AAV Capsid Variants and Their Use

Abstract

To provide AAV capsid variants and their uses. [Solution] Aspects of this disclosure relate to compositions and methods for delivering a transgene (e.g., a transgene encoding one or more gene products) to target cells. This disclosure is partly based on adeno-associated virus (AAV) capsid protein variants characterized by their tropism to certain cell types (e.g., neurons, muscle cells, osteocytes, cardiac cells, etc.). In some embodiments, recombinant AAV (rAAV) containing a capsid protein variant (e.g., AAVv66, SEQ ID NO: 1) is packaged more efficiently than rAAV having a certain wild-type AAV capsid protein. Methods for delivering rAAV containing an AAV capsid protein variant are also described in this disclosure.

Description

[Background technology]

[0001] background Recombinant AAV adeno-associated viruses (rAAVs) can drive stable and sustained transgene expression in target tissues without significant toxicity or host immunogenicity. Therefore, rAAVs are a promising delivery vehicle for long-term therapeutic gene expression. However, the low transduction efficiency and limited histotropy of currently available rAAV vectors may limit their application as a viable and effective therapeutic method. In addition, the reliable clinical transition of major therapeutic AAV serotypes derived from non-human tissues is a concern. Thus, the need for novel AAV vectors for gene delivery remains. [Overview of the project] [Means for solving the problem]

[0002] overview Aspects of this disclosure relate to compositions and methods for delivering a transgene (e.g., a transgene encoding one or more gene products) to target cells. This disclosure is partly based on adeno-associated virus (AAV) capsid protein variants characterized by their tropism to certain cell types (e.g., neurons, muscle cells, osteocytes, cardiac cells, etc.). In some embodiments, recombinant AAV (rAAV) containing a capsid protein variant is packaged more efficiently than rAAV having a certain wild-type AAV capsid protein. Methods for delivering rAAV containing an AAV capsid protein variant are also described in this disclosure.

[0003] In some embodiments, the Disclosure provides a method for delivering a transgene to target cells, comprising the steps of intracranial administration of an isolated nucleic acid comprising a transgene encoding one or more gene products of interest, and recombinant adeno-associated virus (rAAV) comprising an adeno-associated acid (AAV) capsid protein having the sequence shown in SEQ ID NO: 1.

[0004] In some embodiments, intracranial administration includes intrahippocampal injection.

[0005] In some embodiments, the target cells are central nervous system (CNS) cells. In some embodiments, the CNS cells are neurons, oligodendrocytes, astrocytes, or microglia.

[0006] In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans. In some embodiments, the subjects are characterized by the production of anti-AAV2 antibodies. In some embodiments, administration of rAAV does not result in a neutralizing immune response to rAAV by the subjects.

[0007] In some embodiments, the isolated nucleic acid includes an AAV inverted terminal repeat (ITR) adjacent to the transgene. In some embodiments, the nucleic acid sequence encoding one or more gene products is operably ligated to a promoter. In some embodiments, one or more gene products include a protein or inhibitory nucleic acid.

[0008] In some embodiments, the Disclosure provides a method for delivering a transgene to target cells, comprising the steps of intravenously administering to the target a recombinant adeno-associated virus (rAAV) comprising an isolated nucleic acid containing a transgene encoding one or more gene products of interest; and an adeno-associated acid (AAV) capsid protein having the sequence shown in SEQ ID NO: 1, wherein, as a result of the administration, the rAAV crosses the blood-brain barrier (BBB) ​​of the target.

[0009] In some embodiments, the target cells are central nervous system (CNS) cells. In some embodiments, the CNS cells are neurons, oligodendrocytes, astrocytes, or microglia.

[0010] In some embodiments, the subjects are mammals. In some embodiments, the subjects are humans. In some embodiments, the subjects are characterized by the production of anti-AAV2 antibodies. In some embodiments, administration of rAAV does not result in a neutralizing immune response to rAAV by the subjects.

[0011] In some embodiments, the isolated nucleic acid includes an AAV inverted terminal repeat (ITR) adjacent to the transgene. In some embodiments, the nucleic acid sequence encoding one or more gene products is operably ligated to a promoter. In some embodiments, one or more gene products include a protein or inhibitory nucleic acid.

[0012] In some embodiments, recombinant AAVs (rAAVs) containing the capsid protein variant described herein (e.g., AAVv66, SEQ ID NO: 1) are packaged more efficiently than rAAVs having a particular wild-type AAV capsid protein (e.g., AAV2 capsid protein, SEQ ID NO: 2) (e.g., by 2x, 3x, 4x, 5x, 10x, 20x, 30x, 50x, 100x, or higher). In certain embodiments, for example, the following items are provided: (Item 1) A method for delivering a transgene to target cells, (i) an isolated nucleic acid containing a transgene encoding one or more gene products of interest; and (ii) Adeno-associated acid (AAV) capsid protein having the sequence shown in Sequence ID No. 1 A method comprising the step of intracranially administering recombinant adeno-associated virus (rAAV) containing the above to the subject. (Item 2) The method according to item 1, wherein the intracranial administration includes an intrahippocampal injection. (Item 3) The method according to item 1 or 2, wherein the target cell is a central nervous system (CNS) cell. (Item 4) The method according to any one of items 1 to 3, wherein the CNS cell is a neuron, oligodendrocyte, astrocyte, or microglial cell. (Item 5) The method according to any one of items 1 to 4, wherein the subject is a mammal, and optionally the mammal is a human. (Item 6) The method according to any one of items 1 to 5, wherein the subject is characterized by the production of anti-AAV2 antibodies. (Item 7) The method according to item 6, wherein after administration of the rAAV, the subject does not induce a neutralizing immune response against the rAAV. (Item 8) The method according to any one of items 1 to 7, wherein the isolated nucleic acid contains an AAV inverted terminal repeat sequence (ITR) adjacent to the transgene. (Item 9) The method according to any one of items 1 to 8, wherein the nucleic acid sequence encoding the one or more gene products is operably linked to a promoter. (Item 10) The method according to any one of items 1 to 9, wherein the one or more gene products include a protein or an inhibitory nucleic acid. (Item 11) A method for delivering a transgene to a target cell of a subject, comprising: (i) an isolated nucleic acid containing a transgene encoding one or more gene products of interest; and (ii) a recombinant adeno-associated virus (rAAV) containing an adeno-associated virus (AAV) capsid protein having the sequence shown in SEQ ID NO: 1 administering intravenously to the subject, whereby as a result of the administration, the rAAV crosses the blood-brain barrier (BBB) of the subject. (Item 12) The method according to item 11, wherein the target cell is a central nervous system (CNS) cell. (Item 13) The method according to item 12, wherein the CNS cell is a neuron, oligodendrocyte, astrocyte, or microglial cell. (Item 14) The method according to any one of items 11 to 13, wherein, as a result of the administration, transduction of liver cells is reduced as compared with the administration of rAAV having an AAV2 capsid protein. (Item 15) The method according to any one of items 11 to 14, wherein the subject is a mammal, and optionally the mammal is a human. (Item 16) The method according to any one of items 11 to 15, wherein the subject is characterized by the production of anti-AAV2 antibodies. (Item 17) The method according to item 16, wherein after the administration of the rAAV, the subject does not induce a neutralizing immune response against the rAAV. (Item 18) The method according to any one of items 11 to 17, wherein the isolated nucleic acid contains an AAV inverted terminal repeat sequence (ITR) adjacent to the transgene. (Item 19) The method according to any one of items 11 to 18, wherein the nucleic acid sequence encoding the one or more gene products is operably linked to a promoter. (Item 20) The method according to any one of items 11 to 19, wherein the one or more gene products include a protein or an inhibitory nucleic acid. Brief Description of the Drawings

