HK1226768B - Adeno-associated virus vector - Google Patents

Description

Adeno-associated virus vector

Field of the Invention

The present invention relates to the field of recombinant viral vectors. In particular, the present invention relates to recombinant viral vectors suitable for in vivo delivery of therapeutic genes.

Background of the Invention

To date, adeno-associated virus remains one of the most promising vectors for the delivery of therapeutic genes. A significant number of preclinical and clinical studies have firmly established that this approach is suitable for the development of gene-based drugs that can achieve market approval.

Since the development of AAV2 as a vector for gene therapy began in the 1980s, much progress has been made in optimizing this platform for a variety of applications and target tissues. Perhaps the most important of these developments has been the discovery of a wide variety of serotypes, of which 10 to 12 are now commonly studied. One of the most prominent characteristics of these different serotypes is their respective relative tissue tropism and (in some cases) the ability to transport antineurons. Of these serotypes, AAV1-10 is widely used for preclinical and clinical purposes.

Newer platforms involving processes that allow targeting and detargeting of specific tissues and cell subtypes in patients have been developed. The core technology of these approaches is based on trial-and-error evaluation of existing AAV variants (serotypes) and in vivo selection of randomly introduced AAV capsid mutants. Together, these two promising approaches provide dozens (if not hundreds) of potential vectors with different transduction behaviors.

The most attractive aspect of AAV serotypes is their ability to efficiently transduce specific tissues in animal models and humans. To date, a comprehensive molecular understanding of the underlying mechanisms of tissue tropism has yet to be established, and therefore it is generally believed that the availability of tissue-specific receptors for each serotype plays a central role in the efficient transduction of various serotypes.

There remains a need for additional AAV vectors with improved properties in terms of transgene expression and tissue specificity in vivo. In particular, such vectors have the potential to provide greatly enhanced benefits for gene delivery to various target tissues in humans.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV2 capsid protein, wherein the variant AAV2 capsid protein comprises at least four amino acid substitutions relative to the wild-type AAV2 capsid protein; wherein the at least four amino acid substitutions are present at the following positions of the AAV2 capsid protein sequence: 457, 492, 499 and 533; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In one embodiment, the variant AAV capsid protein comprises the sequence of SEQ ID NO: 2, or a sequence having at least 95% sequence identity thereto. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 1.

In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: M457, A492, D499, and Y533. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: Q457M, S492A, E499D, and F533Y.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions relative to the wild-type AAV capsid protein at the following positions of the AAV2 capsid protein sequence: 125, 151, 162, and 205. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, and S205. In another preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S, and T205S.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions relative to the wild-type AAV capsid protein at the following positions of the AAV2 capsid protein sequence: S585 and 588. Preferably, the variant AAV2 capsid protein comprises one or more of the following residues: S585 and T588. More preferably, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: R585S and R588T.

In one embodiment, the variant AAV2 capsid protein further comprises one or more amino acid substitutions relative to the wild-type AAV capsid protein at the following positions of the AAV2 capsid protein sequence: 546, 548, and 593. Preferably, the variant AAV2 capsid protein comprises one or more of the following residues: D546, G548, and S593. More preferably, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: G546D, E548G, and A593S.

In one embodiment, the variant AAV2 capsid protein comprises residue N312, i.e., the residue present in the wild-type AAV2 capsid protein at position 312. In this embodiment, the variant AAV2 capsid protein is not mutated at position 312 compared to the wild-type AAV2 capsid protein sequence.

In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV8 capsid protein, wherein the variant AAV8 capsid protein comprises an amino acid substitution at position 315 of the AAV8 capsid protein sequence relative to the wild-type AAV8 capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In one embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 6.

In one embodiment, the variant AAV8 capsid protein comprises the amino acid substitution S315N relative to the wild-type AAV8 capsid protein. Preferably, the AAV8 capsid protein sequence comprises one or more amino acid substitutions at one or more of the following positions: 125, 151, 163, 206, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

In preferred embodiments, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein: (a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S; and/or (b) T591R.

In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV3B capsid protein, wherein the variant AAV3B capsid protein comprises an amino acid substitution at position 312 of the AAV3B capsid protein sequence relative to the wild-type AAV3B capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In one embodiment, the variant AAV3B capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 11. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO:11.

In one embodiment, the variant AAV3B capsid protein comprises the amino acid substitution S312N relative to the wild-type AAV3B capsid protein.

In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV-LK03 capsid protein, wherein the variant AAV-LK03 capsid protein comprises an amino acid substitution at position 312 relative to the AAV-LK03 capsid protein sequence defined as SEQ ID NO: 12; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In one embodiment, the variant AAV-LK03 capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO:12.

In another aspect, the present invention provides a recombinant adeno-associated virus (AAV) vector comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution relative to the wild-type AAV capsid protein at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In one embodiment, the at least one amino acid substitution occurs at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or more corresponding positions of an alternative AAV capsid protein sequence.

In one embodiment, the vector comprises a variant AAV2 capsid protein. In another embodiment, the variant AAV capsid protein comprises the sequence of SEQ ID NO: 2, or a sequence having at least 95% sequence identity thereto. In another embodiment, the wild-type AAV capsid protein is from AAV2. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 1.

In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, and/or S593. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and/or A593S.

In other embodiments, the variant AAV capsid protein is from AAV1, AAV5, AAV6, AAV8, AAV9, or AAV10.

In one embodiment, the vector comprises a variant AAV1 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 3. In another embodiment, the wild-type AAV capsid protein is from AAV1. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 3.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589, and/or 594. In a preferred embodiment, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV1 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. In an alternative embodiment, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV1 capsid protein: S205T, G549E, S586R, and/or T589R.

In one embodiment, the vector comprises a variant AAV5 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 4. In another embodiment, the wild-type AAV capsid protein is from AAV5. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 4.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions of the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578, and/or 583. In a preferred embodiment, the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV5 capsid protein: V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D, and/or G583S. In an alternative embodiment, the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV5 capsid protein: G537E, S575R, and/or T578R.

In one embodiment, the vector comprises a variant AAV6 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 5. In another embodiment, the wild-type AAV capsid protein is from AAV6. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 5.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions of the AAV6 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589, and/or 594. In a preferred embodiment, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV6 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. In an alternative embodiment, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV6 capsid protein: S205T, G549E, S586R, and/or T589R.

In one embodiment, the vector comprises a variant AAV8 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6. In another embodiment, the wild-type AAV capsid protein is from AAV8. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 6.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591, and/or 596. In a preferred embodiment, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S, and/or G596S. In an alternative embodiment, the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein: S315N and/or T591R.

In one embodiment, the vector comprises an AAV9 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7. In another embodiment, the wild-type AAV capsid protein is from AAV9. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 7.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589, and/or 594. In a preferred embodiment, the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV9 capsid protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T, and/or G594S. In an alternative embodiment, the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV9 capsid protein: S162A, S205T, G549E, and/or S586R.

In one embodiment, the vector comprises a variant AAV10 capsid protein. In another embodiment, the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8. In another embodiment, the wild-type AAV capsid protein is from AAV10. In another embodiment, the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 8.

In one embodiment, at least one amino acid substitution is present at one or more of the following positions in the AAV10 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591, and/or 596. In a preferred embodiment, the variant AAV10 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV10 capsid protein: V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T, and/or G596S. In an alternative embodiment, the variant AAV10 capsid protein comprises the following amino acid substitution relative to the wild-type AAV10 capsid protein: G551E.

In one embodiment, the recombinant AAV vector exhibits increased transduction of neurons or retinal tissue compared to an AAV vector comprising a corresponding wild-type AAV capsid protein.

In another embodiment, the recombinant AAV vector exhibits increased transduction of liver tissue compared to the corresponding wild-type AAV capsid protein.

In one embodiment, the gene product comprises an interfering RNA or an aptamer. In another embodiment, the gene product comprises a polypeptide. Preferably, the gene product comprises a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of neurons or retinal cells. In a preferred embodiment, the gene product comprises glial-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin.

In another aspect, the present invention provides a pharmaceutical composition comprising: (a) a recombinant AAV vector according to any one of the preceding claims; and (b) a pharmaceutically acceptable excipient.

In another aspect, the present invention provides a method for delivering a gene product to a tissue of a subject, the method comprising administering to the subject a recombinant AAV vector or a pharmaceutical composition as defined above.

In some embodiments, the tissue is selected from blood, bone marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue or heart tissue. Preferably, the tissue is neuronal, retinal or liver tissue.

In another aspect, the present invention provides a method for treating a condition in a subject, the method comprising administering to the subject a recombinant AAV vector or pharmaceutical composition as defined above. In some embodiments, the condition is a neurological, ocular, or liver condition.

In another aspect, the present invention provides a recombinant AAV vector or pharmaceutical composition as defined above for use in treating a condition in a subject. In some embodiments, the condition is a neurological, ocular, or hepatic condition. Preferably, the neurological condition is a neurodegenerative disease. In alternative embodiments, the ocular condition is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, or diabetic retinopathy.

In another aspect, the present invention provides an isolated variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution relative to the wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or is present at one or more corresponding positions of the alternative AAV capsid protein sequence.

In another aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as defined above.

In another aspect, the invention provides an isolated host cell comprising a nucleic acid as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the amino acid sequence of wild-type adeno-associated virus 2 capsid protein VP1 (SEQ ID NO: 1; NCBI Reference Sequence: NC_001401). Residues V125, V151, A162, T205, N312, Q457, S492, E499, F533, G546, E548, R585, R588, and A593 are highlighted.

Figure 2 shows the amino acid sequence of the authentic adeno-associated virus 2 (ttAAV2) capsid protein VP1 (SEQ ID NO: 2). Residues I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, and S593 differ from wild-type AAV2 VP1 (SEQ ID NO: 1) and are highlighted.

Figure 3 shows the amino acid sequence of wild-type adeno-associated virus 1 capsid protein VP1 (SEQ ID NO: 3; NCBI Reference Sequence: NC_002077). Highlighted residues: S205 (aligned with S205 in ttAAV2 (SEQ ID NO: 2))-G549 (aligned with G548 in ttAAV2)-S586 (aligned with S585 in ttAAV2)-T589 (aligned with T588 in ttAAV2).

Figure 4 shows the amino acid sequence of wild-type adeno-associated virus 5 capsid protein VP1 (SEQ ID NO: 4; NCBI reference sequence: AF085716). Highlighted residues: G537 (aligned with G548 in ttAAV2)-S575 (aligned with S585 in ttAAV2)-T578 (aligned with T588 in ttAAV2).

Figure 5 shows the amino acid sequence of wild-type adeno-associated virus 6 capsid protein VP1 (SEQ ID NO: 5; NCBI reference sequence: AF028704). Highlighted residues: S205 (aligned with S205 in ttAAV2) - G549 (aligned with G548 in ttAAV2) - S586 (aligned with S585 in ttAAV2) - T589 (aligned with T588 in ttAAV2).

Figure 6 shows the amino acid sequence of wild-type adeno-associated virus 8 capsid protein VP1 (SEQ ID NO: 6; NCBI reference sequence: NC_006261). Highlighted residues: S315 (aligned with S312 in ttAAV2)-T591 (aligned with T588 in ttAAV2).

Figure 7 shows the amino acid sequence of wild-type adeno-associated virus 9 capsid protein VP1 (SEQ ID NO: 7; NCBI reference sequence: AY530579). Highlighted residues: S162 (aligned with S1621 in ttAAV2)-S205 (aligned with S205 in ttAAV2)-G549 (aligned with G548 in ttAAV2)-S586 (aligned with S585 in ttAAV2).

Figure 8 shows the amino acid sequence of wild-type adeno-associated virus 10 capsid protein VP1 (SEQ ID NO: 8). Highlighted residue: G551 (aligned with G548 in ttAAV2).

FIG9 shows an alignment of the amino acid sequences of AAV capsid protein VP1.

Figure 10: The plasmid used to generate AAV2 vectors is the packaging plasmid pDG. Top: pDG harboring the wild-type AAV2 genome. Bottom: pDG-ttAAV2 harboring the authentic AAV2 genome, highlighting two key mutations in the heparan binding domain at positions 585 and 588. MMTV: The promoter driving AAV rep expression; E2a, E4ORF6, and VA are genes expressing adenoviral accessory factors.

Figure 11: Viral titers of rAAV2 authentic (TT) and wild-type (WT) for in vivo injection were quantified by SDS-PAGE showing krypton staining of separated proteins and scanned with an infrared-fluorescence scanner (Odyssey Imaging systems). A: 10 μl of AAV2 viral particles and 62.5 ng-500 ng of BSA were separated on a 12% separating gel containing SDS and stained with krypton protein dye. The image was converted to grayscale. The capsid gene proteins VP1, VP2, and VP3 are labeled on the left. B: Table showing titers from qPCR (vector genomes [vg/ml]) and SDS-PAGE (capsid titer [capsid/ml]).

Figure 12 A: shows a representative example of a rat brain section stained with a GFP-specific antibody. The vector was injected into the striatum as indicated by the arrow. B: shows a representative example of an injection into the substantia nigra.

Figure 13: shows GFP transduction of eyes using ttAAV2 and wtAAV2. A: shows the retina in cross section after ttAAV2 (upper panel) and wtAAV2 (lower panel) vector administration. B: shows an enlarged view of the dotted box in A.

