icosahedron of 25 nm and is composed of three different viral proteins, VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa). Said proteins are encoded in the same open reading frame (ORF) and share a common stop codon. They differ in their aminotermini due to alternative splicing and different initiation codons, resulting in three progressively shorter proteins. In a particular embodiment a chimeric capsid can be formed through insertion of a GAr-domain in the amino acid sequence of a particular capsid protein. In case such a chimeric capsid protein proves to be non-functional in the packaging process (or capsid assembly process) additional wild type capsid can be provided in trans (by a specific capsid-encoding plasmid). Genetic coupling between the GAR domain and capsid proteins can be interrupted by a spacer encoding about 0 to about 30 amino acids. In another particular embodiment the coupling between a capsid protein and a GAr-domain is carried out via a chemical coupling. Methods of chemical coupling also known as cross-linking technologies are known by a person skilled in the art. In case chemical coupling with a GAr- domain is envisaged it is most conveniently carried out on a purified recombinant AAV vector.

By an "AAV vector" is meant a vector derived from any adeno-associated virus serotype isolated from any animal species, including without limitation, vectors mentioned in Gao G. et al (2005) New recombinant serotypes of AAV vectors. Curr. Gene Ther. 3:285-97. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging

(e.g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.

"AAV helper or packaging functions" refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. Thus, AAV helper functions include both of the major AAV open reading frames (ORFs), rep and cap. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. The Cap expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.

The term "AAV helper or packaging construct" refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest. AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither replicate nor package themselves. AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs and vectors that encode Rep and/or Cap expression products have been described. See, e.g., U.S. Pat. Nos. 6,001 ,650, 5,139,941 and 6,376,237, Samulski et al. (1989) J. Virol. 63: 3822-3828; and McCarty et al. (1991 ) J. Virol. 65: 2936- 2945.

The term "accessory functions" refers to non-AAV derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, the term captures proteins and RNAs that are required in AAV replication, including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1 ) and vaccinia virus.

The term "accessory function vector" refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions. An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell. Expressly excluded from the term are functional viral particles as they exist in nature, such as adenovirus, herpesvirus or vaccinia virus particles. Thus, accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. It has been demonstrated that the full-complement of adenovirus genes are not required for accessory helper functions. In particular, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. By "recombinant virus" is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle. By "AAV virus" is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat). In this regard, single-stranded AAV nucleic acid molecules of either complementary sense, e.g., "sense" or "antisense" strands, can be packaged into any one AAV virus and both strands are equally functional. The words 'virus' and 'virion' are herein used interchangeable.

A "recombinant AAV virus" or "rAAV virus" or "rAAV virion" or "rAAV vector" - which are all equivalent terms - is defined herein as a functional, replication-defective virus including an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs. A rAAV virus is produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into (a) functional recombinant virus particle(s) for subsequent gene delivery. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. Such techniques can be used to introduce one or more exogenous DNA moieties, such as an episomal or integrative vector into suitable host cells. The term 'transduction' refers to viral vector mediated gene transfer.

The term DNA "control sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. The term "promoter" refers to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters".

The terms "effective amount" or "therapeutically effective amount" of a composition or agent, as provided herein, refer to a nontoxic but sufficient amount of the composition or agent to provide the desired response. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular macromolecule of interest, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