[0013] [Figure 1-1]Figures 1A–1D show the identification of novel proviral AAV capsid sequences from human surgical specimens. Figure 1A shows that the AAV capsid proviral sequence was initially amplified by PCR from human surgical specimens using primers adjacent to the AAV Cap ORF. The amplicon was subjected to single-molecule real-time (SMRT) sequencing, and the resulting reads were analyzed by BWA-MEM alignment to modern AAV serotype sequences to remove insertions / deletions associated with PCR or SMRT sequencing errors, InDelFixer, and de novo assembly to cluster reads with high sequence similarity. Figure 1B shows that the cap sequence of variant AAVv66 was found to be the most abundant (45%) in the analysis. Figure 1C shows an overview of 13 unique residues in the AAVv66 capsid sequence that differ from AAV2. (d) Phylogenetic tree of AAV2 variants (including AAVv66) and modern serotypes. [Figure 1-2] Same as above. [Figure 2-1] Figures 2A–2D show the extent of transduction of rAAV2 and rAAVv66 after intrahippocampal injection. Figure 2A shows native EGFP expression after unilateral intrahippocampal administration of rAAV2-CB6-Egfp or rAAVv66-CB6-Egfp injection. Scale bar = 700 μm. Figure 2B shows quantification of EGFP-positive surface normalized to DAPI-positive surface. Data are expressed as mean ± SD; n=3. ****P<0.0001. Figure 2C shows a schematic diagram of the coronal brain depicting the target sub-anatomical region in both the contralateral and ipsilateral hemispheres: Ammon's horns (CA1, CA2, CA3, CA4), dentate gyrus (DG), corpus callosum (CC), and cortex (CTX). Figure 2C shows high-magnification images of the sub-anatomical region transductioned with rAAVv66. Scale bar = 50 μm. [Figure 2-2] Same as above. [Figure 3-1]Figures 3A–3P show the transduction of major brain cell types by rAAVv66. Figures 3A, 3E, 3I, and 3M show coronal sections of mouse brains transduced with rAAVv66-CB6-Egfp. IF-stained sections with antibodies against NEUN (Figure 3A, neurons), GFAP (Figure 3E, astrocytes), IBA1 (Figure 3I, microglia), or OLIG2 (Figure 3M, oligodendrocytes) show the distribution of cell types across the brain. Native EGFP expression co-localizing with IF staining indicates positively transduced cell types. Scale bar = 700 μm. Figures 3B, 3F, 3J, and 3N show 3D renderings of the anatomical region of a single representative frame from a dashed rectangular box within the coronal section diagram (top panel) and single cell displays from the field of view defined by dashed square boxes (bottom three panels). The left panel shows the total area of ​​EGFP and cell marker IF staining; the center panel shows EGFP co-localized with the entire cell marker IF staining; and the right panel shows co-localized EGFP and cell marker IF staining. Scale bars = 50 μm (top panel), 5 μm (bottom three panels). Figures 3C, 3G, 3K, and 3O show quantification of cell type-specific IF staining across the indicated hippocampal regions (x-axis), normalized to DAPI signaling. Figures 3D, 3H, 3L, and 3P show quantification of cell type-specific transduction across the indicated regions, normalized to the overall cell type IF and DAPI signaling. Data are expressed as mean ± SD; n=3. Ammon's horns (CA1, CA2, CA3, CA4), dentate gyrus (DG), corpus callosum (CC), and cortex (CTX). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 3-6] Same as above. [Figure 4-1]Figures 4A–4E show the biophysical analysis of AAVv66. Heatmaps of differential scanning fluorescence (DSF) analysis to query capsid protein unfolding (Figure 4A) and DNA accessibility (vector genome release) (Figure 4B) at pH 7, 6, 5, and 4. Each specified amino acid residue of AAVv66 was converted to that of AAV2 by site-directed mutagenesis, and changes in packaging yield (Figure 4C), capsid stability (Figure 4D), and genome release (Figure 4D) at pH 7 were investigated. Values ​​represent mean ± SD. p-values ​​were determined by one-way ANOVA. *p<0.05, *p<0.01, *p<0.001, ****p<0.0001. n33. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] Figures 5A–5E show the cryo-EM primary metrics, map reconstruction, and model generation for AAVv66. Figure 5A shows the density map of AAVv66. The grayscale scheme defines the topological distance (Å) from the center. Figure 5B shows the refined ribbon structure of the AAVv66 capsid monomer. The amino acids that distinguish AAV2 are highlighted. Annotations indicate 2-fold symmetry (elliptic), 3-fold symmetry (triangular), and 5-fold symmetry (pentagonal). Partial electron density (dark gray mesh) and residues of AAVv66 are shown for the region near (Figure 5C) L583, R487, Y533, and K532, (Figure 5D) S446, D499, and S501, and (Figure 5E) N407–T414. [Figure 5-2] Same as above. [Figure 6]Figure 6 shows the structural differences between AAVv66 and AAV2. The center shows the structure of AAVv66 60mer (gray). Amino acid residues unique to AAVv66 are highlighted in green, while amino acid residues of single monomers common to AAV2 are colored. The atomic model shows the residue side chains of the selective region that has substantial differences between AAVv66 and AAV2. Alignments were prepared using monomers of AAV2(1lp3) and AAVv66, and the modeled side chains derived from adjacent residues are shown in gray. The amino acid annotations indicate AAVv66, position number, and then that it belongs to AAV2. [Figure 7-1] Figures 7A–7C show differential capsid surface electrostatics between AAV2 and AAVv66. Figure 7A shows the positive and negative surface charges for the 60mer, trimer (3-fold symmetry), and pentamer (outer and inner 5-fold symmetry) structures of AAV2 and AAVv66. The black arrows in the AAV2 60mer and trimer structures indicate the approximate locations of R585 and R588 in a single 3-fold protrusion. Figure 7B shows magnified views of amino acid residues at 585–588 in AAV2 and AAVv66. Figure 7C shows a bar graph of the zeta potentials of the purified vector measured by a zetasizer. The values ​​represent mean ± SD, n=3. [Figure 7-2] Same as above. [Figure 8] Figure 8 shows the amino acid sequence of the AAVv66 capsid protein, which is a mutation of AAV2. The amino acid differences between AAV2 and AAVv66 are highlighted. Residues in the variable region (VR) are shown with short bars. The aH domain is separated by a dotted bar, and the residues forming the b-sheet are marked with black arrows. The start positions of VP1, VP2, and VP3 are marked with greater-than signs (>). The PLA domain within VP1 is shown with a bar. [Figure 9]Figure 9 shows that AAVv66 yields a higher vector yield than AAV2. PCR assays of crude lysates were performed on cell lysates of HEK239 cells triple-transfected with pAAV and either AAV2 or AAVv66 packaging plasmids, as well as in culture medium. Values ​​represent mean genome copies ± SD, n=3. [Figure 10] Figure 10 shows that AAVv66 lacks strong heparin binding. Heparin competition assay showing the transduction efficiency of AAV2-CB6-FLuc and AAVv66-CB6-FLuc in HEK293 cells in the presence of gradually increasing heparin (x axis). Luminescence values ​​were adjusted to 1 (y axis) for values ​​obtained in heparin-free wells. Values ​​represent mean ± SD, n=3. **, p<0.01 by two-way ANOVA. [Figure 11] Figure 11 shows the in vitro infection efficiencies of AAV2, AAV3b, and AAVv66 in HEK293 cells. The vectors were packaged in CB6-FLuc. To assess the infectivity of the vectors by detecting luciferase activity (RLU, relative luminescence), cells were lysed 48 hours after infection. Data are presented on a logarithmic scale. Values ​​represent mean ± SD, and ***p<0.0001, n=3, by one-way ANOVA. [Figure 12-1]Figures 12A-12D show that intravenous administration of the AAVv66 vector induces hepatic transduction. Systemic injection of AAVv66-CB6-Fluc resulted in hepatic transduction. rAAV2-CB6-Fluc or AAVv66-CB6-Fluc (1.0E11 GC / mouse) was injected into mice via tail vein. After 14 days, mice were intraperitoneally injected with luciferin substrate and imaged (Figure 12A). Quantification of systemic bioluminescence of luciferase activity showed no significant difference in hepatic transduction between AAVv66-CB6-Fluc and AAV2-CB6-Fluc. However, isolation of liver tissue, quantification of luciferase activity, and detection of vector genome copies by qPCR indicated that AAVv66 is a significantly weaker hepatic transduction factor than AAV2. Total abdominal flux was recorded in the acquired images (Figure 12B). Tissue was collected and assayed for luciferase activity (Figure 12C) and vector genome abundance by qPCR (Figure 12D). Values ​​represent mean ± SD, n=3. * indicates p<0.05 by Student's t-test. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 13-1] Figures 13A-13D show that intramuscular administration of the AAVv66 vector induces transduction in muscle. Intramuscular injection of AAVv66 into the tibialis anterior muscle showed little difference in transduction ability compared to transduction with AAV2. Mice were injected intramuscularly into one hind limb (tibialis anterior muscle) with either AAV2-CB6-FLuc or AAVv66-CB6-FLuc (4.0E10 GC / mouse). After 14 days, mice were intraperitoneally administered luciferin substrate and imaged (Figure 13A). The total flux of the injected hind limb in the acquired image was recorded (Figure 13B). Tissue was collected and assayed for luciferase activity (Figure 13C) and vector genome abundance by qPCR (Figure 13D). Values ​​represent mean ± SD, n=3. *, p<0.05 by Student's t-test. [Figure 13-2] Same as above. [Figure 14-1]Figures 14A–14D show the immunological characterization of AAVv66. Mice were intramuscularly administered the AAV2-CB6-Egfp vector (1E11 GC / mouse). Four weeks after administration, serum was collected to test neutralizing antibody (NAb) titers against AAV2 or AAVv66 infection. The NAb50 value for AAV2 (Figure 14A) and AAVv66 (Figure 14B) is defined as the titer dilution that can block 50% of the total transduction achievable by the LacZ reporter gene-packaged vector. The left column is a summary table of NAb values ​​for individual animals tested. The right column plots transduction efficiency against various serum dilutions. Values ​​represent mean ± SD. The dashed line shows the mean NAb50 serum titer. After a 4-week period, AAV2-hA1AT or AAVv66-hA1AT (1E11 GC / mouse) was administered intramuscularly to the contralateral hind limb of mice. Serum A1AT levels were measured by ELISA at weeks 5, 6, 7, and 8 (Figure 14C). Values ​​represent mean ± SD, n=3. ns are not significant; two-way ANOVA on cross-sectional data points*, p<0.05;**, p<0.01; and ***, p<0.001. Figure 14D shows the cross-reactivity of rabbit anti-AAV serum. Rabbit antisera elevated against AAV serotypes were tested against allogeneic AAV serotypes for NAb against AAVv66 to assess relative cross-reactivity. Log2 values ​​represent the highest antibody dilution required to achieve 50% inhibition of transduction. [Figure 14-2] Same as above. [Figure 15] Figures 15A-15B show the cryo-EM primary metrics, map reconstruction, and model generation for AAVv66. Figure 15A shows a cryo-electron micrograph of AAVv66. The scale bar represents 100 Å. Figure 15B shows the Fourier shell correlation (FSC_part) of even and odd particles for AAVv66. [Figure 16]Figure 16 shows RMSD(Å) statistics comparing AAVv66 to AAV2 or AAV3b. A summary of total and regional RMSD(Å) between AAVv66 and AAV2(1LP3) or AAV3b(3KIC), measured across all shown alpha-carbon pairs (AAV2 numbering), calculated using the rms_cur function in PyMOL. The complete capsid structures of AAV2, 3b, and AAVv66 were aligned by optimized fit within a cryo-EM density map of AAVv66. A custom script in PyMOL was used to quantitatively convert the distance(Å) values ​​between individual alpha-carbon pairs for either AAV2 (top) or AAV3b (bottom) and represent them as color and radius thickness of the corresponding residues in AAVv66. [Modes for carrying out the invention]

[0014] Detailed explanation Aspects of this disclosure relate to compositions and methods for delivering a transgene (e.g., a transgene encoding one or more gene products) to target cells. This disclosure is partly based on adeno-associated virus (AAV) capsid protein variants characterized by their tropism to certain cell types (e.g., neurons, muscle cells, osteocytes, cardiac cells, etc.). In some embodiments, recombinant AAV (rAAV) containing a capsid protein variant is packaged more efficiently than rAAV having a certain wild-type AAV capsid protein. Methods for delivering rAAV containing an AAV capsid protein variant are also described in this disclosure.

[0015] AAVv66 Capsid Protein In some embodiments, the Disclosure provides a method for delivering a transgene to target cells (e.g., target cells of the central nervous system (CNS)), comprising the steps of: administering to the target (e.g., intracranially or intravenously) an isolated nucleic acid comprising a transgene encoding one or more gene products of interest; and a recombinant adeno-associated virus (rAAV) comprising an adeno-associated acid (AAV) capsid protein comprising an AAVv66 capsid protein or a capsid protein having substantial homology to the AAVv66 capsid protein. In some embodiments, the AAVv66 protein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0016] In some embodiments, the AAVv66 capsid protein described herein includes a mutation to AAV2 selected from the group consisting of: K39Q, V151A, R447K, T450A, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593T. In some embodiments, the AAVv66 capsid protein described herein includes each of the following mutations to AAV2: K39Q, V151A, R447K, T450A, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593T. In some embodiments, a capsid protein having substantial homology to the AAVv66 capsid protein includes one or more mutations in AAV2 selected from the group consisting of: K39Q, V151A, R447K, T450A, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593T. In some embodiments, the AAVv66 capsid protein or a capsid protein having substantial homology to the AAVv66 capsid protein includes one or more mutations in the VP1, VP2, and / or VP3 regions of AAV2. In some embodiments, the AAVv66 capsid protein or a capsid protein substantially homologous to the AAVv66 capsid protein contains one or more mutations in the variable region (VR)-IV, VR-V, VR-VI, VT-VII, and / or VR-VIII relative to AAV2. In some embodiments, the AAVv66 capsid protein or a capsid protein substantially homologous to the AAVv66 capsid protein contains one or more mutations relative to AAV2, as shown in Figure 1C.

[0017] "Homologousity" refers to the percentage of identity between two polynucleotides or two polypeptide segments. The term "substantial homology," when referring to nucleic acids or fragments thereof, indicates that, when optimally aligned with another nucleic acid (or its complementary chain) with appropriate nucleotide insertions or deletions, approximately 90–100% of the aligned sequence is identical. When referring to polypeptides or fragments thereof, the term "substantial homology" indicates that, when optimally aligned with another polypeptide with appropriate gaps, insertions, or deletions, approximately 90–100% of the aligned sequence is identical. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably more than 97% identity. In some cases, highly conserved may mean 100% identity. Identity can be readily determined by those skilled in the art, for example, by the use of algorithms and computer programs known to those skilled in the art.