Figure 14: Transduction of the mouse brain after neonatal vector injection. i.v.: intravenous vector administration; i.c.: intracranial injection; AAV-2, wtAAV2; AAV-TT, ttAAV2.

Figure 15: 3D representation of the AAV2 capsid. Highlighted residues correspond to amino acid changes between ttAAV2 and wild-type particles and are grouped by color according to their position.

Figure 16: Graphical representation of the triplet peak on AAV2 capsid. Highlighted residues correspond to amino acid changes between authentic and wild-type particles. Heparin binding site residues are highlighted in green.

Figure 17: Schematic representation of the inside of the AAV2 capsid. The light blue highlighted residues correspond to single amino acid changes in ttAAV2 that are located on the inside of the capsid.

Figure 18: Graphic representation of the threefold peaks on the AAV2 capsid. The light brown highlighted residues correspond to two amino acid changes in the authentic vector that are spatially close together and located in the groove between the two threefold-proximal peaks on the AAV capsid.

Figure 19: Schematic representation of the threefold peaks on AAV2 capsid. The brown highlighted residue corresponds to a single isolated amino acid change (S593) in the authentic vector located in the groove between the threefold-proximal peaks.

Figure 20: Schematic representation of the threefold peak on the AAV2 capsid. The four amino acids highlighted in pink are involved in receptor binding and are closely located on the threefold peak.

Figure 21: Three-dimensional representation of the alignment of the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV1 (orange). The residues highlighted in the center left of the figure correspond to G549 in AAV1 (orange spheres) and E548 in AAV2 (cyan spheres). The residues highlighted in the upper right of the figure correspond to S586 and T589 in AAV1 (orange spheres) and R585 and R588 in AAV2 (cyan spheres).

Figure 22: Three-dimensional representation of the alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV5 (purple). The highlighted residues in the middle of the figure correspond to G537 in AAV5 (purple spheres) and E548 in AAV2 (cyan spheres). The highlighted residues in the upper right of the figure correspond to S575 and T578 in AAV5 (purple spheres) and R585 and R588 in AAV2 (cyan spheres).

Figure 23: Three-dimensional representation of the alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV6 (yellow). The highlighted residues at the bottom of the figure correspond to G549 in AAV6 (orange spheres) and E548 in AAV2 (cyan spheres). The highlighted residues at the top right of the figure correspond to S586 and T589 in AAV6 (orange spheres) and R585 and R588 in AAV2 (cyan spheres).

Figure 24: Three-dimensional representation of the alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV8 (pink). The residues highlighted in the upper left of the figure correspond to S315 in AAV8 (red spheres) and N312 in AAV2 (cyan spheres). The residues highlighted in the lower right of the figure correspond to T591 in AAV8 (red spheres) and R588 in AAV2 (cyan spheres).

Figure 25: Three-dimensional representation of the alignment between the VP1 capsid monomer from AAV2 (light blue) and the VP1 monomer from AAV9 (green). The residues highlighted in the middle of the figure correspond to G549 in AAV9 (yellow spheres) and E548 in AAV2 (cyan spheres). The residues highlighted in the lower left of the figure correspond to S586 in AAV9 (yellow spheres) and R585 in AAV2 (cyan spheres).

Figure 26: Analysis of rAAV2TT and WT expression in the parafascicular nucleus after striatal injection in rat brain. A: Representative images of rat brain sections showing the rostral end on the left and the caudal end on the right. The site of injection in the striatum is indicated, and the projection area in the hypothalamus (parafascicular nucleus, pf) observed in B and C is shown. B and C: High magnification images of GFP expression detected in the parafascicular nucleus (pf) after striatal injection of rAAV2WT (B) or TT (C).

Figure 27: Overview of intracranial injection of rAAV2TT and WT in neonatal mice. Representative examples of neonatal mouse brain sections stained with a GFP-specific antibody are shown. ^{5 x 1010} vg of rAAV2TT (top panel) or rAAV2WT (center panel) were injected into the lateral ventricle of the neonatal mouse brain. Simultaneously stained uninjected brains from neonatal mice are shown as negative controls (NT, non-transduced).

Figure 28: High-magnification photographs of neonatal mouse brain sections following intracranial injection of rAAV2TT or WT. Shown are neonatal mouse brain sections stained with a GFP-specific antibody. 5 x ^{10 10} vg of rAAV2TT (left) or rAAV2WT (right) were injected into the lateral ventricle of the neonatal mouse brain. S1BF: Barrel zone primary somatosensory cortex.

Figure 29: Overview of brain transduction after systemic injection of rAAV2TT and WT in neonatal mice. Representative examples of neonatal mouse brain sections stained with a GFP-specific antibody are shown. 2x10 ¹¹ vg of rAAV2TT (top) or rAAV2WT (bottom) were injected into the jugular vein of neonatal mice.

Figure 30: High-magnification photographs of neonatal mouse brain sections following systemic injection of rAAV2TT or WT. Shown are neonatal mouse brain sections stained with a GFP-specific antibody. 2 x 10 ¹¹ vg of rAAV2TT (left) or rAAV2WT (right) were injected into the jugular vein of neonatal mice. S1BF: Barrel zone primary somatosensory cortex.

Figure 31: High-magnification photographs of tissue sections from newborn mice after systemic injection of rAAV2TT or WT. 2x10 ¹¹ vg of rAAV2TT or rAAV2WT were injected into the jugular vein of newborn mice. Organs from uninjected mice served as negative controls.

Figure 32: High-magnification images of adult rat brain sections following striatal injection of rAAV2TT, WT, and HBnull. Representative examples of rat brain sections stained with a GFP-specific antibody are shown. ^{3.5 x 109} vg of rAAV2WT (left), TT (right), or AAV2-HBnull (center) were injected into the striatum of adult rat brains, and representative images were obtained in the thalamus or substantia nigra (SN).

FIG33 : Overview of intracranial injection of full AAV-TT compared to various TT mutants in neonatal mice. Shown are representative examples of neonatal mouse brain sections stained with a GFP-specific antibody. 5×10 ¹⁰ vg of rAAV2TT, TT-S312N, TT-S593A, or TT-D546G/G548E (TT-DG) were injected into the lateral ventricles of neonatal mouse brains. Simultaneously stained uninjected brains from neonatal mice are shown as negative controls (NT).

Figure 34: High-magnification photographs of neonatal mouse brain sections following intracranial injection of various TT mutant vectors. Shown are neonatal mouse brain sections stained with a GFP-specific antibody. 5 x ^{10 10} vg of vector were injected into the lateral ventricle of the neonatal mouse brain. TT-DG: TT-D546G/G548E.

FIG35 : Overview of neonatal intracranial injection of full AAV-TT compared to the TT-S312N mutant and the potential final TT vector containing 10 mutations. Shown are representative examples of neonatal mouse brain sections stained with a GFP-specific antibody. ^{5×10 09} vg of rAAV2TT, TT-S312N, TT, or TT-S312N-D546G/G548E-S593A (TT-S312N-DG-S593A) were injected into the lateral ventricle of the neonatal mouse brain. Simultaneously stained uninjected brains from neonatal mice are shown as negative controls (NT).

Figure 36: High-magnification photographs of neonatal mouse brain sections after intracranial injection of various TT mutant vectors. Shown are neonatal mouse brain sections stained with a GFP-specific antibody. ^{5 x 10 09} vg of vector were injected into the lateral ventricle of the neonatal mouse brain.

Figure 37: ELISA quantification of GFP protein in the brains of newborn mice injected with full AAV-TT, TT-S312N mutant, or TT-S312N-DG-S593A. ^{5 x 10 09} vg of vector were injected into the lateral ventricle of the newborn mouse brain, and total protein was extracted from the whole harvested brain. A GFP-specific antibody was used to detect GFP expression in each brain sample, and a standard GFP protein was used for quantification. N = 5 animals/condition. Error bars represent mean ± SEM.

Figure 38: Amino acid sequence of the VP1 capsid protein of AAV3B. Highlighted residues indicate residues identical to those at corresponding positions in AAV-tt. The internal serine residue at position 312 is underlined.

FIG. 39 : Amino acid sequence of the VP1 capsid protein of AAV-LK03.

Sequence Listing

SEQ ID NO: 1 is the amino acid sequence of wild-type adeno-associated virus 2 capsid protein VP 1 (see FIG1 ).

SEQ ID NO: 2 is the amino acid sequence of the authentic adeno-associated virus 2 (ttAAV2) capsid protein (see FIG2 ).

SEQ ID NO: 3 is the amino acid sequence of wild-type adeno-associated virus 1 capsid protein VP1 (see FIG3 ).

SEQ ID NO: 4 is the amino acid sequence of wild-type adeno-associated virus 5 capsid protein VP1 (see FIG4 ).

SEQ ID NO: 5 is the amino acid sequence of wild-type adeno-associated virus 6 capsid protein VP1 (see FIG5 ).

SEQ ID NO: 6 is the amino acid sequence of wild-type adeno-associated virus 8 capsid protein VP1 (see FIG6 ).

SEQ ID NO: 7 is the amino acid sequence of wild-type adeno-associated virus 9 capsid protein VP1 (see FIG7 ).

SEQ ID NO: 8 is the amino acid sequence of wild-type adeno-associated virus 10 Upenn capsid protein VP1 (see FIG8 ).

SEQ ID NO: 9 is the amino acid sequence of wild-type adeno-associated virus 10 Japan capsid protein VP1 (see FIG9 ).

SEQ ID NO: 10 is the consensus amino acid sequence of the adeno-associated virus shown in FIG9 .

SEQ ID NO: 11 is the amino acid sequence of wild-type adeno-associated virus 3B capsid protein VP1 (see FIG38 ).

SEQ ID NO: 12 is the amino acid sequence of adeno-associated virus LK-03 capsid protein VP1 (see FIG39 ).

Detailed Description of the Invention

In one aspect, the present invention relates to recombinant adeno-associated virus (AAV) vectors. rAAV vectors typically contain variant capsid proteins that are different from wild-type AAV capsid proteins. Variant capsid proteins can advantageously confer enhanced vector infectivity in the brain and/or eye, making the vector particularly suitable for delivering therapeutic agents to these tissues via gene therapy.

Recombinant AAV vector

The present disclosure provides recombinant adeno-associated virus (rAAV) vectors. "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes and naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes, for example, AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10, including AAVrh10), AAV type 12 (AAV-12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and the like.

The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TRs), Rep proteins, and capsid subunits are well known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8); the disclosures of which are incorporated herein by reference. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Application Nos. WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No. 6,156,303.

As used herein, "rAAV vector" refers to an AAV vector comprising a polynucleotide sequence that is not derived from AAV (i.e., a polynucleotide heterologous to AAV), typically a target sequence for genetic transformation of a cell. In some embodiments, the heterologous polynucleotide may be flanked by at least one and sometimes two AAV inverted terminal repeats (ITRs). The term rAAV vector includes rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV).

"AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle that contains at least one AAV capsid protein (usually all capsid proteins of wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is usually referred to as a "rAAV vector particle" or simply "rAAV vector". Therefore, the production of rAAV particles necessarily includes the production of rAAV vectors, because such vectors are contained within rAAV particles.

As used herein, "recombinant" refers to vectors, polynucleotides, polypeptides, or cells that are the product of cloning, restriction, or ligation steps (e.g., involving the polynucleotides or polypeptides contained therein), and/or various combinations of other procedures that result in constructs that are different from those found in nature. A recombinant virus or vector is a viral particle that contains a recombinant polynucleotide. The term includes duplicates of the original polynucleotide construct and progeny of the original viral construct, respectively.

Variant AAV capsid protein

The rAAV vectors described herein comprise variant AAV capsid proteins. "Variant" refers to an AAV capsid protein that differs from a corresponding wild-type AAV capsid protein of the same serotype. For example, a variant AAV capsid protein can comprise one or more amino acid substitutions relative to a corresponding wild-type AAV capsid protein. In this context, "corresponding" refers to a capsid protein of the same serotype, i.e., a variant AAV1 capsid protein comprises one or more amino acid substitutions relative to a corresponding wild-type AAV1 capsid protein, a variant AAV2 capsid protein comprises one or more amino acid substitutions relative to a corresponding wild-type AAV2 capsid protein, and so on.

The variant AAV capsid protein can comprise, for example, 1 to 50, 1 to 30, 1 to 20, or 1 to 15 amino acid substitutions relative to the wild-type AAV capsid protein. Preferably, the variant AAV capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid substitutions relative to the corresponding wild-type AAV capsid protein. In preferred embodiments, the variant AAV capsid protein retains at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type capsid protein.

In an embodiment of the present invention, the variant AAV capsid protein comprises at least one amino acid substitution relative to the wild-type AAV capsid protein at a position corresponding to one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593. In this context, "corresponding" refers to a position in any AAV capsid protein sequence (e.g., in an AAV2 protein sequence or a non-AAV2 capsid protein sequence) that corresponds to one of the aforementioned positions of the AAV2 capsid protein. In one embodiment, the at least one amino acid substitution is present at one or more of the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593; or at one or more corresponding positions in an alternative AAV capsid protein sequence.

Typically, AAV capsid proteins include VP1, VP2, and VP3. In a preferred embodiment, the capsid protein includes AAV capsid protein VP1.