In another particular embodiment the recombinant AAV vectors of the present invention are used for the manufacture of a medicament to treat a variety of diseases. The type of disease depends on the type of heterologous gene present in the recombinant AAV vector. "Treating" or "treatment" of a disease includes: (1 ) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. The invention preferably encompasses rAAV viruses comprising at least one heterologous nucleotide sequence coding for one or more polypeptides, the rAAV viruses preferably administered to one or more cells or tissue of a mammal. Thus, the invention embraces the delivery of at least one heterologous nucleotide sequence encoding one or more peptides, polypeptides, or proteins, which are useful for the treatment or prevention of disease states in a mammalian subject. The invention also includes novel mutant viruses comprising a gene or genes coding for blood coagulation proteins, which proteins may be delivered to the cells of a mammal having hemophilia for the treatment of hemophilia. Thus, the invention includes: delivery of the Factor IX gene to a mammal for treatment of hemophilia B, delivery of the Factor VIII gene to a mammal for treatment of hemophilia A, delivery of the Factor VII gene for treatment of Factor VII deficiency, delivery of the Factor X gene for treatment of Factor X deficiency, delivery of the Factor Xl gene for treatment of Factor Xl deficiency, delivery of the Factor XIII gene for treatment of Factor XIII deficiency, and, delivery of the Protein C gene for treatment of Protein C deficiency. Delivery of each of the above-recited genes to the cells of a mammal is accomplished by first generating a rAAV virus comprising the gene and then administering the rAAV virus to the mammal. Thus, the invention includes rAAV viruses comprising genes encoding any one of Factor IX, Factor VIII, Factor X, Factor VII, Factor Xl, Factor XIII or Protein C. Delivery of the recombinant viruses containing one or more heterologous gene sequences to a mammalian subject may be by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit the recombinant viral vectors into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV viruses. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403 and herein incorporated by reference, can also be employed by the skilled artisan to administer the mutant viruses into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, for certain conditions, it may be desirable to deliver the mutant viruses to the central nervous system (CNS) of a subject. By "CNS" is meant all cells and tissue of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cereobrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like. Recombinant AAV virions or cells transduced in vitro may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11 :2315-2329, 2000). The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: the route of rAAV virus administration, the level of gene (or anti- sense RNA or ribozyme or shRNA) expression required to achieve a therapeutic effect, the specific disease or disorder being treated, a host immune response to the rAAV virus, a host immune response to the gene (or anti-sense RNA or ribozyme) expression product, and the stability of the gene (or anti-sense RNA or ribozyme) product. One of skill in the art can readily determine a rAAV virus dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. Generally speaking, by "therapeutic effect" is meant a level of expression of one or more heterologous gene sequences sufficient to alter a component of a disease (or disorder) toward a desired outcome or clinical endpoint, such that a patient's disease or disorder shows clinical improvement, often reflected by the amelioration of a clinical sign or symptom relating to the disease or disorder. Using hemophilia as a specific disease example, a "therapeutic effect" for hemophilia is defined herein as an increase in the blood-clotting efficiency of a mammal afflicted with hemophilia, efficiency being determined, for example, by well known endpoints or techniques such as employing assays to measure whole blood clotting time. Reductions in either whole blood clotting time are indications of an increase in blood-clotting efficiency. In severe cases of hemophilia, hemophiliacs having less than 1 % of normal levels of Factor VIII or Factor IX have a whole blood clotting time of greater than 60 minutes as compared to approximately 10 minutes for non-hemophiliacs. Expression of 1 % or greater of Factor VIII or Factor IX has been shown to reduce whole blood clotting time in animal models of hemophilia, so achieving a circulating Factor VIII or Factor IX plasma concentration of greater than 1 % will likely achieve the desired therapeutic effect of an increase in blood-clotting efficiency.