[0018] As described herein, alignment between nucleic acid or polypeptide sequences is performed using one of various publicly or commercially available multiplex sequence alignment programs, such as "Clustal W," which is accessible via a web server on the Internet. Alternatively, the Vector NTI utility can also be used. There are also several algorithms known in the art that can be used to measure the identity of nucleotide sequences, including those contained in the above-mentioned programs. As another example, polynucleotide sequences may be compared using BLASTN, which provides alignment and sequence identity percentage of the best overlap region between the query sequence and the search sequence. Similar programs are available for comparing amino acid sequences, such as the "Clustal X" program and BLASTP. Typically, one of these programs is used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize other algorithms or computer programs that provide at least the same level of identity or alignment as those provided by the referenced algorithms and programs. Alignment may be used to identify corresponding amino acids between two proteins or peptides. "Corresponding amino acids" are amino acids of the protein or peptide sequence aligned with amino acids of another protein or peptide sequence. The corresponding amino acids may be identical or different. A corresponding amino acid that is different from the original amino acid is sometimes called a variant amino acid.

[0019] In some embodiments, the disclosure relates to an AAVv66 capsid protein (e.g., an isolated nucleic acid encoding an AAVv66 capsid protein, a recombinant adeno-associated virus (rAAV) containing an AAVv66 capsid protein), or a capsid protein substantially homologous to the AAVv66 capsid protein. In some embodiments, the capsid protein substantially homologous to the AAVv66 capsid protein is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence shown in Sequence ID No. 1. In some embodiments, a capsid protein having substantial homology to the AAVv66 capsid protein includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions, insertions, or deletions from the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, a capsid protein having substantial homology to the AAVv66 capsid protein includes more than 50 amino acid substitutions, insertions, or deletions from the amino acid sequence shown in SEQ ID NO: 1.

[0020] This disclosure relates to the surprising finding that, in some embodiments, rAAVs containing the AAVv66 capsid protein can be produced in greater quantities in mammalian cell lines (e.g., HEK-293 cells) than rAAVs having certain other AAV capsid proteins (e.g., AAV2 capsid protein, AAV3B capsid protein, etc.). In some embodiments, transduced mammalian (e.g., HEK)-producing cells produce rAAVs having about 1.5 to about 5 times (e.g., 1.5, 2, 3, 4, 5 times) more AAVv66 capsids than mammalian (e.g., HEK)-producing cells transduced with the AAV2 capsid protein. In some embodiments, transduced mammalian (e.g., HEK)-producing cells produce rAAVs having approximately 5% to 50% more AAVv66 capsids (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc.) than transduced mammalian (e.g., HEK)-producing cells with AAV3B capsid protein.

[0021] Aspects of this disclosure relate to the transduction efficiency of central nervous system (CNS) cells of AAVv66 capsid protein (e.g., rAAVs containing AAVv66 capsid protein) that is unexpectedly improved compared to rAAVs containing AAV2 capsid protein. In some embodiments, rAAVs containing AAVv66 transduce CNS cells at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 100%, 200%, 500%, 1000%, or more efficiently than rAAVs containing AAV2. In some embodiments, CNS cells include neurons, oligodendrocytes, astrocytes, or microglia.

[0022] Aspects of this disclosure relate to a specific AAV capsid protein (e.g., AAVv66 capsid protein) that is serologically distinct from other AAV capsid proteins (e.g., AAV1, AAV2, AAV3B, AAV8, AAV9, AAVrh.8, AAVrh.10, etc.). While we do not wish to be bound by any particular theory, rAAVs containing AAVv66 capsid protein are not subject to neutralizing antibody reactions in subjects that are serologically positive for antibodies against certain other AAV capsids. Therefore, in some embodiments, rAAVs containing AAVv66 capsid protein may be useful as a second-line therapy for transgene delivery to subjects that have previously received AAV therapy or are serologically positive for certain AAV capsid neutralizing antibodies.

[0023] In some embodiments, this disclosure relates to an rAAV capsid protein (e.g., AAVv66 capsid protein) that exhibits increased thermal stability compared to a particular wild-type AAV capsid protein (e.g., AAV2 capsid protein). In some embodiments, the AAVv66 capsid protein is more thermally stable than the AAV2 capsid protein at pH ranges from about pH 4 to about pH 7. In some embodiments, thermal stability is determined by calculating the melting temperature of the capsid protein. In some embodiments, the AAVv66 capsid protein is characterized by a melting temperature that exceeds the melting temperature of the AAV2 capsid protein at a given pH (e.g., between pH 4 and pH 7) by about 5°C to about 10°C.

[0024] Isolated nucleic acids In some embodiments, the disclosure relates to isolated nucleic acids encoding a particular AAV capsid protein variant (e.g., AAVv66 capsid protein). “Nucleic acid” sequence refers to a DNA or RNA sequence. In some embodiments, the term nucleic acid is not limited to the following, but includes: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxyl-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, and 7-methylguanine. It captures sequences containing any of the known base analogs of DNA and RNA, such as 5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylkeosin, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, oxybutoxosin, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetic acid, pseudouracil, keosin, 2-thiocytosine, and 2,6-diaminopurine.

[0025] In some embodiments, the proteins and nucleic acids of this disclosure are isolated. As used herein, the term “isolated” means artificially obtained or produced. As used herein with respect to nucleic acids, the term “isolated” generally means: (i) amplified in vitro, e.g., by polymerase chain reaction (PCR); (ii) produced by recombination by cloning; (iii) purified, e.g., by cleavage and gel separation; or (iv) synthesized, e.g., by chemical synthesis. Isolated nucleic acids are nucleic acids that can be readily manipulated by DNA recombination techniques well known in the art. Thus, nucleotide sequences contained in vectors in which the 5' and 3' restriction sites are known or in which primer sequences for polymerase chain reaction (PCR) are disclosed are considered isolated, but nucleic acid sequences that exist in their native state in their natural host are not isolated. Isolated nucleic acids may, but do not need to be substantially purified. For example, nucleic acids isolated in a cloning vector or expression vector may not be pure in that they constitute only a small percentage of the material in the cell in which they exist. However, since such nucleic acids can be easily manipulated by standard techniques known to those skilled in the art, they are isolated as used herein. As used herein with respect to proteins or peptides, the term “isolated” generally refers to proteins or peptides that are artificially obtained or produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.).

[0026] It should be recognized that conserved amino acid substitutions may be made to provide functionally equivalent variants or homologs of capsid proteins. In some embodiments, this disclosure encompasses sequence modifications resulting in conserved amino acid substitutions. As used herein, a conserved amino acid substitution refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, such methods can be found, for example, in *Molecular Cloning: A Laboratory Manual*, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or in reference literature compiling such methods, such as *Current Protocols in Molecular Biology*, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Conservative amino acid substitutions can therefore be made to the amino acid sequences of proteins and polypeptides disclosed herein.

[0027] Recombinant AAV (rAAV) In some embodiments, this disclosure provides isolated AAVs. As used herein with respect to AAVs, the term “isolated” refers to artificially obtained or generated AAVs. Isolated AAVs may be generated using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs.” Recombinant AAVs (rAAVs) preferably have tissue-specific targeting capabilities such that the rAAV transgene is delivered specifically to one or more predetermined tissues. The AAV capsid is an important element in determining these tissue-specific targeting capabilities. Thus, an rAAV having a capsid appropriate for the target tissue can be selected. In some embodiments, the rAAV comprises an AAVv66 capsid protein. In some embodiments, the rAAV comprises a capsid protein having the amino acid sequence shown in SEQ ID NO: 1.

[0028] Methods for obtaining recombinant AAV having a desired capsid protein are well known in the art. (See, for example, US2003 / 0138772, the contents of which are incorporated herein by reference in their entirety.) Typically, these methods involve culturing a host cell containing a nucleic acid sequence (e.g., a nucleic acid encoding a polypeptide having the sequence shown in Sequence ID No. 1) or a fragment thereof; a functional rep gene; a recombinant AAV vector comprising an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper functions to enable the packaging of the recombinant AAV vector into the AAV capsid protein. In some embodiments, the capsid protein is a structural protein encoded by the cap gene of AAV. In some embodiments, AAV comprises three capsid proteins, virion proteins 1-3 (named VP1, VP2, and VP3), all of which may be expressed from a single cap gene. Thus, in some embodiments, the VP1, VP2, and VP3 proteins share a common core sequence. In some embodiments, the molecular weights of VP1, VP2, and VP3 are approximately 87 kDa, 72 kDa, and 62 kDa, respectively. In some embodiments, during translation, the capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the protein shell is primarily composed of the VP3 capsid protein. In some embodiments, the function of the capsid proteins is to protect the viral genome, deliver the genome, and interact with the host. In some embodiments, the capsid proteins deliver the viral genome to the host in a tissue-specific manner. In some embodiments, the VP1 and / or VP2 capsid proteins may contribute to the tissue tropism of the packaged AAV. In some embodiments, the tissue tropism of the packaged AAV is determined by the VP3 capsid protein. In some embodiments, the tissue tropism of the AAV is enhanced or altered by mutations occurring in the capsid proteins.

[0029] In some embodiments, the AAV variants described herein are variants of AAV2. AAV2 is known to efficiently transduce human central nervous system (CNS) tissue, kidney tissue, ocular tissue (e.g., photoreceptor cells and retinal pigment epithelium (RPE)), and other tissues. Therefore, in some embodiments, the AAV2 variants described herein may be useful for delivering gene therapy to CNS tissue, kidney tissue, or ocular tissue. In some embodiments, the AAV capsid proteins described herein may be useful for targeting other tissues, such as muscle tissue, liver tissue, or cardiac tissue. In some embodiments, the AAV capsid proteins described herein (e.g., AAVv66 capsid protein) may be able to cross the blood-brain barrier (BBB) ​​of target when delivered by intravenous or systemic injection.

[0030] In some embodiments, the AAV variants described herein may be useful in the treatment of CNS-related disorders. As used herein, “CNS-related disorders” are diseases or conditions of the central nervous system. CNS-related disorders may affect the spinal cord (e.g., myelopathy), the brain (e.g., encephalopathy), or the tissues surrounding the brain and spinal cord. CNS-related disorders may be of genetic origin, either inherited or acquired through somatic mutation. CNS-related disorders may be psychological conditions or disorders such as attention deficit hyperactivity disorder, autism spectrum disorder, mood disorders, schizophrenia, depression, or Rett syndrome. CNS-related disorders may be autoimmune disorders. CNS-related disorders may also be cancers of the CNS, such as brain cancer. CNS-related disorders that are cancers may be primary cancers of the CNS, such as astrocytoma or glioblastoma, or cancers that have metastasized to CNS tissue, such as lung cancer that has metastasized to the brain. Further non-exclusive examples of CNS-related disorders include Parkinson's disease, lysosomal storage disorders, ischemia, neuropathic pain, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Canavan disease (CD).

[0031] In some embodiments, the AAV variants described herein may target liver tissue. Therefore, in some embodiments, the AAV variants described herein may be useful in treating liver disease. As used herein, “liver disease” is a disease or condition of the liver. Liver disease may be of genetic origin, either inherited or acquired through somatic mutation. Liver disease may be cancer of the liver, including but not limited to hepatocellular carcinoma (HCC), fibrolamellar carcinoma, cholangiocarcinoma, angiosarcoma, and hepatoblastoma. Further non-exclusive examples of lung diseases include Alagille syndrome, alpha-1 antitrypsin deficiency, autoimmune hepatitis, biliary atresia, cirrhosis, cystic hepatic disease, fatty liver disease, galactosemia, gallstones, Gilbert's syndrome, hemochromatosis, liver disease in pregnancy, neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Rey syndrome, sarcoidosis, toxic hepatitis, glycogen storage disease type 1, tyrosinemia, viral hepatitis A, B, and C, Wilson's disease, and schistosomiasis.