Nucleic acid and amino acid sequences and sequence identity

The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, the modification of the nucleotide structure may be imparted before or after the assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise noted or required, any embodiment of the present invention described herein that is a polynucleotide includes each of a double-stranded form and two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also includes amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation to a labeling component. Polypeptides, such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering gene products to mammalian subjects, and therefore compositions, refer to the respective intact polypeptides, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for delivering gene products to mammalian subjects (which may be referred to as "transgenes" to be delivered to recipient cells) include polynucleotides encoding the intact polypeptides or any fragment or genetically engineered derivative having the desired biochemical function.

A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that, when aligned, the percentage of bases or amino acids in the sequence when comparing the two sequences is the same. Sequence similarity can be measured in many different ways. To determine sequence identity, sequences can be aligned using methods and computer programs including BLAST (available on the World Wide Web at ncbi.nlm.nih.gov/BLAST/). Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) program package from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc. (a division of Harcourt Brace & Co., San Diego, California, USA). Of particular interest are alignment programs that allow for gaps in sequences. Smith-Waterman is a type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). In addition, the GAP program, which uses the Needleman and Wunsch alignment method, can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

Of interest is the BestFit program that uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489 (1981)) to determine sequence identity. The gap creation penalty will generally be in the range of 1 to 5, typically in the range of 2 to 4, and in many embodiments will be 3. The gap extension penalty will generally be in the range of about 0.01 to 0.20, and in many cases will be 0.10. The program has default parameters determined by the sequences input for comparison. Preferably, sequence identity is determined using the default parameters determined by the program. The program is also available from the Genetics Computing Group (GCG) program package from Madison, Wisconsin, USA.

Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based on the following parameters:

Mismatch penalty: 1.00;

Gap penalty: 1.00;

Gap size penalty: 0.33; and

Adding penalty: 30.0.

Variant AAV2 capsid protein

In one embodiment, the vector comprises a variant AAV2 capsid protein. In this embodiment, the variant AAV2 capsid protein comprises at least one amino acid substitution at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593.

The sequence of the wild-type AAV2 capsid protein VP1 is known and is shown in Figure 1 (SEQ ID NO: 1). The sequence of the wild-type AAV2 capsid protein is also available from database accession numbers: NC-001401; UniProt P03135; NCBI reference sequence: YP_680426.1; and GenBank: AAC03780.1.

Preferably, the variant AAV2 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In a preferred embodiment, the variant AAV2 capsid protein comprises the sequence of SEQ ID NO: 2, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

In one embodiment, the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588, and/or S593. In a preferred embodiment, the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and/or A593S.

Combinations of mutations in the AAV2 capsid protein

The variant AAV2 capsid protein may comprise any combination of the above amino acid substitutions. Thus, in specific embodiments, the variant AAV2 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid substitutions selected from the above list. In one embodiment, the variant AAV2 capsid protein comprises all 14 amino acid substitutions disclosed above, for example, the variant AAV2 capsid protein comprises the sequence of SEQ ID NO: 2 (i.e., ttAAV2 or AAV2-TT as referred to herein).

In other embodiments, the variant AAV2 capsid protein may comprise a subset of the 14 mutations described above. Without being bound by theory, in individual embodiments, the variant AAV2 capsid protein may comprise the following residues (which are divided into functional groups below):

1) S585 and/or T588; these residues may be associated with reduced heparin binding and increased viral spread in brain tissue rich in heparan sulfate proteoglycans;

2) S312; this internal serine residue may play a role in capsid-DNA interaction;

3) D546 and/or G548; these residues may be involved in interactions with neutralizing antibodies, thereby contributing to transduction properties in vivo;

4) S593; this residue is located in the groove between the three-fold proximal peaks;

5) M457, A492, D499 and/or Y533; these four amino acids may be involved in receptor binding and are closely located on the threefold peak;

6) I125, A151, S162 and/or S205; these residues may be involved in PLA2 activity and/or trafficking of invading viruses.

It is to be understood that corresponding subsets comprising mutations corresponding to the above-mentioned residues when present at corresponding positions in other AAV serotypes (see below) are also contemplated herein.

In a preferred embodiment, the variant AAV2 capsid protein comprises four or more mutations at the aforementioned positions associated with receptor binding, namely residues 457, 492, 499, and 533. Thus, it is particularly preferred that the variant AAV2 capsid protein comprises the following residues: M457, A492, D499, and Y533.

In some preferred embodiments, the variant AAV2 capsid protein is not mutated at position 312 relative to the wild-type AAV2 capsid protein, e.g., the variant AAV2 capsid protein comprises residue N312 (which is present in the wild-type AAV2 capsid protein). Thus, in some embodiments, the variant AAV2 capsid protein may comprise 1 to 13 of the above-mentioned specific mutations, but does not comprise the mutation N312S.

Variant AAV capsid proteins from other serotypes

In other embodiments, the variant AAV capsid protein is from an alternative AAV serotype, i.e., an AAV serotype other than AAV2. For example, the variant AAV capsid protein can be derived from an AAV1, AAV3B, AAV-LK03, AAV5, AAV6, AAV8, AAV9, or AAV10 (e.g., AAVrh10) capsid protein.

In these embodiments, the variant AAV capsid protein comprises at least one amino acid substitution at one or more positions corresponding to the positions described above for AAV2. In other words, the variant AAV capsid protein comprises at least one amino acid substitution at positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593 of the AAV2 capsid protein sequence in an alternative (i.e., non-AAV2) AAV capsid protein sequence.

Based on a comparison of the amino acid sequences of capsid proteins from various AAV serotypes, one skilled in the art will know how to identify positions in capsid proteins from alternative AAV serotypes that correspond to positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593 of the AAV2 capsid protein. In particular, such positions can be readily identified by sequence alignments known in the art and described herein. For example, one such sequence alignment is provided in FIG9 .

Of particular relevance in this context are positions in alternative AAV capsid protein sequences that correspond in three-dimensional space to positions 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593 of the AAV2 capsid protein. Methods for three-dimensional modeling and alignment of protein structures are well known in the art and can be used to identify such corresponding positions in non-AAV2 capsid protein sequences. Exemplary 3D alignments of AAV2 capsid protein sequences with capsid protein sequences of alternative AAV serotypes (e.g., AAV1, AAV5, AAV6, AAV8, and AAV9) are shown in Figures 21 to 25 and discussed below. One skilled in the art can perform similar 3D alignments with capsid proteins from other serotypes (e.g., AAV2, AAV3, AAV7, AAV10, and AAV12) and identify positions in such sequences that correspond to the positions defined above in AAV2.

Variant AAV1 capsid protein

In one embodiment, the vector comprises a variant AAV1 capsid protein. In this embodiment, the variant AAV1 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. These positions in the AAV1 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV1 capsid protein VP1 is known and is shown in Figure 3 (SEQ ID NO: 3). The wild-type AAV1 capsid protein sequence is also available from database accession number: NC-002077. Preferably, the variant AAV1 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3.

The wild-type AAV1 capsid protein VP1 already comprises the following residues at positions corresponding to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2); and T589 (aligned with T588 in ttAAV2). Thus, in preferred embodiments, the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV1 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. Typically, such variant AAV1 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV1 capsid protein.

In alternative embodiments, the variant AAV1 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV1 capsid protein may comprise one or more of the following substitutions: S205T, G549E, S586R, and/or T589R. Generally, such variant AAV1 capsid proteins may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV1 capsid protein.

Variant AAV5 capsid protein

In one embodiment, the vector comprises a variant AAV5 capsid protein. In this embodiment, the variant AAV5 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578, and/or 583. These positions in the AAV5 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV5 capsid protein VP1 is known and is shown in Figure 4 (SEQ ID NO: 4). The wild-type AAV5 capsid protein sequence is also available from database accession number: AF085716. Preferably, the variant AAV5 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

The wild-type AAV5 capsid protein VP1 already contains the following residues at positions corresponding to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): G537 (aligned with G548 in ttAAV2); S575 (aligned with S585 in ttAAV2); T578 (aligned with T588 in ttAAV2). Thus, in preferred embodiments, the variant AAV5 capsid protein contains one or more of the following amino acid substitutions relative to the wild-type AAV5 capsid protein: V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D, and/or G583S. Typically, such variant AAV5 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV5 capsid protein.

In alternative embodiments, the variant AAV5 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV5 capsid protein may comprise one or more of the following substitutions: G537E, S575R, and/or T578R. Generally, such variant AAV5 capsid proteins may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV5 capsid protein.

Variant AAV6 capsid protein

In one embodiment, the vector comprises a variant AAV6 capsid protein. In this embodiment, the variant AAV6 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV6 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. These positions in the AAV6 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV6 capsid protein VP1 is known and is shown in Figure 5 (SEQ ID NO: 5). The wild-type AAV6 capsid protein sequence is also available from database accession number: AF028704. Preferably, the variant AAV6 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5.

The wild-type AAV6 capsid protein VP1 already comprises the following residues at positions corresponding to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2); T589 (aligned with T588 in ttAAV2). Thus, in preferred embodiments, the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV6 capsid protein: V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D, and/or G594S. Typically, such variant AAV6 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV6 capsid protein.

In alternative embodiments, the variant AAV6 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV6 capsid protein may comprise one or more of the following substitutions: S205T, G549E, S586R, and/or T589R. Generally, such variant AAV6 capsid proteins may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV6 capsid protein.

Variant AAV8 capsid protein

In one embodiment, the vector comprises a variant AAV8 capsid protein. In this embodiment, the variant AAV8 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596. These positions in the AAV8 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV8 capsid protein VP1 is known and is shown in Figure 6 (SEQ ID NO: 6). The wild-type AAV8 capsid protein sequence is also available from database accession number: NC_006261. Preferably, the variant AAV8 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

The wild-type AAV8 capsid protein VP1 already contains the following residues at positions corresponding to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): S315 (aligned with S312 in ttAAV2); T591 (aligned with T588 in ttAAV2). Thus, in a preferred embodiment, the variant AAV8 capsid protein contains one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein: V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S. Typically, such variant AAV8 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV8 capsid protein.

In alternative embodiments, the variant AAV8 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV8 capsid protein may comprise one or more of the following substitutions: S315N and/or T591R. Generally, such variant AAV8 capsid proteins may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV8 capsid protein.

In one embodiment, the variant AAV8 capsid protein comprises an amino acid substitution at position 315 of the AAV8 capsid protein sequence relative to the wild-type AAV8 capsid protein. For example, the variant AAV8 capsid protein can comprise residue N315. Thus, in one embodiment, the variant AAV8 capsid protein comprises the amino acid substitution S315N relative to the wild-type AAV8 capsid protein.

Variant AAV9 capsid protein

In one embodiment, the vector comprises a variant AAV9 capsid protein. In this embodiment, the variant AAV9 capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594. These positions in the AAV9 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV9 capsid protein VP1 is known and is shown in Figure 7 (SEQ ID NO: 7). The wild-type AAV9 capsid protein sequence is also available from database accession number: AY530579. Preferably, the variant AAV9 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7.

The wild-type AAV9 capsid protein VP1 already contains the following residues at positions corresponding to amino acid residues present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): S162 (aligned with S162 in ttAAV2); S205 (aligned with S205 in ttAAV2); G549 (aligned with G548 in ttAAV2); S586 (aligned with S585 in ttAAV2). Thus, in preferred embodiments, the variant AAV9 capsid protein contains one or more of the following amino acid substitutions relative to the wild-type AAV1 capsid protein: L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and/or G594S. Typically, such variant AAV9 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV9 capsid protein.

In alternative embodiments, the variant AAV9 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV9 capsid protein may comprise one or more of the following substitutions: S162A, S205T, G549E, and/or T589R. Typically, such variant AAV9 capsid proteins may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV9 capsid protein.

Variant AAV10 capsid protein

In one embodiment, the vector comprises a variant AAV10 capsid protein. As used herein, "AAV10" includes AAVrh10. In this embodiment, the variant AAV10 (e.g., AAVrh10) capsid protein comprises at least one amino acid substitution at one or more of the following positions in the AAV10 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591, and/or 596. These positions in the AAV10 capsid protein VP1 correspond to those disclosed above for AAV2.

The sequence of the wild-type AAV10 capsid protein VP1 is known and is shown in Figure 8 (SEQ ID NO: 8). Preferably, the variant AAV10 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 8.

The wild-type AAV10 capsid protein VP1 already contains the following residue at a position corresponding to an amino acid residue present in the variant AAV2 capsid protein disclosed above (SEQ ID NO: 2, ttAAV2) but not in wild-type AAV2 (SEQ ID NO: 1): G551 (aligned with G548 in ttAAV2). Thus, in a preferred embodiment, the variant AAV10 capsid protein contains one or more of the following amino acid substitutions relative to the wild-type AAV10 capsid protein: V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and/or G596S. Typically, such variant AAV10 capsid proteins may share one or more functional properties with a variant AAV2 capsid protein (SEQ ID NO: 2, ttAAV2), for example, may confer increased infectivity and/or transduction of neuronal or retinal tissues compared to wild-type AAV10 capsid protein.

In alternative embodiments, the variant AAV10 capsid protein comprises one or more amino acid substitutions corresponding to mutations present in ttAAV2 that convert back to the wild-type AAV2 sequence. For example, the variant AAV10 capsid protein may comprise the following substitution: G551E. Typically, such a variant AAV10 capsid protein may share one or more functional properties with the wild-type AAV2 capsid protein (SEQ ID NO: 1), for example, may confer reduced infectivity and/or transduction of neuronal or retinal tissues compared to the wild-type AAV10 capsid protein.