Examples

1 ) Production of a rAAV vector comprising a chimeric VP2 capsid-GAr fusion

A (Gly-Ala)-repeat domain (Gar-domain) comprising a contiguous 239 amino-acid sequence composed of G or A was genetically fused to the nucleotide sequence encoding the VP2- p r o t e i n . T h e p l a s m i d p C M V-EBNA (www.addgene.org/pgvec1 ?f=v&cmd=viewvecseq&from=&soid=5205&view=Draw) was obtained from Invitrogen and encodes the EBNA-1 antigen that contains the Gar-domain. pCMV-EBNA was digested with BstXI and an adapter containing a BgI Il site was cloned into this site to generate plasmid pCMV-EBNA(Bglll). The BgIII adapter was created by annealing the synthetic phosphorylated oligonucleotides 5'-TACG/AG/A7C7GAAG-3 (SEQ ID NO: 3) and 5'-ΛG,47C7CGTACTTC-3 (SEQ I D NO: 4) (The BgII I site is denoted in italics). The VP2 sequence was PCR amplified from plasmid pAAV-RC (Stratagene) using primers: 5'- TAAGATCTTGCTCCGGGAAAAAAGAGGCCGGTAG-3' (S E Q I D N O : 5) a n d 5 '- TAA GA TCTTTACAGATTACGAGTCAGGTAT-S' (S EQ I D NO : 6) and cloned after BgIII restriction into the corresponding BgIII site of the Bglll-restricted pCMV-EBNA(Bglll) vector. An extra T was included in the 5' primer next to the BgIII site to make sure the VP2 is in frame with the Gar-domain-containing polypeptide. To avoid translation from the natural VP2 start codon, the translation start codon ACG was deleted. This genetic fusion is depicted in SEQ ID NO: 1 and the corresponding amino acid sequence encoded by this fusion sequence is denoted in SEQ ID NO: 2. The corresponding plasmid comprising SEQ ID NO: 1 is designated as pGar-VP2. An AAV helper plasmid was generated in which the VP2 startcodon is mutated from ACG to ACC. A G-to-C substitution within the wobble position of the VP2 start codon was hereby introduced which consequently abolishes VP2 expression without altering the amino acid sequence of VP1. This was done by PCR cloning using plasmid pAAV-RC (Stratagene) as template, primers and methodology as described previously (Lux K. et al (2005) J. of Virology 79(18): 1 1776, page 1 1777 - materials and methods section. Primers used were 5'- TTCCTGGTTGAGGAACCTGTTAAGACCGCTCCGGG-3' (SEQ ID NO: 7) (containing EcoNI site) and δ'-GAGGACGTACGGGAGCTGGTACTCCGAGTCAG-S' (SEQ ID NO: 8) with BsiWI restriction site. The resulting plasmid is designated as pAAV-RCΔ_sta_rt- For the vector production pGar-VP2, pAAV-RCΔ_sta_rt, pHelper and AAV vector were contransfected in HEK293 cells at a 1 :1 :2:1 w/w ratio. AAV vectors encoded green fluorescent protein (GFP) or a therapeutically relevant protein (e.g. clotting factor IX) (designated as AAV-GFP or AAV-FIX, see also VandenDriessche et al. (2007). J. Thromb. Hemost; 5(1 ): 16-24). The AAV-GFP vector (SEQ ID NO: 9) expresses GFP from a CMV promoter, whereas the AAV-FIX vector (SEQ ID NO: 10) expresses human FIX cDNA driven from a chimeric promoter/enhancer composed of the transthyretin promoter along with a serpin enhancer. The AAV-FIX vector also contains a truncated FIX intron A.