[0032] In some embodiments, the AAV variants described herein may be useful for delivering gene therapy to ocular tissue (e.g., ocular tissue or cells). Therefore, in some embodiments, the AAV variants described herein may be useful for treating ocular disorders. As used herein, “ocular disorder” is a disease or condition of the eye. Ocular disorders may affect the eye, sclera, cornea, anterior chamber, posterior chamber, iris, pupil, lens, vitreous fluid, retina, or optic nerve. Ocular disorders may be of genetic origin, either inherited or acquired through somatic mutation. Non-exclusive examples of ocular diseases and disorders include, but are not limited to, age-related macular degeneration, retinopathy, diabetic retinopathy, macular edema, glaucoma, retinitis pigmentosa, and cancer of the eye.

[0033] The components cultured in host cells to package the rAAV vector into the AAV capsid may be provided to the host cells in trans. Alternatively, one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper function) may be provided by stable host cells manipulated to contain one or more of the required components using methods known to those skilled in the art. Most preferably, such stable host cells would contain the required components under the control of an inductive promoter. However, the required components may also be under the control of a constitutive promoter. Examples of suitable inductive and constitutive promoters are provided herein in the discussion of suitable regulatory elements for use with transgenes. In another alternative, selected stable host cells may contain selected components under the control of a constitutive promoter, and other selected components under the control of one or more inductive promoters. For example, stable host cells can be generated that originate from 293 cells (containing E1 helper function under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inductive promoter. Other stable host cells can also be generated by those skilled in the art.

[0034] The recombinant AAV vector, rep sequence, cap sequence, and helper function required to generate the rAAV of this disclosure can be delivered to a packaging host cell using any suitable gene element (vector). In some embodiments, a single nucleic acid encoding all three capsid proteins (e.g., VP1, VP2, and VP3) is delivered into the packaging host cell in a single vector. In some embodiments, the nucleic acids encoding the capsid proteins are delivered into the packaging host cell by two vectors; the first vector contains a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2), and the second vector contains a second nucleic acid encoding a single capsid protein (e.g., VP3). In some embodiments, three vectors, each containing nucleic acids encoding different capsid proteins, are delivered into the packaging host cell. The selected gene element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those skilled in the art of nucleic acid manipulation, and these include genetic engineering, recombination, and synthetic techniques. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the selection of a preferred method is not a limitation of this disclosure. See, for example, K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0035] In some embodiments, recombinant AAV may be generated using a triple transfection method (described in detail in U.S. Patent No. 6,001,650). Typically, recombinant AAV is generated by transfecting host cells with a recombinant AAV vector (containing the transgene), an AAV helper function vector, and an accessory function vector, which are packaged in AAV particles. The AAV helper function vector encodes “AAV helper function” sequences (e.g., rep and cap) that function in trans for productive AAV replication and capsid formation. Preferably, the AAV helper function vector supports efficient AAV vector generation without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with this disclosure include pHLP19, described in U.S. Patent No. 6,001,650, and the pRep6cap6 vector, described in U.S. Patent No. 6,156,303 (both in their entirety are incorporated herein by reference). Accessory function vectors encode nucleotide sequences relating to non-AAV-derived viral and / or cellular functions (e.g., “accessory functions”) on which AAV depends for replication. Accessory functions include, but are not limited to, those functions required for AAV replication, such as activation of AAV gene transcription, step-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions may originate from any known helper viruses, such as adenoviruses, herpesviruses (other than herpes simplex virus type 1), and vacciniaviruses.

[0036] In some embodiments, the disclosure provides transfected host cells. The term “transfection” is used to refer to the uptake of foreign DNA by a cell, and a cell is “transfected” when exogenous DNA is introduced into the inside of the cell (e.g., across the cell membrane). Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Using such techniques, one or more exogenous nucleic acids, such as nucleotide insertion vectors and other nucleic acid molecules, can be introduced into suitable host cells.

[0037] "Host cell" refers to any cell that harbors or is capable of harboring the substance of interest. In many cases, the host cell is a mammalian cell. The host cell may be used as the recipient of an AAV helper construct, an AAV minigene plasmid, an accessory functional vector, or other transfer DNA associated with the production of recombinant AAV. The term includes the offspring of the transfected original cell. Thus, as used herein, "host cell" may refer to a cell transfected with an exogenous DNA sequence. It is understood that the offspring of a single parental cell may not necessarily be completely identical to the original parent in morphology or in complementarity of the genome or DNA as a whole due to natural, accidental, or intentional mutations.

[0038] As used herein, the term “cell line” refers to a population of cells capable of continuous or long-term proliferation and division in vitro. Often, a cell line is a clonal population derived from a single progenitor cell. Furthermore, it is known in the art that spontaneous or induced changes in karyotype can occur during the storage or transport of such clonal populations. Therefore, cells derived from a cell line referred to may not be exactly identical to the ancestral cells or culture, and the cell line referred to may include such variants.

[0039] As used herein, the term “recombinant cell” refers to a cell into which an exogenous DNA segment, such as a DNA segment resulting in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid, such as RNA, has been introduced.

[0040] Cells may be transfected with a vector that imparts helper functions to AAV (e.g., a helper vector). Helper function-imparting vectors may impart adenovirus functions including, for example, E1a, E1b, E2a, and E4ORF6. The sequences of these functional adenovirus genes can be obtained from any known adenovirus serotype, e.g., serotypes 2, 3, 4, 7, 12, and 40, and may further include any of the currently identified human types known in the art. Thus, in some embodiments, the method involves transfecting cells with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0041] As used herein, the term “vector” includes any genetic element that is replicable when associated with an appropriate regulatory element and capable of transferring a gene sequence between cells, such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, and virions. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are intended to be these vectors in which the nucleic acid segment to be transcribed (e.g., nucleic acid sequence) is placed under the transcriptional control of a promoter. “Promoter” refers to a DNA sequence that is recognized by the cell’s synthetic mechanism, or introduced synthetic mechanism, and is required to initiate a specific transcription of a gene. The phrases “operatably positioned,” “under control,” or “transcriptionally controlled” mean that the promoter is in the correct position and orientation in relation to the nucleic acid and controls the initiation of RNA polymerase and gene expression. The term “expression vector or construct” means any type of genetic construct containing a nucleic acid in which part or all of the sequence encoding the nucleic acid is transcribable. In some embodiments, expression involves the transcription of nucleic acids, for example, to produce biologically active polypeptide products or inhibitory RNAs (e.g., shRNA, miRNA, miRNA inhibitors) from the transcribed gene.

[0042] In some embodiments, the promoter is the cytomegalovirus initial enhancer / chicken β-actin (CB6) promoter.

[0043] In some cases, isolated capsid genes can be used to construct and package recombinant AAVs using methods well known in the art, and the functional characteristics associated with the capsid protein encoded by the gene can be determined. For example, isolated capsid genes can be used to construct and package recombinant AAVs (rAAVs) containing reporter genes (e.g., β-galactosidase, GFP, luciferase, etc.). The rAAVs may then be delivered to animals (e.g., mice), and the tissue targeting characteristics of the novel isolated capsid gene may be determined by examining the expression of the reporter gene in various tissues of the animal (e.g., heart, liver, kidney). Methods for characterizing novel isolated capsid genes are disclosed herein, and others are well known in the art.

[0044] The method described above for packaging a recombinant vector within a desired AAV capsid to generate the rAAV of this disclosure is not limited, and other suitable methods will be apparent to those skilled in the art.

[0045] rAAV vector The “recombinant AAV (rAAV) vector” of this disclosure typically consists of, at a minimum, a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeats (ITRs). This recombinant AAV vector is packaged within a capsid protein and delivered to selected target cells. In some embodiments, the transgene is a nucleic acid sequence heterogeneous to the vector sequence that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product of interest. The nucleic acid-encoding sequence is operably ligated to the regulatory components to enable transcription, translation, and / or expression of the transgene within the cells of the target tissue.

[0046] The AAV sequence of the vector typically contains cis-acting 5' and 3' inverted terminal repeats (see, e.g., BJ Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155-168 (1990)). The ITR sequence is approximately 145 bp long. Preferably, substantially the entire sequence encoding the ITR is used in the molecule, although some minor modifications to these sequences are permissible. The ability to modify these ITR sequences is within the scope of the art (see, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and texts such as K. Fisher et al., J Virol., 70:520-532 (1996)). An example of such a molecule used in this disclosure is a “cis-acting” plasmid containing a transgene, where the sequence of the selected transgene and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types.

[0047] In some embodiments, this disclosure provides self-complementary AAV vectors. As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome resulting from the absence of a terminal segregation site (TR) from one of the ITRs of an AAV. The absence of a TR prevents the initiation of replication at the TR-less vector ends. Generally, scAAV vectors generate a single-stranded inverted repeat sequence genome with wild-type (wt) AAV TRs at both ends and a mutant TR (mTR) in the middle.

[0048] In some embodiments, the rAAV of this disclosure is a pseudotyped rAAV. Pseudotyping is the process of generating a virus or viral vector by combining it with a foreign viral envelope protein. The result is a pseudotyped viral particle. In this method, the foreign viral envelope protein can be used to increase / decrease the host tropism or the stability of the viral particle. In some embodiments, the pseudotyped rAAV comprises nucleic acids from two or more different AAVs, where the nucleic acid from one AAV encodes a capsid protein and the nucleic acid from at least one other AAV encodes other viral proteins and / or viral genomes. In some embodiments, the pseudotyped rAAV refers to an AAV containing an inverted terminal repeat sequence (ITR) of one AAV serotype and a capsid protein of a different AAV serotype. For example, a pseudotyped AAV vector containing an ITR of serotype X that forms a capsid with the protein of Y would be denoted as AAVX / Y (e.g., AAV2 / 1 has the ITR of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs are useful for combining the tissue-specific targeting ability of capsid proteins from one AAV serotype with viral DNA from another AAV serotype, thereby enabling targeted delivery of transgenes to target tissues.

[0049] In addition to the key elements identified above with respect to recombinant AAV vectors, the vector also includes necessary conventional regulatory elements operably ligated to the transgene to enable its transcription, translation, and / or expression in cells transfected with a plasmid vector or infected with a virus generated by this disclosure. As used herein, “operably ligated” sequence includes both an expression regulatory sequence adjacent to the gene of interest and an expression regulatory sequence acting in trans or at a distance to control the gene of interest.

[0050] Expression regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (poly-A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance the secretion of encoded products. Numerous expression regulatory sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known and available in the art.

[0051] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be “operably linked” if they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. Two DNA sequences are said to be operably linked if it is desirable that the nucleic acid sequence be translated into a functional protein, if the induction of a promoter in the 5' regulatory sequence results in the transcription of the coding sequence, and the nature of the linkage between the two DNA sequences is such that (1) it does not result in the introduction of a frameshift mutation, (2) it does not interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) it does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a nucleic acid sequence if the promoter region is capable of resulting in the transcription of its DNA sequence such that the resulting transcript is translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins translated within a frame. In some embodiments, operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequence yields a functional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

[0052] For protein-coding nucleic acids, the polyadenylation sequence is generally inserted after the transgene sequence and before the 3'AAV ITR sequence. Useful rAAV constructs in this disclosure may preferably contain an intron located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40 T intron sequence. Another vector element that can be used is an intra-sequence ribosome entry site (IRES). IRES sequences are used to produce two or more polypeptides from a single gene transcript. IRES sequences will be used to produce proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional, and many such sequences are available [see, for example, Sambrook et al. and the references cited therein, e.g., pp. 3.18, 3.26 and 16.17, 16.27, as well as Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, New York, 1989]. In some embodiments, the foot-and-mouth disease virus 2A sequence is contained within a polyprotein; this is a small peptide (approximately 18 amino acids long) that has been shown to mediate the cleavage of the polyprotein (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459).The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0053] The exact nature of the regulatory sequences required for gene expression in host cells may vary between species, tissues, or cell types, but generally, they will include, as needed, 5' untranscribed and 5' untranslated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, enhancer elements, etc. In particular, such 5' untranscribed regulatory sequences will include a promoter region containing a promoter sequence for the transcriptional control of the operably conjugated gene. The regulatory sequences may also include enhancer sequences or upstream activator sequences, if desired. The vectors of this disclosure may include 5' leader sequences or signal sequences, as needed. The selection and design of appropriate vectors are within the scope of the skill and discretion of those skilled in the art.