Variant AAV3B capsid protein

In one embodiment, the vector comprises a variant AAV3B capsid protein. In this embodiment, the variant AAV3B capsid protein may comprise an amino acid substitution at position 312 relative to the wild-type AAV3B capsid protein. For example, the variant AAV3B capsid protein may comprise residue N312. Thus, in one embodiment, the variant AAV8 capsid protein comprises the amino acid substitution S312N relative to the wild-type AAV8 capsid protein. In other embodiments, the variant AAV3B capsid protein may comprise one or more additional mutations at positions corresponding to those disclosed above for AAV2.

The sequence of the wild-type AAV3B capsid protein VP1 is known and is shown in Figure 38 (SEQ ID NO: 11). The wild-type AAV3B capsid protein sequence is also available from the NCBI database under Accession No. AF028705. Preferably, the variant AAV3B capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11.

Variant AAV-LK03 capsid protein

In one embodiment, the vector comprises a variant AAV-LK03 capsid protein. In this embodiment, the variant AAV-LK03 capsid protein may comprise an amino acid substitution at position 312 relative to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12. For example, the variant AAV-LK03 capsid protein may comprise residue N312. Thus, in one embodiment, the variant AAV-LK03 capsid protein comprises the amino acid substitution S312N relative to the AAV-LK03 capsid protein sequence as defined in SEQ ID NO: 12. In other embodiments, the variant AAV-LK03 capsid protein may comprise one or more additional mutations at positions corresponding to those disclosed above for AAV2.

The sequence of the wild-type AAV-LK03 capsid protein VP1 is known and is shown in Figure 39 (SEQ ID NO: 12). The wild-type AAV-LK03 capsid protein sequence is also disclosed in WO 2013/029030 as Sequence No. 31. Preferably, the variant AAV-LK03 capsid protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12.

gene products

In one embodiment, the rAAV further comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a specific protein after transcription and translation. A "gene product" is a molecule produced by the expression of a specific gene. Gene products include, for example, polypeptides, aptamers, interfering RNAs, mRNAs, and the like.

"Heterologous" means derived from an entity that is genotypically different from the rest of the entity being compared. For example, a polynucleotide that is introduced into a plasmid or vector from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally found is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising a nucleic acid that is not normally included in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV.

In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide.

interfering RNA

When the gene product is an interfering RNA (RNAi), suitable RNAi includes RNAi that reduces the level of apoptosis or angiogenesis factors in the cell. For example, RNAi can be shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in the cell. Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes" and the products (mRNA; proteins) of these genes are referred to as "pro-apoptotic gene products". Pro-apoptotic gene products include, for example, Bax, Bid, Bak and Bad gene products. See, for example, U.S. Patent No. 7,846,730.

Interfering RNA can also be directed against angiogenic products, such as VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication No. 2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al. (2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther. 13:225), or VEGFR2 (Kou et al. (2005) Biochem. 44:15064)). See also, U.S. Patent Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; and U.S. Patent Nos. 7,947,659 and 7,919,473.

"Small interfering" or "short interfering RNA" or siRNA is an RNA duplex of nucleotides targeted to a target gene ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA is "targeted" to a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the duplex of the siRNA is less than 30 nucleotides in length. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can also include a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may further comprise a 3' or 5' overhang. In some embodiments, the overhang is a 3' or 5' overhang nucleotide of 0, 1, 2, 3, 4, or 5 nucleotides in length.

"Short hairpin RNA" or shRNA is a polynucleotide construct that can be generated to express interfering RNA, such as siRNA.

Aptamer

When the gene product is an aptamer, exemplary aptamers of interest include aptamers to vascular endothelial growth factor (VEGF). See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902. Also suitable for use are aptamers specific for PDGF, e.g., E10030; see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol. 207:407).

peptides

In one embodiment, the gene product is a therapeutic protein. A "therapeutic" peptide or protein is one that alleviates or reduces symptoms caused by a protein deficiency or defect in a cell or subject. Alternatively, a "therapeutic" peptide or protein is one that confers a benefit to a subject in other ways, such as an anti-degenerative effect.

When the gene product is a polypeptide, the polypeptide is typically one that enhances the function of a cell, e.g., a cell present in neuronal, retinal, or liver tissue, such as a hepatocyte, a neuron, a glial cell, a rod or cone photoreceptor cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell.

Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptor; VEGF-binding antibody; VEGF-binding antibody fragment (e.g., single-chain anti-VEGF antibody); endostatin; tumstatin; angiostatin; soluble Fit polypeptide (Lai et al. (2005) Mol. Ther. 12:659); Fc fusion proteins comprising soluble Fit polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); soluble Tie-2 receptor; etc.); tissue inhibitor of metalloproteinase-3 (TIMP-3); light-responsive opsin, such as rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); etc. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; nerve growth factor (neurturin) (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and sonic hedgehog. Suitable polypeptides are disclosed, for example, in WO 2012/145601.

Exemplary polypeptides for gene delivery to the liver include, for example, PBGD (porphobilinogen deaminase) IDUA (iduronidase) Fah (fumaryl acetoacetate hydrolase) A1AT (alpha(1)-antitrypsin), 1A1 (hUGT1A1) (uridine diphosphate glucuronosyltransferase), HCCS1 (hepatocellular carcinoma suppressor protein 1), CD (cytosine deaminase), SOCS3 (suppressor of cytokine signaling 3), TNF (tumor necrosis factor), thymidine kinase, IL-24 (interleukin 24), IL-12 (interleukin-12), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand).

Regulatory sequences

In some embodiments, the nucleotide sequence encoding the target gene product is operably connected to a constitutive promoter. In other embodiments, the nucleotide sequence encoding the target gene product is operably connected to an inducible promoter. In some cases, the nucleotide sequence encoding the target gene product is operably connected to a tissue-specific or cell type-specific regulatory element.

For example, in some cases, the nucleotide sequence encoding the target gene product is operably linked to a hepatocyte-specific, neuron-specific, or photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers selective expression of an operably linked gene in neurons or photoreceptor cells. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter; the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); the beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); the retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); the interphotoreceptor retinoid binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225). Suitable neuron-specific promoters include the neuron-specific enolase (NSE) promoter, Andersen et al. Cell. Mol. Neurobiol., 13: 503-15 (1993); the neurofilament light chain gene promoter, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88: 561 1-5 (1991); and the neuron-specific vgf gene promoter, Piccioli et al., Neuron, 15: 373-84 (1995); etc. Suitable hepatocyte-specific promoters include the albumin promoter (Heard et al., Mol Cell Biol 1987; 7: 2425) or the α1-antitrypsin promoter (Hafenrichter et al. Blood 1994; 84, 3394-404).

"Control elements" or "control sequences" are nucleotide sequences that participate in the interaction of molecules that contribute to the regulation of the function of a polynucleotide, including replication, multiplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, speed, or specificity of a process and can be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region, typically located downstream (3' direction), from the promoter.

"Operably linked" or "operably linked" refers to the juxtaposition of genetic elements wherein the elements are in a relationship that permits them to operate in their intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

As used herein, the term "promoter" may refer to a DNA sequence located adjacent to a DNA sequence encoding a recombinant product. A promoter is preferably operably linked to an adjacent DNA sequence. A promoter typically increases the amount of recombinant product expressed from a DNA sequence compared to the amount of recombinant product expressed when no promoter is present. A promoter from one organism may be used to enhance the expression of a recombinant product derived from a DNA sequence of another organism. For example, a vertebrate promoter may be used for the expression of jellyfish GFP in vertebrates. In addition, a promoter element may increase the amount of recombinant product expressed from a plurality of DNA sequences in series. Therefore, a promoter element may increase the expression of one or more recombinant products. Various promoter elements are well known to those of ordinary skill in the art.

As used herein, the term "enhancer" can refer to a DNA sequence located adjacent to the DNA sequence encoding the recombinant product. An enhancer element is typically located upstream of a promoter element or can be located downstream of or among the coding DNA sequence (for example, transcribed or translated into the DNA sequence encoding the recombinant product). Therefore, an enhancer element can be located upstream or downstream 100 base pairs, 200 base pairs, or 300 or more base pairs of the DNA sequence encoding the recombinant product. An enhancer element can increase the amount of the recombinant product expressed from the above-mentioned DNA sequence that has increased expression by the promoter element. A variety of enhancer elements are easily available to those of ordinary skill in the art.

Pharmaceutical composition

The present disclosure provides pharmaceutical compositions comprising: a) an rAAV vector as described above; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for human use.

Such excipients, carriers, diluents, and buffers include any agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol.

Pharmaceutically acceptable salts may be included therein, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances may be present in such vehicles, such as wetting or emulsifying agents, pH buffering substances, etc. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are fully described in various publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7(th) ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

In a specific embodiment, the present invention provides a pharmaceutical composition comprising a rAAV vector as described above in a pharmaceutically acceptable carrier or other medicament, drug, carrier, adjuvant, diluent, etc. For injection, the carrier will generally be a liquid. For other methods of administration, the carrier can be a solid or a liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. For administration by inhalation, the carrier will be inhalable and will preferably be in the form of solid or liquid particles. As an injection medium, water containing additives commonly used for injection solutions, such as stabilizers, salts or saline and/or buffers is preferably used.

"Pharmaceutically acceptable" means not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing any undesirable biological effect. Thus, such pharmaceutical compositions can be used, for example, for ex vivo transfection of cells or for direct administration of viral particles or cells to a subject.

Methods and therapeutic methods for delivering gene products to tissues or cells (e.g., liver, neuronal, or retinal tissues or cells)

The methods of the present invention provide means for delivering heterologous nucleic acid sequences to host tissues or cells (including dividing and non-dividing cells). The vectors and other reagents, methods and pharmaceutical preparations of the present invention can additionally be used to administer proteins or peptides to subjects in need thereof, as a method for treating or otherwise. In this way, proteins or peptides can therefore be produced in vivo in a subject. A subject may need the protein or peptide because the subject has a defect in the protein or peptide, or because the production of the protein or peptide in the subject can impart a certain therapeutic effect, thereby as a method for treating or otherwise, and as further explained below.

As used herein, the terms "treat", "treating" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects attributable to the disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease or is at risk of developing the disease but has not yet been diagnosed with it; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

In general, the present invention can be used to deliver any foreign nucleic acid with a biological effect to treat or ameliorate symptoms associated with any disorder related to gene expression in any organ, tissue, or cell, particularly those associated with, for example, the liver, brain, or eye. Exemplary disease states include, but are not limited to, lysosomal storage diseases, acute intermittent porphyria, ornithine carbamyltransferase deficiency, alpha(1)-antitrypsin deficiency, acute liver failure, Pompe disease, tyrosinemia, Crigler-Najjar syndrome, hepatitis, cirrhosis, hepatocellular carcinoma, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancers (e.g., brain cancer), retinal degenerative diseases, and other eye diseases.

Gene transfer has substantial potential for understanding and providing treatments for disease states. There are many genetic diseases in which the defective genes are known and have been cloned. In some cases, the functions of these cloned genes are known. In general, the above-mentioned disease states are divided into two categories: defective states, usually enzymatic, which are usually inherited in a recessive manner, and imbalanced states, at least sometimes involving regulatory or structural proteins, which are inherited in a dominant manner. For defective disease states, gene transfer can be used to introduce normal genes into affected tissues for replacement therapy, as well as to create animal models of the disease using antisense mutations. For imbalanced disease states, gene transfer can be used to create the disease state in a model system, which can then be used to attempt to eliminate the disease state. The methods of the present invention thus allow for the treatment of genetic diseases. As used herein, a disease state is treated by partially or completely remedying the defect or imbalance that causes the disease or makes it more severe. It is also possible to use site-specific integration of nucleic acid sequences to induce mutations or correct defects.

In one aspect, the present invention provides a method for delivering a gene product to a tissue or cell (e.g., liver, neuron or retinal tissue or cell) of a subject, the method comprising administering to the subject an rAAV vector as described above. The gene product can be a polypeptide or interfering RNA (e.g., shRNA, siRNA, etc.), or an aptamer, such as described above. The cell can be, for example, a blood cell, a stem cell, a bone marrow (e.g., hematopoietic) cell, a liver cell, a cancer cell, a vascular cell, a pancreatic cell, a nerve cell, a glial cell, an eye or retinal cell, an epithelial or endothelial cell, a dendritic cell, a fibroblast, a lung cell, a muscle cell, a cardiomyocyte, an intestinal cell or a kidney cell. Similarly, tissue can be, for example, selected from blood, bone marrow, muscle tissue (e.g., skeletal muscle, myocardium or smooth muscle including vascular smooth muscle), central or peripheral nervous system tissue (e.g., brain, neuronal tissue or retinal tissue), pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue or heart tissue.

Delivery of gene product to retinal cells can provide the treatment of retinal diseases.Retinal cells can be photoreceptors, retinal ganglion cells, Mueller cells, bipolar cells, amacrine cells, horizontal cells or retinal pigment epithelial cells.In some cases, retinal cells are photoreceptors, for example, rods or cone cells.Similarly, delivery of gene product to neuronal tissue or cells can provide the treatment of neurological disorders.Gene product can be delivered to the various cell types present in neuronal tissue, for example, neurons or glial cells (for example, astrocytes, oligodendrocytes etc.).Delivering gene product to the liver can provide the treatment of liver disease.Gene product can be delivered to, for example, hepatocytes.