As controls, the cognate AAV2 vectors are packaged by cotransfecting pAAVRC and pHelper. The recombinant AAV vector comprising SEQ ID NO: 1 are then purified according to Lux K. et al (2005) J. of Virology 79(18): 1 1776, page 1 1777 - materials and methods section - here incorporated by reference or by cesium chloride purification (VandenDriessche et al. (2007). J. Thromb. Hemost.; 5(1 ):16-24). The determination of AAV titers is based on the same previous references and is determined by real-time quantitative PCR using vector-specific primers. 1 ,5 μl of AAV vector was first subjected to DNAse I treatment to remove any traces non- encapsidated DNA by 1 hour incubation at 37°C with 4 U DNAse, 10 μl DNAse buffer and topped up to 100 μl with H₂O. After heat-inactivation at 99°C for 5 min to inactivate DNase, disrupt the AAV vector particles and release the single-stranded genomes, samples were diluted 50-fold and 8.5 μl of the diluted sample were assayed in triplicate by qPCR after adding 16,5 μl ABI (Perkin Elmer) Q-PCR Master Mix and TaqMan probe and primers, according to the manufacturer's instructions (i.e. UDP-qPCR Master Mix 12,5 μl; 0,8 μl of each primers (1 OmM); 0.5 μl of the FAM-TAMRA probe (1 OmM) and H₂O topped up till 16,5 μl). Vector titer was performed by quantitative (q)PCR on a ABI7500 FAST (Sequencer Detector Unit from ABI). The PCR probes/primers that were used to titer the Gar-modified AAV-GFP vector (or wild-type AAV2) or are: 5'-FAM-CTT TCC AAA ATG TCG TAA CAA CTC CGC CC- 3'-TAMRA (probe) (SEQ ID NO: 1 1 ), δ'-TGGGAGTTTGTTTTGCACCAA-S' (forward) (SEQ ID NO: 12), 5'- CGCCTACCGCCCATTTG-3' (reverse) (SEQ ID NO: 13). The PCR probes/primers that was u sed to tite r th e wi l d-type AAV2 or Gar-modified AAV-F I X vecto r a re : 5'-FAM- AACCATGACATTGCCCTTCTGGAACTGG-3' TAM RA (probe) (S E Q I D N O : 1 4 ), 5 '- CACCACAACTACAATGCAGCTATTAA-3 ' (fo rwa rd ) ( S E Q I D N O : 1 5 ), 5 '- TGCAAATAGGTGTAACGTAGCTGTT-S' (reverse) (SEQ I D NO: 16). TaqMan qPCR was performed in accordance with the manufacturer's recommendations (50⁰C for 2 min, 95°C for 1 0 min and 40 cycles of 95°C 1 5 sec, 60⁰ C 1 min ), using a serially diluted plasmid corresponding to a known amount of vector as standard. The number of genome copies (gc) in 8.5 microliter of a 10-fold serially diluted reference samples of known quantitaties is shown in Fig. 1A & B and ranges from 10⁸ to 10² gc/8.5 microliter. The qPCR profiles of the experimental samples Gar-AAV-GFP (Fig.1A) and Gar-AAV-FIX are also shown (Fig. 1 B) and did not differ from non Gar-modified AAV. The qPCR signal was multiplied with a dilution factor (i.e. x 100 x 1000 x 50)/(8,5 x 1 ,5) to obtain the particle titer expressed in gc/ml. The particle titers for Gar-AAV-GFP corresponded to 1 ,08x10¹² gc/ml and for Gar-AAV-FIX to 2,15x10¹² gc/ml. This indicates that the Gar-modified AAV is capable of packaging viral vector genomes. 2) Effect of Gar-modification of VP2 on rAAV function

The functionality of Gar-modified AAV is tested by in vitro or in vivo transduction (and compared to non-modified wt AAV) using reporter genes (e.g. green fluorescent protein, GFP) and a therapeutically relevant protein (particularly FIX), as described (VandenDriessche et al. (2007). J. Thromb. Hemost.; 5(1 ):16-24). Briefly, GFP expression in transduced cells in vitro or in vivo is determined by fluorescence or confocal microscopy following transduction with Gar- modified AAV2 vectors versus unmodified wild-type AAV2 vectors as controls. Subconfluent (50% confluency) AAV293, 293T and HeLa cellines were exposed to different doses of Gar- AAV-GFP vectors (50 μl, 30μl, 20μl, 10μl, 5μl, 2.5μl in a total volume of 1 ml of culture medium (DMEM + 10% fetal bovine serum). Cells were kept in incubator at 37°C, 5% CO₂ and after 2 days GFP expression was determined by fluorescence microscopy. The results shown in Fig. 2 indicate that the Gar-AAV-GFP vector were fully functional and capable of transferring a gene (in casu the GFP reporter gene) into various different cell types leading to robust expression. This reflects the broad tropism of the Gar-modified AAV vectors. Similarly, when the vector was injected into mice (150 μl, i.e. 1 .6x10¹¹ vg) it was possible to readily detect GFP-positive hepatocytes, confirming the functionality of the Gar-modified vector in vivo, whereas no fluorescence was apparent in negative control animals injected with PBS (phosphate buffer saline) (Fig. 3). Most importantly, when the Gar-AAV-FIX vector (180 μl, i.e. 4x10¹¹ vg) was administered intravenously by tail vein injection into mice, it was possible to detect robust and sustained circulating human FIX levels in the plasma of recipient mice in the therapeutic range (216 +/- 51 ng/ml human FIX) (Fig. 4, see also Table 1 ), which would be sufficient to obtain a therapeutic effect in patients suffering from hemophilia. Blood was collected by retro-orbital bleeding under general anesthesia. The presence of human FIX in plasma samples with 20% 0.1 M sodium citrate (to prevent clotting) was determined using an enzyme-linked immunosorbent assay (Asserachrome FIX ELISA, Diagnostica Stago, Parsippany, NJ, USA) on mouse plasma. The GFP and FIX expression confirms the functionality of the Gar-modified AAV2 vectors in vivo and underscores their therapeutic potential.