[0054] Examples of constitutive promoters include, but are not limited to, the retroviral Roussarcoma virus (RSV) LTR promoter (with RSV enhancer as needed), the cytomegalovirus (CMV) promoter (with CMV enhancer as needed) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter [Invitrogen].

[0055] Inducible promoters allow for the regulation of gene expression, which can be controlled only by exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as the acute phase, the presence of specific cell differentiation states, or in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inductive promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)); the tetracycline inhibitory system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); and the tetracycline-inducible system (Gossen et al, Science, 268:1766-1769 (1995), Harvey et al, Curr. Opin. Chem. Biol., 2:512-518). Examples include RU486-inducible systems (see also 1998), RU486-inducible systems (Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)), and rapamycin-inducible systems (Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Another type of inducible promoter that may be useful in this context is one that is regulated only under specific physiological conditions, such as temperature, acute phase, specific differentiation states of cells, or in replicating cells.

[0056] In another embodiment, the native promoter of the transgene is used. A native promoter may be preferred when it is desirable for the expression of the transgene to mimic native expression. A native promoter may also be used when the expression of the transgene must be regulated transiently, developmentally, tissue-specifically, or in response to a specific transcriptional stimulus. In further embodiments, other native expression regulatory elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.

[0057] In some embodiments, the regulatory sequence confers tissue-specific gene expression ability. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces tissue-specific transcription. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, gastrointestinal tract-specific mucin-2 promoter, eye-specific retinosuxin promoter, eye-specific K12 promoter, respiratory tissue-specific CC10 promoter, respiratory tissue-specific surfactant protein C (SP-C) promoter, breast tissue-specific PRC1 promoter, breast tissue-specific RRM2 promoter, urinary tract-specific uroplakin 2 (UPII) promoter, uterine tissue-specific lactoferrin promoter, or cardiac troponin T (cTnT) promoter.Other exemplary promoters, which are obvious to those skilled in the art, include the beta-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996); the bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)); the CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998)); the immunoglobulin heavy chain promoter; the T cell receptor α-chain promoter; and neuronal promoters such as neuron-specific enolase (NSE) (Andersen et al. Examples include the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)).

[0058] In some embodiments, the tissue-specific regulatory sequence is a CNS-specific promoter. Examples of CNS-specific promoters include, but are not limited to, 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, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In some embodiments, the CNS-specific promoter is an astrocyte-specific promoter, such as the glial filament acid protein promoter. In some embodiments, the CNS-specific promoter is a neuron promoter, such as the synapsin (Syn) promoter. In some embodiments, the CNS-specific promoter is a promoter of a gene selected from neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis (APC), and ionized calcium-binding adapter molecule 1 (Iba-1). In some embodiments, the CNS-specific promoter is a tissue-specific promoter in the CNS, as described by Kuegler S. (2016) in Manfredsson F. (eds) Gene Therapy for Neurological Disorders. Methods in Molecular Biology, vol 1382. Humana Press, New York, NY.

[0059] In some embodiments, one or more binding sites for one or more miRNAs are incorporated into the transgene of the rAAV vector to inhibit the expression of the transgene in one or more target tissues that carry the transgene (e.g., cell-type specific detargeting of transgene expression). Those skilled in the art will recognize that binding sites may be selected to control transgene expression in a tissue-specific manner. For example, a binding site for liver-specific miR-122 may be incorporated into the transgene to inhibit the expression of this transgene in the liver. The target site in mRNA may be in the 5'UTR, 3'UTR, or coding region. Typically, the target site is located in the 3'UTR of mRNA. Furthermore, the transgene may be designed so that multiple miRNAs regulate mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites can lead to the coordinated action of multiple RISCs, resulting in highly efficient inhibition of expression. The target site sequence may contain a total of 5 to 100, 10 to 60, or more nucleotides. The target site sequence may include at least five nucleotides from the target gene binding site sequence.

[0060] In some embodiments, the transgene includes one or more (e.g., 1, 2, 3, 4, 5, or more) miRNA binding sites that detarget the expression of the transgene from immune cells (e.g., antigen-presenting cells (APCs), such as macrophages, dendrites, etc.). Incorporation of miRNA binding sites relating to immunity-related miRNAs can detarget the expression of the transgene from antigen-presenting cells, as described, for example, in US 2018 / 0066279 (the entire content of which is incorporated herein by reference), thereby reducing or eliminating the immune response (cellular and / or humoral) that arises in a target against the product of the transgene. In some embodiments, the miRNAs associated with immunity are selected from the following: miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, miR-21, miR-29a / b / c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a / b, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424, miR-221, miR-222, let-7i, miR-148 and miR-152.

[0061] The sequence composition of the transgene in an rAAV vector varies depending on the intended use of the resulting vector. For example, one type of transgene sequence may include a reporter sequence that produces a detectable signal upon expression. In another example, the transgene encodes a therapeutic protein or therapeutic functional RNA. In yet another example, the transgene encodes a protein or functional RNA intended for research purposes, for example, to create somatic cell transgenic animal models possessing the transgene, or to study the function of the transgene product. In yet another example, the transgene encodes a protein or functional RNA intended for use in creating animal models of disease. The sequences encoding appropriate transgenes will be obvious to those skilled in the art.

[0062] Reporter sequences that may be provided in a transgene include, but are not limited to, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art. When associated with regulatory elements that drive their expression, the reporter sequence produces a signal detectable by conventional means, including enzymatic, radiometric, colorimetric, fluorescence or other spectroscopic assays, fluorescence-activated cell preservation assays, and immunological assays including enzyme-coupled immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of a signaling vector can be detected by assays for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signaling vector can be visually measured by color or light generation in a luminometer. Such reporters may be useful, for example, for verifying the tissue-specific targeting ability and tissue-specific promoter-modulating activity of rAAVs.

[0063] In certain embodiments, the Disclosure provides rAAV vectors for use in methods for preventing or treating one or more genetic defects or dysfunctions in mammals, such as polypeptide deficiency or polypeptide excess in mammals, and in particular in methods for treating or reducing the severity or extent of a defect in a human who manifests one or more disorders associated with such polypeptide deficiency in cells and tissues. The method involves administering to a subject an amount and duration sufficient to treat the defect or disorder in a subject suffering from such a disorder, an rAAV vector encoding one or more therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc., in a pharmaceutically acceptable carrier.

[0064] Therefore, this disclosure encompasses the delivery of rAAV vectors encoding one or more peptides, polypeptides, or proteins useful for treating or preventing disease conditions in mammalian subjects. Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptotic factors, cytokines, anti-diabetic factors, anti-apoptotic agents, coagulation factors, and antitumor factors. Other non-exclusive examples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropins, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.

[0065] rAAV vectors may contain a gene to be transferred to a target in order to treat diseases associated with reduced, absent, or dysfunctional gene expression. In some embodiments, rAAV vectors are used to treat diseases related to the central nervous system. Exemplary genes and associated disease conditions include, but are not limited to: glucose-6-phosphatase, associated with glycogen storage deficiency type 1A; phosphoenolpyruvate carboxykinase, associated with Pepck deficiency; galactose-1-phosphate uridyltransferase, associated with galactosemia; phenylalanine hydroxylase, associated with phenylketonuria; branched-chain alpha-keto acid dehydrogenase, associated with maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium-chain acyl-CoA dehydrogenase, associated with medium-chain acetyl-CoA deficiency; ornithine transcarbamylase, associated with ornithine transcarbamylase deficiency; argininosuccinate synthase, associated with citrullinemia; low-density lipoprotein receptor protein, associated with familial hypercholesterolemia; and associated with Crigler-Nadjar disease. UDP-glucuronosyltransferase; adenosine deaminase, associated with severe combined immunodeficiency; hypoxanthine guanine phosphoribosyltransferase, associated with gout and Lesch-Nyhan syndrome; biotinidase, associated with biotinidase deficiency; beta-glucocerebrosidase, associated with Gaucher disease; beta-glucocerebrosidase, associated with Sly syndrome; peroxisome membrane protein 70kDa, associated with Zellweger syndrome; acute intermittent Polphobilinogen deaminase associated with sexual porphyria; alpha-1 antitrypsin for the treatment of alpha-1 antitrypsin deficiency (emphysema); erythropoietin for the treatment of anemia due to thalassemia or renal failure; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitors for the treatment of occluded vessels, for example, seen in atherosclerosis, thrombosis, or embolism;Aromatic amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; beta-adrenergic receptor, antisense or variant forms of phospholamban, sarcoplasmic reticulum (ER) adenosine triphosphatase-2 (SERCA2), and cardiac adenylyl cyclase for the treatment of congestive heart failure; tumor suppressor genes such as p53 for the treatment of various cancers; cytokines such as one of various interleukins for the treatment of inflammatory and immune disorders as well as cancer; dystrophin or mini-dystrophin and utrophin or mini-utrophin for the treatment of muscular dystrophy; and insulin for the treatment of diabetes.

[0066] Those skilled in the art will also understand that, in the case of a transgene encoding a protein or polypeptide, mutations resulting in conserved amino acid substitutions may be made in the transgene to provide a functionally equivalent variant or homolog of the protein or polypeptide. In some embodiments, the disclosure encompasses sequence modifications resulting in conserved amino acid substitutions in the transgene. In some embodiments, the transgene includes a gene having a dominant-negative mutation. For example, the transgene may express a mutant protein that interacts with the same elements as the wild-type protein, thereby blocking some aspects of the function of the wild-type protein.

[0067] Useful transgene products include miRNAs. miRNAs and other small interfering nucleic acids regulate gene expression by cleaving / degrading the transcript of the target RNA or by repressing the translation of the target messenger RNA (mRNA). miRNAs are typically expressed natively as ultimately 19–25 untranslated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated region (UTR) of the target mRNA. These endogenously expressed miRNAs form hairpin precursors, which are then processed into miRNA double helixes and further into “mature” single-stranded miRNA molecules. These mature miRNAs guide the miRISC, a multiprotein complex, which, based on their complementarity with the mature miRNA, identifies the target site on the target mRNA, such as the 3'UTR region.