The present disclosure provides a method for treating a disease (e.g., a liver, neurological, or ocular disease) comprising administering to a subject in need thereof an effective amount of a rAAV vector as described above. The subject rAAV vector can be administered intracranially, intracerebrally, intraocularly, intravitreally, subretinally, intravenously, or by any other convenient mode or route of administration.

Other exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection. Injections can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. Alternatively, the virus can be administered in a local rather than systemic manner, for example, in a reservoir or sustained-release formulation.

The recombinant viral vector is preferably administered to the subject in an amount sufficient to cause infection (or transduction) of the heterologous nucleic acid sequence in the subject's cell (e.g., liver, neural or retinal cells) and expression. Preferably, the target cell is a hepatocyte, a neural cell (comprising cells of the central and peripheral nervous systems, particularly brain cells) or a retinal cell. In some cases, the retinal cell is a photoreceptor cell (e.g., rod and/or cone cell). In other cases, the retinal cell is an RGC cell. In other cases, the retinal cell is an RPE cell. In other cases, the retinal cell can include amacrine cells, bipolar cells, and horizontal cells.

Preferably, the vector is administered in a therapeutically effective amount. As used herein, a "therapeutically effective" amount is an amount sufficient to alleviate (e.g., alleviate, reduce, reduce) at least one symptom associated with a disease state. Alternatively, a "therapeutically effective" amount is an amount sufficient to provide a certain improvement in the condition of the subject. A "therapeutically effective amount" will fall within a relatively wide range that can be determined by experiments and/or clinical trials. For example, for in vivo injection, a therapeutically effective dose will be about 10 ⁶ to about 10 ¹⁵ rAAV virions, such as about 10 ⁸ to 10 ¹² rAAV virions on the order of magnitude. For in vitro transduction, the rAAV virions to be delivered to an effective amount of cells will be about 10 ⁸ to about 10 ¹³ rAAV virions on the order of magnitude. Other effective doses can be easily established by those of ordinary skill in the art by establishing a dose-response curve through routine experiments.

In some embodiments, more than one administration (e.g., two, three, four, or more administrations) may be used to achieve desired levels of gene expression over varying time intervals (e.g., daily, weekly, monthly, yearly, etc.).

Neurological diseases that can be treated include any disease related to the brain or CNS, including psychiatric disorders. Diseases of the brain fall into two general categories: (a) pathological processes such as infection, trauma, and tumors; and (b) diseases specific to the nervous system, which include myelin diseases and neuronal degenerative diseases. Diseases in any of these categories can be treated. For example, the neurological disease can be selected from neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy and cerebellar degeneration; schizophrenia; epilepsy; ischemia-related diseases and stroke; demyelinating diseases such as multiple sclerosis, perivenous encephalitis, leukodystrophy such as metachromatic leukodystrophy caused by deficiency of arylsulfatase A, Krabbe disease caused by deficiency of galactocerebroside β-galactosidase, adrenoleukodystrophy and adrenomedullary neuropathy; post-viral diseases such as progressive multifocal leukoencephalopathy, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis; mitochondrial encephalomyopathy; neurocancer, such as primary brain tumors, including gliomas, meningiomas, schwannomas, pituitary adenomas, medulloblastomas, craniopharyngiomas, hemangiomas, epidermoid tumors, sarcomas and intracranial metastases from other tumor sources; neuroinfectious or neuroinflammatory diseases.

Ocular diseases that can be treated using the methods of the present invention include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or location; ocular tumors; retinal diseases, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinal disease; sympathetic ophthalmia; Vogt-Koyanagi (V KH) syndrome; uveal foveal ectasia; posterior eye disorders caused by or affected by ocular laser therapy; posterior eye disorders caused by or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinopathy; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (yellow spots); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and disorders of color vision, including achromatopsia, protanopia, deuteranopia, and tritanopia.

Liver diseases that can be treated include, for example, lysosomal storage diseases, such as acute intermittent porphyria, ornithine transcarbamylase deficiency, Wilson's disease, mucopolysaccharidoses (such as MPS type I or MPS type VI), Sly syndrome, Pompe disease, tyrosinemia, alpha(1) antitrypsin deficiency, Crigler-Najjar syndrome; hepatitis A, B, or C; cirrhosis; liver cancer, such as hepatocellular carcinoma; or acute liver failure.

The present invention finds use in both veterinary and medical applications. Suitable subjects include birds and mammals, with mammals being preferred. As used herein, the term "birds" includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. As used herein, the term "mammal" includes, but is not limited to, humans, cattle, sheep, goats, equines, felines, canines, lagomorphs, and the like. Human subjects are most preferred. Human subjects include fetuses, newborn mice, infants, adolescents, and adult subjects.

Transduction of tissues (e.g., liver, neurons, or retinal tissue)

In some embodiments, the rAAV vectors disclosed herein exhibit increased transduction of tissues (e.g., liver, neurons, and/or retinal tissues), e.g., compared to a corresponding AAV vector (from the same serotype) comprising a wild-type AAV capsid protein. For example, the rAAV vector can exhibit at least 10%, 50%, 100%, 500%, or 1000% increased infectivity compared to the infectivity of an AAV virion comprising a corresponding wild-type AAV capsid protein.

In other embodiments, the rAAV vectors disclosed herein can selectively or specifically infect tissues (e.g., liver, neuronal or retinal tissue), for example, showing increased transduction of liver, neuronal or retinal cells compared to other types of cell types. For example, the rAAV vectors can exhibit at least 10%, 50%, 100%, 500% or 1000% increased infectivity of a particular cell type (e.g., liver, neuronal or retinal cells) compared to another cell type (e.g., non-liver, non-neuronal and/or non-retinal cells). For example, rAAV vectors can selectively infect hepatocytes, neurons and/or photoreceptor cells compared to cells in the liver, brain and/or outside the eye.

When the recombinant AAV vector exhibits increased transduction of neurons or retinal tissue, such as when the vector is used to treat a neurological or ocular disorder, the vector preferably comprises a variant AAV2 capsid protein.

When the recombinant AAV vector exhibits increased transduction of liver tissue, for example when the vector is used to treat liver disease, the vector preferably comprises variant AAV3B, AAV-LK03 or AAV 8 capsid protein.

Nucleic acids and host cells

The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein as described above. The isolated nucleic acid can be contained in an AAV vector, such as a recombinant AAV vector.

Recombinant AAV vectors comprising such variant AAV capsid protein coding sequences can be used to produce recombinant AAV virions (i.e., recombinant AAV vector particles). Thus, the present disclosure provides recombinant AAV vectors that, when introduced into suitable cells, can provide for the production of recombinant AAV virions.

The present invention also provides host cells, such as isolated (genetically modified) host cells, which contain target nucleic acid. The subject host cell can be an isolated cell, such as a cell cultured in vitro. The subject host cell can be used to produce subject rAAV virions, as described below. When the subject host cell is used to produce subject rAAV virions, it is referred to as a "packaging cell". In some embodiments, the subject host cell is stably genetically modified with the subject nucleic acid. In other embodiments, the subject host cell is transiently genetically modified with the subject nucleic acid.

The subject nucleic acid is stably or transiently introduced into the host cell using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, the subject nucleic acid will typically also include a selectable marker, e.g., any of several well-known selectable markers, such as neomycin resistance, etc.

The subject host cells are produced by introducing the subject nucleic acid into any of various cells, such as mammalian cells, including, for example, mouse cells, primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, wherein suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, etc. Non-limiting examples of suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, etc. The subject host cells can also be prepared using baculovirus to infect insect cells, such as Sf9 cells, which produce AAV (see, eg, US Patent No. 7,271,002; US Patent Application 12/297,958).

In some embodiments, in addition to the nucleic acid comprising the nucleotide sequence encoding the variant AAV capsid protein as described above, the genetically modified subject host cell also includes a nucleic acid comprising the nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the subject host cell also includes a rAAV vector. The subject host cell can be used to produce rAAV virions. Methods for producing rAAV virions are described in, for example, U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.

As used herein, "packaging" refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles. The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes." Assembly-associated protein (AAP) is the product of the open reading frame within the cap gene and may be required for packaging.

A "helper virus" for AAV refers to an AAV that allows AAV (e.g., wild-type AAV) to replicate and be packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses, and pox viruses such as vaccinia. Adenoviruses include many different subgroups, although type 5 of adenovirus subgroup C is the most commonly used. A large number of adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); they are also available from depositories such as the ATCC.

"Helper virus function" refers to a function encoded in the helper virus genome that allows AAV replication and packaging (in combination with the other requirements for replication and packaging described herein). As described herein, "helper virus function" can be provided in a variety of ways, including by providing a helper virus or providing, for example, a polynucleotide sequence encoding the desired function to the producer cell. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins is transfected into the producer cell together with the rAAV vector.

"Infectious" viruses or viral particles are viruses or viral particles that contain a properly assembled viral capsid and are capable of delivering a polynucleotide component to cells for which the viral species is tropic. The term does not necessarily imply any replication ability of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are well known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11: S337 (describing the TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6: 973.

"Replication-competent" viruses (e.g., replication-competent AAV) refer to phenotypically wild-type viruses that are infectious and also capable of replicating in infected cells (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (particularly in human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility that replication-competent AAV will be generated by recombination between the AAV packaging genes and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein are rAAV vector preparations that contain little, if any, replication-competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV/10 ² rAAV particles, less than about 1 rcAAV/10 ⁴ rAAV particles, less than about 1 rcAAV/10 ⁸ rAAV particles, less than about 1 rcAAV/10 ¹² rAAV particles, or no rcAAV).

An "isolated" nucleic acid, vector, virus, viral particle, host cell or other substance refers to a preparation of the substance that is free of at least some other components that may also be present when the substance or a similar substance is naturally present or initially prepared therefrom. Thus, for example, an isolated substance can be prepared by using purification techniques to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured relative to a second, potentially interfering substance present in the source mixture. Incremental enrichment of embodiments of the present disclosure is incrementally more isolated. An isolated nucleic acid, vector, virus, host cell or other substance is, in some embodiments, purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure or purer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with the cited publications.

It should be understood that certain features of the present invention that are described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for the sake of clarity may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and disclosed herein as if each combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically encompassed by the present invention and disclosed herein as if each combination were individually and explicitly disclosed.

The following examples are provided to illustrate certain embodiments of the present invention. They are not intended to limit the invention in any way.

Example

Example 1

In this example, the inventors designed and constructed a novel AAV2 vector, designated ttAAV2 (as in authentic). Furthermore, this novel vector was tested in a number of animal models (rat, mouse, and neonatal mice) to assess whether ttAAV2 behaves differently compared to tissue culture-adapted (wild-type) AAV2. The inventors demonstrated that ttAAV2 has advantages over AAV2 for gene delivery and is particularly useful for in vivo transduction of brain or eye tissue using heterologous sequences.

method

1. Cloning: The capsid gene of wtAAV2 was obtained from our producer plasmid pDG (Figure 10). This plasmid contains the wtAAV2 rep and cap genes. Subfragments of the capsid gene (pDG nucleotides A: 3257-3759, B: 4025-4555, C: 4797-5287, and D: 5149-5425, respectively) were subcloned into pBS for subsequent mutagenesis. Four mutations were introduced into segment A by site-directed mutagenesis, resulting in a construct encoding amino acid (AA) changes V125I, V151A, A162S, and T205S. Segment B was mutated to encode a single AA change, N312S. Segment C was mutated to encode AA changes Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593S. After confirmation of successful mutagenesis, fragments A-C were recloned into pDG, generating a producer plasmid (pDG-ttAAV2) that would support the production of recombinant viruses encapsidated by ttAAV2 capsids.

2. Production, purification and titration of ttAAV2-GFP viral vector.

Vector production was set up according to a standard protocol using co-transfection of rAAV plasmids with pDG (which provides Ad helper functions and AAV rep and cap genes).Various rAAV plasmids were used to generate recombinant plasmids.

pTR-UF11 (CAG-GFP) was used as the rAAV plasmid. 8x10 ⁸ 293 cells were inoculated per cell factory (CF10). After 14-18 hours, the cells were transfected with pDG or pDG-rrAAV2 and prAAV (e.g., PTR-UF11) using a CaPO ₄ co-precipitation method. The cells were harvested after 72 hours and resuspended in lysis buffer (20mMTris-HCl, pH 8, 150mM NaCl, 0.5% deoxycholate). The cell pellet was lysed by four freeze-thaw cycles to release the virus, each cycle consisting of -80°C for 30 minutes followed by 37°C for 30 minutes. After the final thawing, the lysate was treated with benzonase at a concentration of 50U/ml and incubated at 37°C for 30 minutes. The recombinant virus was purified using a gravity flow column.

Purification. As a first step, the crude lysate was clarified by centrifugation at 4000 g for 15 minutes and applied to a preformed iodixanol step gradient. The viral fraction was then collected and rebuffered into lactated Ringer's solution and concentrated using an Amicon centrifugal filter.