Table 1. Circulating human FIX levels in plasma of recipient mice (see Fig. 4).

ng/ml, STDEV: 51.07 ng/ml, Average: 215.70

3) Effect of Gar-modification on recognition of rAAV by CTL

Although AAV capsids are not expressed de novo in transduced target cells , it is possible to assess the MHC class I restricted CTL response using target cells that are transfected with expression constructs encoding the cognate capsid antigens. Hence, to test whether the recognition of Gar-modified AAV vectors by CTLs is reduced, we first generate CTL targets that express Gar-modified AAV capsid proteins. To achieve this and direct presentation of antigenic peptides derived from Gar-modified VP2 (vs. unmodified VP2, as control) to MHC class I, Gar-VP2 expression constructs are transfected into MHC-l-positive cells (vs. unmodified VP2 expression construct, as control). The expression of Gar-VP2 vs. unmodified VP2 in the transfected cells ("target" cells) is tested by RT-PCR. AAV capsid-specific CTLs ("effector" cells) are generated by immunizing mice with AAV capsid expression constructs, as described previously (Sabatino et al., MoI. Ther., 12(6):1023-33., 2005). The splenocytes from immunized mice containing the "effector" CTLs are co-cultured with the "target" cells during an in vitro stimulation phase. Subsequently, IFN-gamma production by the "effector" cells is assessed following coincubation of effector and target cells at different effectoπtarget ratios (Ossevoort et al., Gene Ther. 2003; Sabatino et al. MoI Ther 2005). The Gar domain prevents recognition of Gar-VP2 expressing cells by VP2-specific CTLs consistent with a reduction in IFN-gamma production. RT-PCR analysis shows that expression of Gar-modified VP2 is more prolonged compared to unmodified VP2.

Claims

1. A recombinant adeno-associated vector (AAV) comprising a chimeric capsid protein which is fused to a domain of about 25-250 amino acids consisting of a repeat domain of (GIy-AIa).

2. An AAV vector according to claim 1 wherein said capsid protein is selected from the list consisting of VP1 and/or VP2 and/or VP3.

3. An AAV vector according to claims 1 and 2 wherein said AAV is AAV-2.

4. A recombinant adeno-associated vector (AAV) comprising a chimeric capsid protein having an amino acid sequence depicted in SEQ ID NO: 2.

5. An AAV vector according to claims 1-4 further comprising a transgene operably linked to regulatory sequences which direct its expression in a host cell.

6. An AAV vector according to claim 5 wherein said transgene encodes for a blood clotting factor.

7. A composition comprising an AW vector according to claims 1-6 and a physiologically compatible carrier.

8. An isolated chimeric VP2 gene comprising the nucleotide sequence as depicted in SEQ ID NO: 1 which encodes a chimeric VP2 protein depicted in SEQ ID NO: 2.