[0068] The following non-exclusive list of miRNA genes and their homologs may be used as transgenes or small interfering nucleic acids encoded by transgenes in certain embodiments of this method (e.g., miRNA sponges, antisense oligonucleotides, TuD hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa -let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-101, hsa-miR- 101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-106a, hsa-miR-106a* , hsa-miR-106b, hsa-miR-106b*, hsa-miR-107, hsa-miR-10a, hsa-miR-10a*, hsa- miR-10b, hsa-miR-10b*, hsa-miR-1178, hsa-miR-1179, hsa-miR-1180, hsa-miR-1 181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-1207-3p, hsa-miR-1207-5p, h sa-miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-122 6*, hsa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233, hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa- miR-1238, hsa-miR-124, hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR-1247, hsa-miR-1248, hsa-miR-1249 hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b, hsa-miR-1256, hsa-miR-1257, hsa-mi R-1258、hsa-miR-1259、hsa-miR-125a-3p、hsa-miR-125a-5p、hsa-miR-12 5b、hsa-miR-125b-1*、hsa-miR-125b-2*、hsa-miR-126、hsa-miR-126*、hsa -miR-1260、hsa-miR-1261、hsa-miR-1262、hsa-miR-1263、hsa-miR-1264、hsa-miR-1265、hsa-miR-1266、hsa-miR-1267、hsa-miR-1268、hsa-miR-126 9, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-1275, hsa-miR-127-5phsa-miR-1276, hsa-miR-1277, hsa-miR-1278, hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282, hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287, hsa-miR-1288, hs a-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299 hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR- 130a, hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*, hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hs a-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b, hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-13 7、hsa-miR-138、hsa-miR-138-1*、hsa-miR-138-2*、hsa-miR-139-3p、hsa-miR-139-5p、hsa-miR-140-3p、hsa-miR-140-5p、hsa-miR-141、hsa-miR-1 41*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa -miR-146a*、hsa-miR-146b-3p、hsa-miR-146b-5p、hsa-miR-147、hsa-miR-147b、hsa-miR-148a、hsa-miR-148a*、hsa-miR-148b、hsa-miR-148b*、hsa -miR-149, hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p, hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154* hsa-miR-155 hsa-miR-155* hsa-miR-15a hsa-miR-15a* hsa-miR-15b hsa-miR-15b* hsa-miR-16 hsa-miR-16-1* hsa-miR-16-2* hsa-miR-17hsa-miR-17*, hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*, hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-1 83, hsa-miR-183*, hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*, hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a, hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b* hsa-miR-190 hsa-miR-190b hsa-miR-191 hsa-miR-191* hsa-miR-192 hsa-miR-192* hsa-miR-193a-3p hsa-miR-193a-5p hsa-miR-193b hsa-miR-193b* hsa-miR-194 hsa-miR-194* hsa-miR-195 hsa-miR-19 5*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p, hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*, hsa-miR-19b-2* hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b, hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*, hsa-miR-203, hsa-mi R-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a, hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*, hsa-miR-21, hsa-miR- 21*, hsa-miR-210, hsa-miR-211, hsa-miR-212, hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-2 18, hsa-miR-218-1*, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR -220b, hsa-miR-220c, hsa-miR-221, hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*, hsa-miR-224, hsa-miR-23a, hsa-mi R-23a*, hsa-miR-23b, hsa-miR-23b*, hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*, hsa-miR-26a, hsa-miR-26a-1*, hsa- miR-26a-2*, hsa-miR-26b, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296 -3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298, hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b, hsa-miR-29b-1*hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300, hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b, hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*, hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30 b、hsa-miR-30b*、hsa-miR-30c、hsa-miR-30c-1*、hsa-miR-30c-2*、hsa- miR-30d、hsa-miR-30d*、hsa-miR-30e、hsa-miR-30e*、hsa-miR-31、hsa-m iR-31*, hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324- 3p, hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329, hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331- 5p, hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p, hsa-mi R-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, h sa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p, hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3phsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*, hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b hsa-miR-374b*, hsa-miR-375, hsa-miR-376a, hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377, hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384 hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411, hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hsa-miR-429, hsa-mi R-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*, hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, hsa-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa -miR-454*, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p, hsa-miR-487a, hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-4 89, hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495, hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa -miR-505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510, hs a-miR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b, hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p hsa-miR-515-5p hsa-miR-516a-3p hsa-miR-516a-5p hsa-miR-516b hsa-miR-517* hsa-miR-517a hsa-miR-517b hsa-miR-517c hsa-miR-518a -3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、hs a-miR-518d-3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa-m iR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p, hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*, hsa-miR-520a-3p, hs a-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、hsa -miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR-520 h, hsa-miR-521, hsa-miR-522, hsa-miR-523, hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-526b, hsa-miR-526b*,hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p, hsa-miR-543, hsa-miR-544, hsa -miR-545, hsa-miR-545*, hsa-miR-548a-3p, hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p, hsa-miR-548c-5p, hsa-miR -548d-3p, hsa-miR-548d-5p, hsa-miR-548e, hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j, hsa-miR-548k, hsa-miR-5 48l, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a, hsa-miR-551b, hsa-mi R-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa-miR-559, hsa -miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570, hsa-miR-571, hsa-miR- 572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578, hsa-miR-579, h sa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR-588hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595, hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606 hsa-miR-607 hsa-miR-608 hsa-miR-609 hsa-miR-610 hsa-miR-611 hsa-miR-612 hsa-miR-613 hsa-miR-614 hsa-miR-615-3p hsa-miR-615- 5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621, hsa-miR-622, hsa-miR-623, hsa-miR-624 hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-64 0, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hs a-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668, hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675 hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744, hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766, hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p, hsa-miR-769-3p, hsa-miR-769-5p, hsa -miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*, hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-88 9, hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923, hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hs a-miR-933, hsa-miR-934, hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939, hsa-miR-940, hsa-miR-941, hsa-miR-942, hs a-miR-943、hsa-miR-944、hsa-miR-95、hsa-miR-96、hsa-miR-96*、hsa- miR-98、hsa-miR-99a、hsa-miR-99a*、hsa-miR-99b、およびhsa-miR-99b*。

[0069] miRNAs inhibit the function of the mRNA they target, thereby inhibiting the expression of the polypeptide encoded by that mRNA. Therefore, by blocking (partially or completely) the activity of a miRNA (e.g., silencing the miRNA), the expression of the polypeptide whose expression is inhibited can be effectively induced or restored (polypeptide repression can be released). In one embodiment, the depression of the polypeptide encoded by the mRNA target of a miRNA is achieved by inhibiting intracellular miRNA activity by one of several methods. For example, blocking the activity of a miRNA can be achieved by hybridization with a small interfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge, TuD RNA) that is complementary or substantially complementary to the miRNA, thereby blocking the interaction of the miRNA with its target mRNA. As used herein, a small interfering nucleic acid that is substantially complementary to the miRNA is one that hybridizes with the miRNA and can block its activity. In some embodiments, a small interfering nucleic acid that is substantially complementary to miRNA is a small interfering nucleic acid that is complementary to miRNA in all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases. In some embodiments, a small interfering nucleic acid sequence is a small interfering nucleic acid sequence that is substantially complementary to miRNA or complementary to miRNA in at least one base.

[0070] "miRNA inhibitors" are drugs that block the function, expression, and / or processing of miRNAs. These molecules include, but are not limited to, microRNA-specific antisense, microRNA sponges, tough decoy RNA (TuD RNA), and microRNA oligonucleotides (double-stranded, hairpin, and short oligonucleotides) that inhibit the interaction of miRNAs with the Drosha complex. MicroRNA inhibitors can be expressed in cells from the transgene of an rAAV vector, as discussed above. MicroRNA sponges specifically inhibit miRNAs through complementary heptamer seed sequences (Ebert, MS Nature Methods, Epub August, 12, 2007). In some embodiments, an entire family of miRNAs may be silenced using a single sponge sequence. TuD RNA achieves efficient and long-term suppression of specific miRNAs in mammalian cells (see, for example, Takeshi Haraguchi, et al., Nucleic Acids Research, 2009, Vol. 37, No. 6 e43, which is incorporated herein by reference). Other methods for silencing miRNA function within cells (de-suppression of miRNA targets) will be obvious to those skilled in the art.

[0071] In some embodiments, the cloning capability of recombinant RNA vectors may be limited to the desired coding sequence, requiring the complete replacement of a 4.8-kilobase viral genome. Therefore, large genes may, in some cases, be unsuitable for use in standard recombinant AAV vectors. Those skilled in the art will recognize that options are available to overcome limited coding capability. For example, AAV ITRs from two genomes can anneal to form a head-to-tail concatemer, nearly doubling the vector's capability. Insertion of a splice site allows for the removal of ITRs from the transcript. Options for overcoming limited cloning capability will be obvious to those skilled in the art.

[0072] Administration rAAV may be delivered to a subject in a composition by any suitable method known in the art. Preferably, rAAV suspended in a physiologically compatible carrier (e.g., in a composition) may be administered to a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cattle, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., macaque). In some embodiments, the host animal does not include humans.

[0073] The delivery of rAAV to mammalian subjects may be, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, artery, or any other vascular conduit. In some embodiments, rAAV is administered into the bloodstream by isolated limb perfusion, a technique well known in the surgical art, by which a person skilled in the art can essentially separate the limb from the systemic circulation before administration of the rAAV virion. A variation of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 may be used by a person skilled in the art to administer the virion into the vascular system of the isolated limb to potentially enhance transduction into muscle cells or tissues. Furthermore, in certain specific cases, it may be desirable to deliver the virion to the CNS of the subject. "CNS" means all cells and tissues of the brain and spinal cord of vertebrates. Thus, the term includes, but is not limited to, nerve cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial space, bone, cartilage, etc. Recombinant AAV may be delivered directly to the CNS or brain by injection with a needle, catheter or related device into the ventricular region, as well as the striatum (e.g., the caudate nucleus or putamen of the striatum), the spinal cord and neuromuscular junction, or the cerebellar lobe, using neurosurgical techniques known in the art, such as stereotactic injection (see, for example, Stein et al., J Virol 73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

[0074] The compositions of this disclosure may include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding or having one or more different transgenes). In some embodiments, the compositions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0075] A suitable carrier can be readily selected by those skilled in the art, taking into account the applications for which rAAV is intended. For example, one suitable carrier is saline solution, which may be formulated with various buffer solutions (e.g., phosphate-buffered saline). Other exemplary carriers include sterile saline solution, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The choice of carrier is not limited to this disclosure.

[0076] If necessary, the compositions of this disclosure may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0077] rAAV is administered in an amount sufficient to transfect cells of the desired tissue and to result in sufficient levels of gene transfer and expression without excessive adverse effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to selected organs (e.g., intra-portal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, intracranial (e.g., intrahippocampal), and other parenteral routes of administration. Routes of administration may be combined if desired.

[0078] The dose of rAAV virions required to achieve a particular "therapeutic effect", e.g., the unit of dose of genomic copies per kilogram of body weight (GC / kg), varies based on several factors including, but not limited to, the route of administration of the rAAV virions, the level of gene or RNA expression required to achieve the therapeutic effect, the particular disease or disorder being treated, and the stability of the gene or RNA product. One of ordinary skill in the art can readily determine the dosage range of rAAV virions to treat a patient having a particular disease or disorder based on the foregoing factors, and other factors well known in the art.

[0079] An effective amount of rAAV is an amount sufficient to target or infect an animal and to target the desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to generate a stable somatic transgenic animal model. The effective amount will vary primarily according to factors such as the species, age, weight, health, and tissue to be targeted of the subject, and thus may vary between animals or between tissues. For example, an effective amount of rAAV generally ranges from about 1 ml to about 100 ml of a solution containing from about 10 9 ~10 16 genomic copies. In some embodiments, rAAV is administered at a dose of 10 10 、10 11 、10 12 、10 13 、10 14 、or 10 15 genomic copies. In some embodiments, rAAV is administered at a dose of 10 10 、10 11 、10 12 、10 13 、or 10 14 genomic copies per kg. In some cases, a dosage of between about 10 11 ~10 12 rAAV genomic copies is appropriate. In certain embodiments, 10 12A single rAAV genome copy is effective in targeting heart, liver, and pancreatic tissues. In some cases, stable transgenic animals are generated by multiple doses of rAAV.