The purity of the viral preparation was then assessed by SDS-polyacrylamide electrophoresis and titrated using real-time PCR. The crude extract contained 4.5 x 10 ¹² particles; the collected viral fraction contained 1.5 x 10 ¹² particles. At this point, the purification process recovered approximately 33% of the virus present in the crude extract.

3. rAAV vector production and purification (alternative method)

In an alternative embodiment of point 2 above, the rAAV2 vector is produced as follows. To produce rAAV2 virions, 5x10 ⁸ 293T cells are inoculated per cell factory (CF10). After 14-18 hours, the cells are double-transfected with the GFP-containing vector PD10-pST2-CMV-GFP and pDG or pDG capsid mutants (pDG-ttAAV2) to produce AAV2-CMV-GFP wild-type or true-type vectors, respectively. Double transfection is achieved using PEI-max from Polysciences at a ratio of 3.5 ml PEI/mg DNA. After incubation at 37°C for 72 hours, the cells are harvested by centrifuging the culture medium and cells at 2200 rpm for 10 minutes at 4°C. The supernatant is removed and retained for further processing, and the cell pellet is resuspended in lysis buffer (0.15M NaCl, 50mM Tris-HCl [pH 8.8]).

The cell pellet was then lysed by four freeze-thaw cycles to release the virus, each cycle consisting of -80°C for 30 minutes followed by 37°C for 30 minutes. After the final thawing, the lysate was treated with 150 U/ml benzonase and incubated at 37°C for 30 minutes. The lysate was then centrifuged at 2000 rpm for 20 minutes to clarify the lysate. The supernatant was filtered using a 0.22 μm cellulose acetate filter, and the recombinant AAV2 virus preparation was purified by FPLC using a chromatography system (GE Healthcare) and an AVB agarose affinity column (bfr.A: PBS, pH 8; bfr B: 0.5 M glycine, pH 2.7). The collected fractions were dialyzed against PBS overnight, and the virus preparation was then titrated by SDS polyacrylamide electrophoresis and real-time PCR.

result

ttAAV2 delivery and spread in vivo

The ttAAV2 vector was tested in vivo to assess the biological activity of the modified virus in this setting. Samples of AAV2-CMV-GFP WT and TT virus were prepared for injection into a number of in vivo models. For this purpose, we concentrated the virus, as only a limited volume of vector can be injected in vivo. We then performed qPCR and SDS-PAGE to assess the fresh titer of the concentrated vector (Figure 11).

After qPCR and protein gel analysis, we obtained the following novel titers: 1.33 x 10 ¹² viral genomes/ml for AAV2-CMV-GFP TT and 1.25 x 10 ¹² viral genomes/ml for AAV2-CMV-GFP WT. Capsid titers were as follows: 8.89 x 10 ¹² capsids/ml for AAV2-CMV-GFP TT and 7.83 x 10 ¹² capsids/ml for AAV2-CMV-GFP WT. Titers differed between genome copies and capsid copies because SDS-PAGE also revealed empty capsids, which are often generated during recombinant AAV vector production. Therefore, capsid titers were higher than viral genome titers obtained from qPCR.

Transduction in rat brain tissue.

rAAV2TT and WT viruses were injected into the substantia nigra or striatum of wild-type rats, with three rats injected per condition at a dose of 2 x ¹⁰⁹ vg or 3.5 x ¹⁰⁹ vg per injection. After 28 days, brains were dissected and tissue sections were prepared for immunofluorescence analysis. The main data are shown in Figures 12 and 26.

Both rAAV2TT and WT viruses were able to transduce neurons and glial cells at each injection site, although with different efficiencies. By comparison, we observed that the TT vector was more efficient in transducing brain tissue and diffused more from the injection site than the WT vector. In addition, we observed the presence of transduced neurons in the parafascicular nucleus (a region of the hypothalamus) after striatal injection of rAAV2TT. This indicates that the TT vector is able to be transferred from the transduced cell bodies at the injection site to the hypothalamus along the neuronal processes by active transport, indicating a strong ability for retrograde transport. This retrograde transport ability has been lost in tissue culture-adapted WT rAAV2 vectors, as untransduced cells can be observed in the same region (see Figure 26).

Taken together, these results in rat brain demonstrate significantly increased spread and transduction efficiency of ttAAV2 compared to titer-matched wtAAV2. Furthermore, AAV2TT exhibits evidence of very good retrograde transport capacity, which is lost in AAV2WT virus.

Transduction in a mouse eye model.

ttAAV2 and wt-AAV2 from the same batch used for our rat brain studies were injected into adult mouse eyes at a dose of ^2x109 vg per eye. To avoid inter-animal variability, each mouse received an injection of rAAV2TT in one eye and rAAV2WT in the contralateral eye. Three different routes of intraocular injection were analyzed: intracameral, intravitreal, and subretinal. Animals were harvested and GFP expression was assessed by immunofluorescence 6 weeks later. The results are shown in Figure 13. Taken together, these data indicate a significant enhancement of ttAAV2 transduction of photoreceptor cells (in terms of both the level and number of transduced photoreceptor cells) after subretinal injection, if compared to wtAAV2 (which was used in the successful RPE65 clinical trial).

Transduction in neonatal mouse models.

In summary, ttAAV2 and wtAAV2 GFP vectors were injected into newborn mice. Two routes of injection were tested, intravenous and intracranial. After 4 weeks, the animals were sacrificed and all tissues were harvested from all mice. We analyzed the brain, which was visualized by direct fluorescence of the organ on a fluorescent microscope after harvest. The results are shown in Figure 14. The results of intracranial and systemic injections are discussed in more detail below.

Intracranial injection

5x10 ¹⁰ vg of either vector were injected into the lateral ventricle of P1 newborn mice. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained with anti-GFP antibody. The results are shown in FIG27 .

As observed in the adult rat brain, these data indicate that AAV2TT shows enhanced transduction and higher spread of mouse brain tissue compared to AAV2WT vectors after intracranial injection. The difference in transduction efficiency between the two vectors was further highlighted when the stained sections were observed at higher magnifications: the TT vector performed better in terms of both expression levels and the number of transduced cells. AAV2TT and WT appear to have the same cell type affinity, each showing transduction of neurons and glial cells, indicating that the observed differences are in efficiency rather than differences in cell type specificity (Figure 28). Taken together, these data indicate that ttAAV2 shows more enhanced transduction of mouse brain tissue after intracranial injection compared to wtAAV2-based vectors. In addition, some evidence suggests transduction of the ependymal cell layer lining the ventricle when using ttAAV2 vectors. This phenomenon is not visible for wtAAV2 vectors.

Systemic injection

2 x ¹⁰ vg of either vector were injected intrajugularly into P1 newborn mice. Four weeks after injection, the animals were sacrificed and various organs were harvested and assessed for GFP transduction (brain, liver, heart, muscle, lung, spleen, and kidney) by immunohistochemistry using anti-GFP antibodies. The results of brain staining are shown in Figure 29 and high-magnification photographs are shown in Figure 30.

We observed good transgene expression in the CNS following systemic injection of AAV2TT. AAV2WT vectors performed poorly in comparison, with only a few transduced neurons observed.

To assess the overall biodistribution of AAV2TT vectors, we evaluated the transduction levels achieved in different tissues following systemic injection. Harvested organs were fixed, paraffin-embedded, sectioned, and stained for GFP expression (Figure 31).

These data suggest that AAV-TT vectors do not appear to have strong affinity for other organs, but rather display specificity primarily for neuronal tissue. This observation could confirm the benefits of intravenous injection of AAV for the treatment of neuronal genetic diseases, as it ensures that the vectors do not transduce non-target peripheral organs but primarily transduce the brain via this injection route.

In summary, our in vivo data demonstrate that ttAAV2 has remarkable transduction properties in both ocular and brain tissues, showing almost exclusive specificity for neuronal tissue.

Example 2

Other considerations

Without wishing to be bound by theory, it is believed that the mutations present in ttAAV2 compared to wtAAV2 include the following functional groups:

1) Heparin-binding residues (S585 and T588) located on the AAV2 capsid triplet; these residues are believed to be responsible for heparin binding in the wtAAV2 capsid. These residues are replaced in ttAAV2 and we hypothesize that this replacement supports viral spread in brain tissue rich in heparan sulfate proteoglycans.

2) A single amino acid change (S312) in ttAAV2 located on the inside of the capsid; this internal serine residue may play a role in capsid-DNA interactions, potentially promoting viral stability, genome packaging, or genome release during infection.

3) Two spatially close amino acids (D546 and G548) located in the groove between the threefold proximal peaks of the AAV capsid structure; these residues may be involved in the interaction with neutralizing antibodies, thereby contributing to the transduction properties in vivo.

4) a single isolated amino acid change (S593) located in the groove between the threefold proximal peaks;

5) four amino acids (M457, A492, D499, and Y533) that are thought to be involved in receptor binding and are closely located on the threefold peak;

6) The remaining four amino acid changes (I125, A151, S162, and S205) are located in the VP1/VP2 primary sequence; these residues are within a capsid region that is not part of VP3. A specific region of VP1/VP2 within the viral capsid is known to contain PLA2 activity that may be involved in the trafficking of incoming viruses. Alterations in this region have been shown to affect viral infectivity.

The three-dimensional positions of these mutations in the AAV2 capsid protein are known and are shown in Figures 15 to 20. Corresponding positions can be identified in AAV capsid proteins from other serotypes (see below). To further characterize ttAAV2 and the role of individual amino acid changes responsible for the improved ttAAV2 phenotype, ttAAV2 capsids can be mutated to reverse selected amino acids (or groups of amino acids, e.g., based on Groups 1 to 6 discussed above) to their corresponding sequences in wild-type AAV2 capsids. Each mutant vector can then be analyzed using the methods described in the above examples.

For example, various mutant vectors expressing GFP can be submitted to a first screening in an animal model by intracranial (IC) injection into CD1 newborn mice. The GFP signal obtained in the injected brain from each mutant vector is then observed by fluorescence microscopy and compared with the signal obtained from the original wtAAV2 and ttAAV2 vectors expressing GFP. After identifying a new phenotype (i.e., the disappearance of ttAAV2-specific strong GFP expression upon mutation of a specific amino acid group), the injected brain is then further sectioned and analyzed by immunohistochemistry.

Concurrently, these selected mutant vectors were submitted to a secondary screen via intracranial injection in adult rats. Furthermore, relevant mutant combinations were analyzed by intravenous (IV) injection in neonatal mice to assess vector biodistribution. Selected organs (heart, lung, liver, spleen, kidney, muscle) were subsequently processed for immunohistochemistry and assessment of GFP expression.

Analysis of the contribution of each TT-specific residue to the efficiency and biodistribution of the vector

To further characterize AAV2TT and pinpoint the amino acid changes responsible for the improved TT phenotype, the AAV2TT capsid was stepwise mutagenized to revert selected amino acids to their corresponding sequences in wild-type AAV2 capsid. This strategy allowed us to more specifically discern the contribution of each of the 14 amino acid mutations to the AAV2TT phenotype and define a minimal authentic vector containing only the essential mutations.

As discussed above, we grouped the 14 TT-specific residues according to their location on the AAV capsid and their relative potential contribution to transduction characteristics. Various AAV-TT mutants were screened by intracranial injection in the brains of newborn mice or adult rats to see whether reversion of some TT-specific residues to their wild-type equivalents was associated with loss of phenotype, thereby identifying important amino acid changes among the 14 TT residues.

Heparin binding site (HBS)

Residues 585 and 588 have been shown to be responsible for heparin binding in the AAV2 WT capsid. These are replaced in AAV-TT and we hypothesize that this replacement supports viral spread in brain tissue rich in heparan sulfate proteoglycans. Both residues are located on the threefold proximal peak of the AAV2 capsid structure (see Figure 16).

We eliminated AAV2WT heparin binding capacity (AAV2-HBnull) by engineering changes R585S and R588T in the WT capsid, mutating the residues to their authentic equivalents. This first analysis was performed to determine that AAV-TT was not simply a more diffuse AAV2 without the heparin binding site, but that some of the other 12 amino acid changes also played a role in improving AAV-TT transduction characteristics.

Intracranial injection in adult rats

Titer-matched AAV2-TT, AAV2-WT, and AAV2-HBnull vectors were injected into the substantia nigra or striatum of wild-type rats at a dose of 3.5×10 ⁹ vg per injection. Brains were dissected 28 days later and tissue sections were prepared for immunofluorescence analysis of GFP expression ( FIG. 32 ).

We observed strong GFP expression in the thalamus and substantia nigra after striatal injection of AAV-TT virus, indicating strong retrograde transport of the vector. In contrast, AAV2-HBnull and AAV2WT showed much less (if any) retrograde transport than AAV-TT, as very few cell bodies were transduced in these regions after striatal injection. This observation suggests that AAV2-HBnull is different from AAV-TT and that the absence of heparin binding capacity on the AAV2 capsid contributes to the good spread of authentic vectors in the brain. However, this is not sufficient to explain its improved transduction characteristics.

Residues S312, D546-G548, and S593

The S312 residue is the only TT-specific alteration located on the inner side of the AAV2 capsid. Our hypothesis is that this internal residue may play a role in capsid-DNA interactions, potentially promoting viral stability, genome packaging, and genome release during infection.

The D and G residues at positions 546 and 548, respectively, are located in the groove between the proximal 3-fold peaks.

A single isolated serine, S593, is located in the groove between the threefold proximal peaks.

The positions of these TT-specific residues on the three-dimensional structure of the AAV2 capsid are shown in Figures 17 to 19. Considering their less prominent positions in the structure, we hypothesized that these residues may not contribute to the ttAAV2 transduction phenotype.

Intracranial injection in neonatal mice

The mutation S312N was engineered into the AAV2-TT capsid to create the TT-S312N mutant. The mutations D546G and G548E were engineered into the AAV2-TT capsid to generate the AAV-TT-DG mutant. The mutation S593A was engineered into the AAV2-TT capsid to create the AAV-TT-S593A mutant.

By reversing these selected TT amino acids to their corresponding sequences in wild-type AAV2 capsid, we aimed to determine the contribution of these residues to the improved AAV-TT phenotype.

5x10 ¹⁰ vg of each mutant vector was injected into the lateral ventricle of P1 newborn mice. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained with anti-GFP antibody. The results are shown in Figure 33.

Interestingly, these data demonstrate that AAV TT-S312N displays enhanced transduction of mouse brain tissue compared to the full AAV2TT vector. On the other hand, amino acid changes S593A or D546E/G548D do not appear to affect the TT phenotype, as similar transduction profiles can be observed throughout the brain. The differences in transduction efficiency were further highlighted when stained sections were viewed at higher magnifications ( FIG. 34 ).

From the high-magnification images, we can observe that AAV TT-S312 appears to transduce neuronal tissue with greater efficiency than the original AAV-TT with 14 amino acid changes. In particular, we can see stronger transgene expression in the rostral part of the brain (cortex, striatum, hippocampus) after TT-S312N vector injection, both in terms of the level and number of transduced cells. This observation was confirmed in all animals analyzed, despite high variability in injected neonatal mouse brains due to difficulties associated with targeting the injection site. On the other hand, reversing S593A or D546E/G548D did not seem to have much effect on the TT vector-transduced phenotype.

Example 3

Targeted amino acid mutations on the authentic AAV2 capsid, selected from the results obtained using the mutant combination in Example 2

Based on the results from the amino acid panel mutations on the complete AAV TT capsid, we determined that the mutation S312N appears to be favorable for the TT phenotype, further improving its transduction efficiency in the brain. Furthermore, we observed that reversing S593A and D546G/G548E did not appear to affect the neuronal phenotype of AAV-TT. Therefore, we hypothesized that the TT-specific residues S593, D546, and G548 could be excluded from the authentic capsid sequence, leaving the AAV2 WT residues at these positions to obtain a final TT vector with only 10 amino acid changes.

To test these hypotheses, we engineered the TT-S312N-D546G-G548D-S593A vector and tested its transduction efficiency by neonatal mouse brain injection. Because the final intracranial injection in neonatal mice appeared to result in saturated signals for GFP expression, we decided to also inject TT and the TT-S312N vector with this "final pre-" TT, using a 10-fold lower dose than previously used. By using this lower dose, we aimed to avoid reaching saturated levels of GFP staining in the transduced brain and circumvent the difficulty of comparing transduction efficiencies between different mutants.

5x10 ⁰⁹ vg of each mutant vector was injected into the lateral ventricle of P1 newborn mice. Four weeks after injection, the animals were sacrificed and the brains were dissected, sectioned, and stained with anti-GFP antibody. The results are shown in Figure 35.

As previously observed, these data indicate that AAV TT-S312N exhibits enhanced transduction of mouse brain tissue compared to the full AAV2 TT vector. On the other hand, the minimal TT-S312N-D546G/G548E-S593A vector did not appear to achieve these higher levels of transduction, despite also containing the internal S312N mutation. This suggests that one or more amino acid changes play some role in the TT phenotype. By reversing these residues back to the AAV2 WT equivalent, we lost some of the increased transduction capacity. These observations were confirmed when the stained sections were viewed at higher magnification (Figure 36).

We decided to further investigate the transduction efficiency achieved by each of these vectors by quantifying the total GFP expression obtained in the injected brains using an enzyme-linked immunosorbent assay (ELISA) of whole brain protein extracts. Briefly, 4 weeks after injection of ^5x1009 vg of vector, the animals were sacrificed, the whole brains harvested and lysed, and total brain protein extracted. By using a GFP protein standard, we were then able to quantify the amount of GFP protein expressed in each injected brain (Figure 37).

We can confirm that the TT-S312N internal mutant transduces mouse brains with greater efficiency than the full TT vector, as it results in greater GFP expression in all injected brains. On the other hand, the minimal TT vector, TT-S312N-D546G/G548E-S593A, appears to result in lower levels of transduction: although the average amount of GFP expressed per brain appears higher in this figure, this is due to the extreme GFP value measured in one brain, as indicated by the high error bar for this condition. Using this minimal TT vector, inter-animal variability was very high, with only one in five animals performing better than those transduced with TT-S312N. This high variability prompts caution regarding this provisional minimal TT vector, particularly since immunohistochemical analysis also showed that the TT-S312N variant performed better than TT-S312N-DG-S593A.

Therefore, we selected the TT-S312N variant as our most preferred AAV TT vector, which contains the following 13 amino acid mutations compared to wild-type AAV2: V125I, V151A, A162S, T205S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and A593S.

Although the above studies indicate TT-S312N as the most preferred AAV-TT vector, these studies show the individual functions and contributions of many TT-specific residues. In particular, the four amino acids located closely on the threefold peak of the capsid are likely to be involved in receptor binding. In some embodiments, these residues are reversed back to the corresponding residues of AAV2WT in AAV-TT. For example, mutations M457Q, A492S, D499E and Y533F can be engineered on the AAV-TT capsid and this mutant vector can be analyzed as described above to show the role of these residues. In other embodiments, four amino acids located on the VP1/VP2 primary sequence are likely to be involved in viral transport and can be reversed back to the corresponding residues of AAV2WT (I125V, A151V, S162A, S205T) in AAV-TT and analyzed similarly.

Example 4

Construction of variant AAV vectors in other serotypes

The functionality of ttAAV2-specific amino acid changes can also be assayed in the context of other non-AAV2 serotypes.

The capsid amino acid sequences of the major adeno-associated viruses (AAVs) (i.e., AAV1, 5, 6, 8, 9, and rh10) were aligned with the amino acid sequence from ttAAV2 (e.g., as shown in FIG9 ). This allowed identification of which ttAAV2-specific amino acids were present at the same positions in the other serotypes. The relevant residues in each serotype were then mutated to the corresponding residues in wtAAV2. These changes demonstrated the importance of the ttAAV2-specific residues for the efficacy and biodistribution of each serotype.

As discussed above, various mutant vectors expressing GFP can be submitted to a first screen by intracranial (IC) injection into CD1 neonatal mice. The GFP signal obtained in the injected brain is then compared with the signal obtained from the appropriate serotype control expressing GFP. The decrease or increase in GFP expression when the identified amino acids are mutated to their corresponding AAV2 residues confirms the importance of these specific residues at these specific positions. Where applicable, the injected brains are then further sectioned and analyzed by immunohistochemistry. In addition, the selected mutant serotypes are analyzed by intravenous (IV) injection into neonatal mice to assess the biodistribution of the vector and compare it with the original, non-mutated counterpart.

In other embodiments, relevant amino acids identified in ttAAV2 are inserted as key mutations for introduction into other major serotypes at relevant positions. The insertion of ttAAV2-specific residues into other AAV subtypes enables us to improve the transduction and biodistribution characteristics of each serotype.

Example 5

Modification of ttAAV2-specific residues to corresponding AAV2 residues that are conserved in other AAV serotypes.

Comparative analysis of the capsid amino acid sequences of existing adeno-associated serotypes with that of ttAAV2 first allowed us to identify ttAAV2-specific residues that are conserved among other serotypes (see Figure 9). These residues consist of S162, S205, S312, G548, S585, and T588. Each non-AAV2 serotype contains one or a combination of these residues at the corresponding amino acid position in its sequence.

In a specific embodiment, each of these residues in various serotypes is converted to the corresponding wild-type AAV2 amino acid and these new mutants are tested for transduction efficiency.

1) Modification of AAV1 serotypes

AAV1 contains residues S205, G549, S586, and T589 that correspond to residues S205, G548, S585, and T588 in ttAAV2. When the VP1 monomer from AAV1 was three-dimensionally aligned with the VP1 from AAV2, we could verify that the corresponding residues in wtAAV2 (i.e., T205, E548, R585, and R588) were in positions that perfectly matched in terms of 3D structure (Figure 21). Therefore, we concluded that it is important to convert each ttAAV2-specific residue in AAV1 to its wild-type AAV2 counterpart without affecting the three-dimensional structure of the protein. In a specific embodiment, the following mutations were made in the AAV1 capsid sequence: S205T, G549E, S586R, and T589R.

2) Modification of AAV5 serotype

AAV5 contains residues G537, S575, and T578, which correspond to residues G548, S585, and T588 in ttAAV2. Residues R585 and R588 in AAV2 are located in positions that perfectly match S575 and T578 in AAV5 based on the 3D structure. Although E548 in AAV2 does not perfectly match residue G537 in AAV5 based on the 3D structure (Figure 22), we decided to include it in our study because the two residues are relatively close together. Therefore, in a specific embodiment, the following mutations were made in the AAV5 capsid sequence: G537E, S575R, and T578R.

3) Modification of AAV6 serotype

AAV6 contains residues S205, G549, S586, and T589 that correspond to residues S205, G548, S585, and T588 in ttAAV2. The corresponding residues in wtAAV2 (i.e., T205, E548, R585, and R588) ( FIG. 23 ) are in positions that are perfectly aligned on the VP1 3D structure. Therefore, in a specific embodiment, the following mutations are made in the AAV6 capsid sequence: S205T, G549E, S586R, and T589R.

4) Modification of AAV8 serotype

AAV8 contains residues S315 and T591 that correspond to residues S312 and T588 in ttAAV2. The corresponding residues in wtAAV2 (i.e., N312 and R588) are in positions that perfectly match the VP1 3D structure ( FIG. 24 ). Therefore, in a specific embodiment, the following mutations are made in the AAV8 capsid sequence: S315N, T591R.

In one embodiment, the improved transduction efficiency conferred by the S312N mutation in TT AAV2 can be transferred to the AAV8 serotype by applying the amino acid change S315N.

We mutated the AAV8 capsid sequence by site-directed mutagenesis and generated the AAV8-S315N vector plasmid. This plasmid was used to generate a recombinant AAV8-S315N vector expressing a CMV-GFP transgene containing an ITR by double transfection of 293T cells. The vector was subsequently purified from cell lysates and harvested culture supernatants by FPLC affinity chromatography using AVB agarose resin. Capsid titers and vector genome titers were assessed by SDS-PAGE and qPCR, respectively.

Mutant AAV8-S315N vectors expressing GFP were screened by systemic injection in CD1 neonatal mice. A titer-matched AAV8 vector was used as a control. GFP expression in various organs, obtained after intrajugular injection of 2 x 10 ¹¹ vg, was subsequently analyzed, focusing on the liver, where AAV8 has previously demonstrated strong transduction efficiency.

5) Modification of AAV9 serotype

AAV9 contains residues S162, S205, G549, and S586 that correspond to residues S162, S205, G548, and S585 in ttAAV2. The corresponding residues in AAV2 (i.e., A162, T205, E548, and R585) are in positions that are perfectly matched on the VP1 3D structure ( FIG. 25 ). Therefore, in a specific embodiment, the following mutations are made in the AAV9 capsid sequence: S162A, S205T, G549E, and S586R.

6) Modification of AAVrh10 serotype

AAVrh10 contains residue G551, which corresponds to residue G548 in ttAAV2. Given the degree of conservation of this residue and position across various serotypes, we hypothesized that G551 in AAVrh10 would align three-dimensionally with E548 in wtAAV2. Therefore, in one embodiment, the following mutation is made in the AAVrh10 capsid sequence: G551E.

7) Modification of AAV3B and AAV-LK03 serotypes

Similar to the AAV8 serotype, after aligning the capsid protein VP1 sequence with that of AAV-TT, we observed that the AAV3B serotype also contains an internal serine at position 312 (see AAV3B capsid sequence in Figure 38).

The newly described LK03 AAV vector (a chimeric capsid comprising five different parental AAV capsids engineered by M.A. Kay through DNA shuffling) also contains residue S312 on the inner side of the capsid (see Lisowski et al., Selection and evaluation of clinically relevant AAV variants in a xenograft liver model, Nature 506, 382–386 (2014)). The capsid sequence of AAV-LK03 is disclosed in WO2013/029030 and is shown in FIG39 .

Therefore, in other embodiments, AAV3B and AAV-LKO3 vectors were mutated by introducing the amino acid change S312N in both vectors. These new mutants and their corresponding AAV control serotypes were also tested by intrajugular injection in newborn mice. GFP expression was then analyzed in the harvested organs.

Example 6

Identification of ttAAV2-specific amino acids transferable between AAV serotypes

In other embodiments, key amino acids identified during the characterization of ttAAV2 are inserted into other major serotypes at relevant positions. The newly engineered vectors are then tested using the appropriate non-mutated serotype as a control. This validates the importance of individual amino acids at specific positions in the AAV capsid, independent of serotype.