[0080] In some embodiments, the rAAV composition is particularly high in concentration (e.g., about 10%). 13 If rAAV is present at a concentration of GC / ml or higher, the composition is formulated to reduce the aggregation of AAV particles. Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of surfactants, pH adjustment, and salt concentration adjustment (see, for example, Wright FR, et al., Molecular Therapy (2005) 12, 171-178, which are incorporated herein by reference).

[0081] Formulations of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as is the development of suitable administration and treatment regimens for the use of the specific compositions described herein in various treatment regimens.

[0082] Typically, these formulations may contain at least about 0.1% or more of the active compound, but the percentage of the active ingredient may, of course, be modified and may be, for convenience, between about 1 or 2% and about 70% or 80% or more of the total weight or volume of the formulation. Naturally, the amount of the active compound in each therapeutically useful composition may be adjusted so that a suitable dosage is obtained at a given unit dose of any of the compounds. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will also be taken into account by those skilled in the art preparing such pharmaceutical formulations, and such as various administration and treatment regimens may be desirable.

[0083] Under certain circumstances, it may be desirable to deliver rAAV-based therapeutic constructs in the suitably formulated pharmaceutical compositions disclosed herein by subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intracranial (e.g., hippocampal), intramuscular, intrathecal, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities described in U.S. Patents 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference as a whole) may be used to deliver rAAV. In some embodiments, the preferred method of administration is by portal vein injection.

[0084] Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Dispersions may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In many cases, the form is sterile and fluid to the extent that syringability is present. The form must be stable under manufacturing and storage conditions and must be protected from microbial contamination such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof, as well as / or vegetable oils. Appropriate fluidity can be maintained, for example, by coating, e.g., by the use of lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Extended absorption of the injectable composition can be achieved by using absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0085] For the administration of injectable aqueous solutions, for example, the solution may be preferably buffered if necessary, and the liquid diluent may be isotonic first with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion, or injected into the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in the dose will inevitably occur depending on the host's condition. The person administering the drug will determine the appropriate dose for each individual host in any event.

[0086] Sterile injectable solutions are prepared by incorporating the required amount of active rAAV in a suitable solvent, along with various other components listed herein, and subsequently sterilizing by filtration if necessary. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredients and any additional desired components from a pre-sterile filtered solution.

[0087] The rAAV compositions disclosed herein may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Once formulated, the solution is administered in a form suitable for the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in various dosage forms, such as injectable solutions and drug-release capsules.

[0088] As used herein, “carrier” includes all kinds of solvents, dispersions, vehicles, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and active ingredients for pharmaceutically active substances is well known in the art. Complementary active ingredients may also be incorporated into the composition. The phrase “pharmaceutically acceptable” means molecular entities and compositions that, when administered to a host, do not produce an allergic reaction or a similar adverse reaction.

[0089] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles may be used for the introduction of the compositions of this disclosure into suitable host cells. In particular, the transgene delivered by the rAAV vector may be formulated for delivery by being encapsulated in any of the following: lipid particles, liposomes, vesicles, nanospheres, or nanoparticles.

[0090] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Recently, liposomes with improved stability in serum and circulating half-life have been developed (U.S. Patent No. 5,741,516). Furthermore, various methods for liposomes and liposome-like preparations as potential drug carriers have been described (U.S. Patents No. 5,567,434; No. 5,552,157; ​​No. 5,565,213; No. 5,738,868 and No. 5,795,587).

[0091] Liposomes have been successfully used for several cell types that are typically resistant to transfection by other procedures. In addition, liposomes are not subject to the DNA length constraints typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapies, viruses, transcription factors, and allosteric effectors into various cultured cell lines and animals. Furthermore, several clinical trials investigating the efficacy of liposome-mediated drug delivery have been successfully completed.

[0092] Liposomes are composed of phospholipids that are dispersed in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also known as multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of smaller monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 ANG. containing an aqueous solution in the core.

[0093] Alternatively, nanocapsule formulations of rAAV may be used. Nanocapsules can generally capture substances in a stable and renewable manner. To avoid side effects due to polymer overload in cells, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet this requirement are intended for use.

[0094] In addition to the delivery methods described above, the following techniques are also being considered as alternative methods for delivering rAAV compositions to the host: Sonophoresis (i.e., ultrasound) is used as a device to enhance the rate and efficacy of drug penetration into and through the circulatory system, as described in U.S. Patent No. 5,656,016. Other proposed alternatives to drug delivery include intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Patents No. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899). [Examples]

[0095] (Example 1) Recombinant adeno-associated viruses (rAAVs) have recently attracted considerable attention in the field of human gene therapy as safe and reliable gene delivery vehicles. Currently, AAV2 is the most commonly used in preclinical and clinical research. However, Luxturna, an AAV2-based drug, is the only virus-based biotherapy drug approved by the FDA, and therefore, improving the pharmaceutical properties of AAVs is extremely important.

[0096] AAV2 is known to be "low productivity" in vector generation and "poor performance" in many tissues and cell types. We isolated a viral variant with improved properties.

[0097] A variant named AAVv66 was identified as the most abundant proviral capsid variant in clinical pancreatic neoplasm samples. The AAVv66 capsid contains 13 residues different from AAV2 (mutations to AAV2 including K39Q, V151A, R447K, T450A, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593T). This variant exhibits favorable tropism in the CNS after intracranial (e.g., subcranial) injection. Furthermore, AAVv66 demonstrates better packaging efficiency than the prototype AAV2. Using differential scanning fluorescence (DSF), the melting temperature of AAVv66 was observed to be approximately 6°C higher than that of AAV2 over a pH range of pH 4 to pH 7. Furthermore, DSF analysis shows that at pH 4, AAVv66 explodes its vector DNA at a higher temperature than AAV2.

[0098] AAVv66 was also confirmed to confer superior CNS transduction compared to AAV2. Structural differences between AAV2 and AAVv66 were revealed at the interface of the 3-turn projection and the 5-turn symmetry axis from the cryo-EM structure at a resolution of 2.9 Å, indicating that residues at these locations contribute to improved stability and function for vector transduction.

[0099] (Example 2) AAVs have recently attracted attention as effective and proven gene therapy vectors. Current classifications of AAVs are stable, confer long-term gene expression, exhibit broad tissue tropism, and have relatively low pathogenicity. To date, three serotype capsids (AAV1, AAV2, and AAV9) have received regulatory approval for commercial use in patients. Unfortunately, the current libraries of discovered and engineered AAV capsids are insufficient for certain clinical applications requiring targeting specific tissues or cell types. Furthermore, patients may have pre-existing immunities to the vector via neutralizing antibodies, which can limit therapeutic efficacy. Additionally, certain capsids are known to have problems in standard production schemes that fail to produce the high yield titers required to meet therapeutic doses. In response to these shortcomings, there is a need for the discovery and development of novel capsids that exhibit better vector yields, evade innate immunity, and possess a unique tropism profile.

[0100] This example describes AAVv66 (SEQ ID NO: 1), a capsid protein variant identified by high-throughput single-molecule real-time (SMRT) sequencing, whose properties are substantially different from those of AAV2 despite high sequence similarity (98%). Firstly, AAVv66 exhibits better vector yield and is more thermally stable than the prototype AAV2. Secondly, AAVv66 has a wider distribution in brain tissue when administered by intracranial injection. Finally, AAVv66 is antigenically distinct from AAV2.

[0101] To better understand how AAVv66 differs from AAV2, cryo-electron microscopy (cryo-EM) was performed to explore the structural and functional characteristics that define AAVv66. Our structural analysis of the AAVv66 capsid at 2.5 Å resolution reveals differences from the AAV2 structure and provides insights into the functional properties of the capsid. In summary, these observations explain the mechanistic characteristics of AAVv66.

[0102] material and method DNA extraction A sample of a pancreatic neoplasm was obtained from a 71-year-old female patient after tumor resection and histopathology by examination of frozen sections and intraoperative frozen section diagnosis. The sample was stored in liquid nitrogen until DNA extraction. To avoid DNA cross-contamination of AAV, DNA extraction and PCR procedures were performed in a sterile, UV-irradiated biosafety cabinet. All surfaces and instruments were sprayed with DNA-Exitus Plus (Applichem, catalog no. A7089) and wiped clean with Milli-Q water after 15 minutes. The frozen tissue was then thawed at room temperature, and approximately 25 mg of tissue was quickly cut with a disposable scalpel and placed in a 2 mL tube. DNA extraction from the tissue was performed using the QIAamp DNA Mini Kit (Qiagen, no. 51306) according to the manufacturer's recommended procedure.

[0103] SMRT sequencing An amplicon library was prepared from genomic DNA using a standard PCR procedure. To amplify the AAV genome, PCR was performed using Platinum® PCR SuperMix High Fidelity (Invitrogen) under the following cycle conditions: 1 minute at 97°C; 46 cycles of 10 seconds at 98°C, 15 seconds at 60°C, and 2 minutes 30 seconds at 68°C; and 10 minutes at 68°C. Correctly sized PCR products were gel-purified using PureLink® PCR Purification Kit (Thermo Fisher) and used for a second 15-cycle PCR for barcoding. The primer pairs used were: First primer: CapF 5'-GACTGCATCTTTGAACAATAAATGA-3' (SEQ ID NO: 3) and CapR 5'-GAAACGAATTAACCGGTTTATTGATTAA-3' (SEQ ID NO: 4) Second primer: EF 5'-CATCACTACGCTAGATGACTGCATCTTTGAACAATAAATGA-3' (SEQ ID NO: 5) and ER 5'-TAGTATATCGAGACTCGAAACGAATTAACCGGTTTATTGATTAA-3' (SEQ ID NO: 6)

[0104] Amplicons representing the ORF of the capsid variant were subjected to standard SMRT sequencing library preparation. Sequencing was performed on the RSII platform. SMRT sequencing returned 17,727 DNA reads mapped to the AAV2 Cap ORF using the BWA-MEM algorithm. Reads were then filtered to exclude artifact sequences, removing sequences shorter than 1,800 nt and longer than 2,500 nt, and then filtered for read quality (Phred score > 30). This filtering reduced the number of reads to 14,500. Finally, the reads were processed with InDelFixer to remove single nucleotide insertions and deletions that could result from error-prone PCR or sequencing errors. De novo assembly (Geneious R9) was performed on the filtered reads to consider only unique capsid sequences and exclude unreliable variants, and reads with 99% sequence similarity were clustered. Only clusters of reads represented by at least 10 reads were considered unique DNA capsid sequences. These DNA sequences were then translated into amino acid sequences to determine the final list of unique AAV capsids.

[0105] Complete AAV Cap ORFs derived from modern AAV serotypes (hu.2, used for AAV2 / 3) were obtained from NCBI, and the predicted amino acid sequences were iteratively aligned using the MUSCLE algorithm until convergence was achieved. Phylogenetic trees were then constructed using PhyML with default parameters within SeaView55, and subsequently visualized using the Interactive Tree of Life online tool.