1) Residues S585, T588, S312, D546, G548, and S593

AAV1, AAV5, and AAV6 naturally contain identical amino acid residues in their capsid protein sequences at positions corresponding to G548, S585, and T588 in ttAAV2. Thus, in other embodiments, the capsid proteins of these serotypes are mutated at matching positions to include the additional residues S312, D546, and S593 present in ttAAV2. Similarly, AAV8, which already contains the same amino acid residues as ttAAV2 at positions corresponding to S312 and T588 in ttAAV2, is further mutated to contain residues corresponding to S585, D546, G548, and S593 in ttAAV2. AAV9, which already contains residues corresponding to G548 and S585 in ttAAV2, is mutated to contain residues corresponding to T588, S312, D546, and S593 in ttAAV2. Finally, AAV10, which already contained the residue corresponding to G548, was further modified to also contain residues corresponding to S585, T588, S312, G548, and S593.

2) Residues I125, A151, S162, S205, M457, A492, D499, and Y533

AAV1 and AAV6 already naturally contain residue S205. Therefore, in other embodiments, these serotypes are mutated at positions corresponding to residues I125, A151, S162, M457, A492, D499, and Y533 in ttAAV2. Similarly, AAV9, which already contains residues S162 and S205, is further mutated to contain residues corresponding to I125, A151, M457, A492, D499, and Y533 in ttAAV2. Finally, AAV5, 8, and 10 are modified to display residues corresponding to I125, A151, S162, S205, M457, A492, D499, and Y533 in ttAAV2.

The positions of the mutations present in ttAAV2 and the corresponding residues present in the wild-type capsid protein VP1 sequence of other AAV serotypes are shown in Table 1 below. In general, variant non-AAV2 vectors can be constructed by mutating any of the residues shown in Table 1 for these serotypes. Residues shown in italics are residues that already exist in ttAAV2 at the corresponding positions. In preferred embodiments, non-AAV2 serotypes are mutated at one or more residues shown in non-italics. In this way, the favorable properties exhibited by ttAAV2 can be transferred to alternative AAV serotypes.

Table 1

ttAAV2 AAV1 AAV5 AAV6 AAV8 AAV9 AAV10 I125 V125 V124 V125 V125 L125 V125 A151 Q151 K150 Q151 Q151 Q151 Q151 S162 T162 K153 T162 K163 S162 K163 S205 S205 A195 S205 A206 S205 A206 S312 N313 R303 N313 S315 N314 N315 M457 N458 T444 N458 T460 Q458 T460 A492 K493 S479 K493 T495 V493 L495 D499 N500 V486 N500 N502 E500 N502 Y533 F534 T520 F534 F536 F534 F536 D546 S547 P533 S547 N549 G547 G549 G548 G549 G537 G549 A551 G549 G551 S585 S586 S575 S586 Q588 S586 Q588 T588 T589 T578 T589 T591 A589 A591 S593 G594 G583 G594 G596 G594 G596

Other embodiments of the present invention

The present invention also relates to further aspects, as defined in the following concluding paragraphs:

1. A recombinant adeno-associated virus (AAV) vector comprising:

(a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588, and/or 593; and

(b) A heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

2. The recombinant AAV vector of paragraph 1, wherein (i) the vector comprises a variant AAV2 capsid protein; (ii) the variant AAV capsid protein comprises the sequence of SEQ ID NO: 2, or a sequence having at least 95% sequence identity thereto; (iii) the wild-type AAV capsid protein is derived from AAV2; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 1.

3. The recombinant AAV vector according to paragraph 2, wherein the variant AAV2 capsid protein comprises one or more of the following residues: I125, A151, S162, S205, S312, M457, A492, D499, Y533, D546, G548, S585, T588 and/or S593.

4. The recombinant AAV vector according to paragraph 2 or paragraph 3, wherein the variant AAV2 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein: V125I, V151A, A162S, T205S, N312S, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T and/or A593S.

5. The recombinant AAV vector according to any one of paragraphs 1 to 3, wherein the variant AAV capsid protein is from AAV1, AAV5, AAV6, AAV8, AAV9 or AAV10.

6. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAV1 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 3; (iii) the wild-type AAV capsid protein is from AAV1; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 3;

And wherein at least one amino acid substitution is present at one or more of the following positions of the AAV1 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594.

7. The recombinant AAV vector according to paragraph 6, wherein the variant AAV1 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV1 capsid protein:

(a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and/or G594S; and/or

(b) S205T, G549E, S586R and/or T589R.

8. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAV5 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 4; (iii) the wild-type AAV capsid protein is from AAV5; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 4;

and wherein at least one amino acid substitution is present at one or more of the following positions of the AAV5 capsid protein sequence: 124, 150, 153, 195, 303, 444, 479, 486, 520, 533, 537, 575, 578 and/or 583.

9. The recombinant AAV vector according to paragraph 8, wherein the variant AAV5 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV5 capsid protein:

(a) V124I, K150A, K153S, A195S, R303S, T444M, S479A, V486D, T520Y, P533D and/or G583S; and/or

(b) G537E, S575R and/or T578R.

10. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAV6 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 5; (iii) the wild-type AAV capsid protein is from AAV6; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 5;

And wherein at least one amino acid substitution is present at one or more of the following positions of the AAV6 capsid protein sequence: 125, 151, 162, 205, 313, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594.

11. The recombinant AAV vector according to paragraph 10, wherein the variant AAV6 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV6 capsid protein:

(a) V125I, Q151A, T162S, N313S, N458M, K493A, N500D, F534Y, S547D and/or G594S; and/or

(b) S205T, G549E, S586R and/or T589R.

12. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAV8 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 6; (iii) the wild-type AAV capsid protein is from AAV8; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 6;

And wherein at least one amino acid substitution is present at one or more of the following positions of the AAV8 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

13. The recombinant AAV vector according to paragraph 12, wherein the variant AAV8 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV8 capsid protein:

(a) V125I, Q151A, K163S, A206S, T460M, T495A, N502D, F536Y, N549D, A551G, Q588S and/or G596S; and/or

(b) S315N and/or T591R.

14. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAV9 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 7; (iii) the wild-type AAV capsid protein is from AAV9; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 7;

And wherein at least one amino acid substitution is present at one or more of the following positions of the AAV9 capsid protein sequence: 125, 151, 162, 205, 314, 458, 493, 500, 534, 547, 549, 586, 589 and/or 594.

15. The recombinant AAV vector according to paragraph 14, wherein the variant AAV9 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAV9 capsid protein:

(a) L125I, Q151A, N314S, Q458M, V493A, E500D, F534Y, G547D, A589T and/or G594S; and/or

(b) S162A, S205T, G549E and/or S586R.

16. The recombinant AAV vector of paragraph 5, wherein (i) the vector comprises a variant AAVrh10 capsid protein; (ii) the variant AAV capsid protein comprises a sequence having at least 95% sequence identity to SEQ ID NO: 8; (iii) the wild-type AAV capsid protein is from AAVrh10; and/or (iv) the wild-type AAV capsid protein comprises the sequence of SEQ ID NO: 8;

And wherein at least one amino acid substitution is present at one or more of the following positions of the AAV10 capsid protein sequence: 125, 151, 163, 206, 315, 460, 495, 502, 536, 549, 551, 588, 591 and/or 596.

17. The recombinant AAV vector according to paragraph 16, wherein the variant AAVrh10 capsid protein comprises one or more of the following amino acid substitutions relative to the wild-type AAVrh10 capsid protein:

(a) V125I, Q151A, K163S, A206S, N315S, T460M, L495A, N502D, F536Y, G549D, Q588S, A591T and/or G596S; and/or

(b)G551E.

18. The recombinant AAV vector according to any preceding paragraph, wherein the recombinant AAV vector exhibits increased transduction of neurons or retinal tissue compared to an AAV vector comprising a corresponding wild-type AAV capsid protein.

19. The recombinant AAV vector according to any preceding paragraph, wherein the gene product comprises an interfering RNA or an aptamer.

20. The recombinant AAV vector according to any one of paragraphs 1 to 18, wherein the gene product comprises a polypeptide.

21. The recombinant AAV vector of paragraph 20, wherein the gene product comprises a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of neurons or retinal cells.

22. The recombinant AAV vector of paragraph 21, wherein the gene product comprises glial-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripherin.

23. A pharmaceutical composition comprising:

(a) a recombinant AAV vector according to any preceding paragraph; and

(b) a pharmaceutically acceptable excipient.

24. A method for delivering a gene product to neurons or retinal tissue in a subject, the method comprising administering to the subject a recombinant AAV vector or pharmaceutical composition according to any preceding paragraph.

25. A method for treating a neurological or ocular disorder in a subject, the method comprising administering to the subject a recombinant AAV vector or pharmaceutical composition according to any preceding paragraph.

26. The recombinant AAV vector or pharmaceutical composition according to any one of paragraphs 1 to 23, for use in treating a neurological or ocular disorder.

27. The method, recombinant AAV vector or pharmaceutical composition for use according to any one of paragraphs 24 to 26, wherein the neurological disorder is a neurodegenerative disease.

28. The method, recombinant AAV vector or pharmaceutical composition for use according to any one of paragraphs 24 to 26, wherein the ocular disorder is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis or diabetic retinopathy.

29. An isolated variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein; wherein the at least one amino acid substitution is present at one or more of the following positions of the AAV2 capsid protein sequence: 125, 151, 162, 205, 312, 457, 492, 499, 533, 546, 548, 585, 588 and/or 593; or at one or more corresponding positions of an alternative AAV capsid protein sequence.

30. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as defined in paragraph 29.

31. An isolated host cell comprising a nucleic acid as defined in paragraph 30.

Claims (23)

1. A recombinant adeno-associated virus (AAV) vector, comprising: (a) Variant AAV2 capsid protein, The variant AAV2 capsid protein described therein contains at least four amino acid substitutions relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1; The at least four amino acid substitutions are: Q457M, S492A, E499D, and F533Y; and The variant AAV2 capsid protein further comprises residue N312; and (b) Heteronucleotides, which contain nucleotide sequences encoding gene products. 2. The recombinant AAV vector of claim 1, wherein the variant AAV2 capsid protein comprises the sequence of SEQ ID NO: 2. 3. The recombinant AAV vector of claim 1, wherein the variant AAV2 capsid protein, relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1, further comprises one or more amino acid substitutions at the following positions in the AAV2 capsid protein sequence: 125, 151, 162, and 205. 4. The recombinant AAV vector of claim 3, wherein the variant AAV2 capsid protein further comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1: V125I, V151A, A162S and T205S. 5. The recombinant AAV vector of claim 1, wherein the variant AAV2 capsid protein, relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1, further comprises one or more amino acid substitutions at the following positions in the AAV2 capsid protein sequence: 585 and 588. 6. The recombinant AAV vector of claim 5, wherein the variant AAV2 capsid protein further comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1: R585S and R588T. 7. The recombinant AAV vector of claim 1, wherein the variant AAV2 capsid protein, relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1, further comprises one or more amino acid substitutions at the following positions in the AAV2 capsid protein sequence: 546, 548, and 593. 8. The recombinant AAV vector of claim 7, wherein the variant AAV2 capsid protein further comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1: G546D, E548G, and A593S. 9. The recombinant AAV vector of claim 1, wherein the variant AAV2 capsid protein, relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1, further comprises one or more amino acid substitutions at the following positions in the AAV2 capsid protein sequence: 125, 151, 162, 205, 585, 588, 546, 548, and 593. 10. The recombinant AAV vector of claim 9, wherein the variant AAV2 capsid protein further comprises one or more of the following amino acid substitutions relative to the wild-type AAV2 capsid protein of SEQ ID NO: 1: V125I, V151A, A162S, T205S, R585S, R588T, G546D, E548G, and A593S. 11. The recombinant AAV vector according to any one of claims 1 to 10, wherein the recombinant AAV vector exhibits increased transduction of neurons or retinal tissue compared to an AAV vector containing the corresponding wild-type AAV capsid protein. 12. The recombinant AAV vector according to any one of claims 1 to 10, wherein the gene product comprises interfering RNA or an aptamer. 13. The recombinant AAV vector according to any one of claims 1 to 10, wherein the gene product comprises a polypeptide. 14. The recombinant AAV vector of claim 13, wherein the gene product comprises a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of neurons or retinal cells. 15. The recombinant AAV vector of claim 14, wherein the gene product comprises glial neurotrophic factor, fibroblast growth factor, nerve growth factor, brain-derived neurotrophic factor, rhodopsin, retinoschisin, RPE65, or peripheral protein. 16. A pharmaceutical composition comprising: (a) the recombinant AAV vector according to any one of claims 1 to 15; and (b) Pharmaceutically acceptable excipients. 17. Use of the recombinant AAV vector of any one of claims 1 to 15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for delivering a gene product to a subject's tissue. 18. The use according to claim 17, wherein the tissue is selected from blood, bone marrow, muscle tissue, neuronal tissue, retinal tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, intestinal tissue, or heart tissue. 19. The use according to claim 18, wherein the tissue is a neuron, retina, or liver tissue. 20. Use of the recombinant AAV carrier according to any one of claims 1 to 15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating a condition of a subject. 21. The use according to claim 20, wherein the condition is a neurological, ocular, or hepatic condition. 22. The use according to claim 21, wherein the neurological condition is a neurodegenerative disease. 23. The use according to claim 21, wherein the eye condition is glaucoma, retinitis pigmentosa, macular degeneration, retinal schisis, or diabetic retinopathy.