[0106] Virus vector generation In HEK293 cells, viruses were generated using a triple transfection method and purified by CsCl gradient centrifugation. All described vectors were packaged in one of the following: a self-complementary AAV vector expressing enhanced green fluorescent protein (scAAV-CB6-EGFP), a single-stranded vector expressing firefly luciferase (ssAAV-CB6-Fluc), a single-stranded vector expressing secreted human alpha-1 antitrypsin (ssAAV-CB6-hA1AT), or a single-stranded vector expressing LacZ. All transgenes were driven by the CMV early enhancer / chicken β-actin (CB6) ubiquitous promoter.

[0107] animal Male C57BL / 6J mice (The Jackson Laboratory) aged 6-8 weeks were administered the test vector intravenously (IV), intramuscularly (IM), or intracranially. Mice administered via IV injection were given a vector containing the ssAAV-CB6-Fluc transgene (1.0E11vg / mouse), and were sacrificed 14 days after injection. Mice administered via IM injection into the TA muscle were given a vector containing the ssAAV-CB6-Fluc transgene (4.0E10vg / mouse), and were sacrificed 28 days after injection. D-luciferin substrate was injected intraperitoneally into the animals weekly leading up to sacrifice, and at the time of sacrifice. The animals were sedated with isoflurane, and luciferase activity was quantified using the IVIS SpectrumCT imaging platform with a 1-minute exposure. Images were acquired using Living Image software. Mice subjected to intrahippocampal injection were administered a vector containing the scAAV-CB6-Egfp transgene (3.6E9vg / mouse). Unilateral injections were performed into the right hemisphere using a stereotactic frame (Stoelting Co. Wood Dale, IL), Hamilton Syringe (1207K95, Thomas Scientific), and Hamilton Needle (77602-06, Hamilton). The following relative coordinates were used for all intrahippocampal injections: x:-1.5mm, y:-2mm, z:-2mm.

[0108] immunostaining Four weeks after injection, the animals were perfused intracardiacly with 1× phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). The brain was extracted and then fixed overnight in 4% PFA at 4°C. The brain was then immersed in 30% sucrose (prepared in 1× PBS) at 4°C until equilibrated in a sucrose mixture. The brain was embedded in a 1:2 mixture of OCT (Tissue Tek, Torrance, CA) and 30% sucrose, and frozen sections were prepared at 40 μm (Cryostar NX70, Thermo Scientific, Waltham, MA). Sections were permeabilized with 0.5% Triton® X-100 for 1 hour, blocked with 5% goat serum (10% normal goat serum, 50062Z, Life Technologies) for 1 hour, and then incubated overnight at 4°C in primary antibodies (anti-NeuN, 1:1000, EMD Millipore MAB377; anti-Gfap, 1:500, EMD Millipore MAB360; anti-Olig2, 1:200, Abcam ab109186; anti-Iba1, 1:1000, Wako Chemicals NC9288364). Sections were washed three times with 1×PBS and incubated at room temperature for 1 hour in secondary antibodies (anti-mouse, Invitrogen A32744; or anti-rabbit, Invitrogen A32740). Sections were washed three times with 1×PBS and mounted using Vectashield (Vector Laboratories, Burlingame, CA) containing DAPI.

[0109] Microscopy Brain section images were obtained using a Leica SP8 Lightning High Resolution Confocal (Leica Microsystems, Wetzlar, Germany). Whole brain images (10× tiled brain sections) and high-magnification images (63× region-specific areas) were acquired at the same intensity and exposure threshold for each magnification. For high-magnification images, 40-50 z-stack steps were acquired at 0.29 z-size. Analysis was performed using Imaris 9.3 Software (Bitplane Inc., Zurich, Switzerland). Each image was 3D rendered and thresholds were manually established. To ensure consistency, unbiased 3D renderings of the total EGFP volume under dissection, co-localized with DAPI volume, and cell type-specific staining were used as proxies for cell counting and the number of positively transduced cells. Percentage quantification of different cell types within each ipsilateral dissection region was performed, followed by percentage quantification of each cell type. The transduction percentage was determined by normalizing the volume of co-localized EGFP relative to the total volume of cell-type specific staining within each region. n=3 mice were analyzed per cell-specific stain. Statistical calculations for Figure 2B were performed using Prism 7 (GraphPad Software, Inc., San Diego, CA), and the analysis was performed using Student's unpaired t-test.

[0110] DSF analysis For capsid stability experiments, 5 μL of SYPRO Orange 5000X (Thermo Fisher Scientific) was diluted in 495 μL of PBS (Corning) to prepare a 50× stock. 45 μL of virus was mixed with 5 μL of 50× SYPRO Orange (the final concentration of SYPRO Orange was 5×). Fluorescence was quantified using a ViiA 7 real-time PCR instrument (Thermo Fisher Scientific) with the following parameters: The sample was incubated at 25°C for 2 minutes, followed by a temperature gradient (0.4°C at each step, held for 2 minutes at each step, from 25°C to 99°C). A ROX filter without passive reference was used to monitor the fluorescence of SYPRO Orange at each temperature step. To investigate the effect of pH on the melting temperature of the AAV vector, 5 μL of viral vector, 5 μL of 50× SYPRO Orange, and 40 μL of 0.6 M acetate buffer adjusted to pH 7 to pH 4 were mixed. The Tm values ​​reported in this study are defined as the maximum Dsignal / Dtemp detected between 25 and 95°C. To investigate the release of the vector genome, the SYBRO Orange dye was switched to SYBR Gold (Thermo Fisher Scientific).

[0111] Site-directed mutagenesis To induce point mutations in the ORF of the AAVv66 capsid, the Q5 site-directed mutagenesis kit (New England Biolabs) and the following mutagenesis primer pair were used: [Table 1-1] [Table 1-2]

[0112] scoreo-EM AAVv66 with racy carbon support film grid (01824G, Ted Preparations for cryo-EM were made on a Pella, Inc. grid. First, the grid was washed with acetyl acetate and dried overnight. Next, the grid was glow-discharged for 60 seconds with a negative polarity current of 20 mA using a PELCO easiGlow glow discharge unit. 3 μL of AAVv66-CB6-Egfp vector at 1E13 vg / mL in buffer (5% sorbitol and 0.001% Pluronic acid F68 in PBS) was placed on the grid mounted on a Vitrobot Mark IV (Thermo Fisher) cryo-EM plunging apparatus. Before rapid freezing in liquid ethane, the grid was blotted for 6–6.5 seconds with Whatman No. 1 filter paper at 10°C and 95% relative humidity.

[0113] A dataset consisting of 2,033 videos was collected using a SerialEM on a Titan Krios electron microscope (FEI) operating at 300kV and equipped with a Gatan Image Filter (GIF) and a K2 Summit direct electron detector (Gatan Inc.), with an underfocus of 0.5–2.2 μm. 50 frames were collected per video, with a dose of 1.43 e- / Ų per frame and a total dose to the sample of 48.62 e- / Ų, and 34 frames were used. The pixel size was 1.0588 Å with respect to the sample. The videos were imported into cisTEM, aligned by dose filtering, and the CTF parameters were determined. Next, a total of 52,874 particles were automatically picked within cisTEM (characteristic radii and maximum radii: 130 Å and 140 Å). The inventors note that both particles encapsulating vector-transgenes and empty capsids with a low percentage were used to determine the final structure. In cisTEM, an initial reference for alignment was created from all particles using the Ab initio 3D reconstruction function. This reference and all particles were iteratively refined using auto-refine to obtain a map with a resolution of 2.95 Å, determined from the FSC_part cutoff of 0.143. A single CTF refinement per particle in manual mode improved the map resolution to 2.62 Å. Finally, a single beam tilt refinement and reconstruction improved the map resolution to 2.46 Å. 3D classification did not improve the map. The final map was sharpened with a B factor by applying a B factor of -32.92 Ų using the PHENIX auto-sharpening function.

[0114] The cryo-EM structure of AAV2 (PDB ID: 1LP3) was used as the starting model for structural refinement. Variant residues were modeled using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schroedinger, LLC.). The resulting AAVv66 model, containing 60 copies of VP3, was refined using PHENIX59 against the cryo-EM map. Refinement using PHENIX's real-space simulated annealing and B-factor yielded a stereochemically optimal model. The refinement results are summarized in Table 2. This model was examined, and figures were created using PyMOL.

[0115] Using the Zetasizer Nano ZS system (Malvern), the vector was diluted to a concentration of approximately 1.0E9vg / mL for zeta potential analysis. 500 μL of the sample was added to the universal dip cell (Malvern). The system was stabilized for 2 minutes before measurement. Three measurements were recorded for each sample. [Table 2]

[0116] immunological research 1.0E11vg / mouse of scAAV-CB6-Egfp was intramuscularly administered to the left / right tibialis anterior muscle of C57BL / 6J mice. Four weeks later, ssAAVv66-CB6-hA1AT or ssAAV2-CB6-hA1AT was delivered to the contralateral limb at 1.0E11vg / mouse. Serum was collected by facial venous aspiration at weeks 4, 5, 6, 7, and 8, and neutralizing antibody titers and A1AT levels were evaluated by ELISA.

[0117] Huh-7.5 (5.0E4 cells / well) was seeded in 96-well plates at 37°C 24 hours prior to transduction. Ad helper virus was then added to the cell monolayer at a 100:1 multiplicity of infection (MOI) and incubated for at least 1 hour. Serial dilutions of serum-AAV2-LacZ or ssAAVv66-LacZ mixtures were prepared in V-bottom 96-well plates and incubated at 37°C for 1 hour. The serum-AAV mixture was then added to the cells and incubated at 37°C for 24 hours. The cells were lysed and treated with a beta-galactosidase substrate using the Galacto-Star One-Step Assay System (Invitrogen). Luminescence signals were detected using a Synergy HT microplate reader (BioTek, Winooski, VT).

[0118] A1AT ELISA A 96-well plate was first coated with anti-A1AT antibody overnight at 4°C, and the wells were incubated at room temperature for 1 hour using blocking buffer (1% nonfat milk and 0.05% Tween®-20 in PBS buffer). 1 / 20, 1 / 200, and 1 / 2,000 serum dilutions were performed with positive controls (A1AT at 100, 50, 25, 12.5, 6.25, and 3.125 ng / mL) using the sample buffer in the 96-well plate (0.05% Tween-20 in PBS buffer). After washing the plate three times, serum was added to each well and incubated overnight at 4°C. The plate was then washed three times and incubated with goat anti-trypsin-HRP antibody (1:5,500 dilution in sample buffer) for 2 hours. Before reacting with the substrate, the plate was washed six times to remove all residual proteins. Finally, the ABTS substrate was added to the wells, and the signal was read using a Synergy HT microplate reader (BioTek).

[0119] Identification of novel AAV variants in human tissue samples using long-read sequencing. To identify novel full-length capsid sequences from human tissue, SMRT sequencing was performed to obtain long DNA reads across the entire open reading frame of the capsid (Figure 1A). This method allows for the sequencing of long DNA fragments without the need for sequence assembly required by short-read sequencing approaches. In this way, capsid diversity, defined by both point mutations and recombination events, can be evaluated at the individual intact molecules across the entire capsid ORF. To investigate AAV diversity, a single tissue was selected from approximately 800 human surgical specimens. Targeted PCR amplicons for SMRT sequencing analysis were generated using primers adjacent to the capsid ORF in sequences conserved across known serotypes. One capsid sequence constituting approximately 45% of the total sequences identified from the single tissue was isolated (Figure 1B). This dominant capsid was named "Variant 66" (AAVv66) and exhibits the closest homology to AAV2 (98% sequence similarity; Figures 1C-1D). AAVv66 was observed to contain 13 amino acid residues distinct from AAV2 (Figures 1C and 8): one in the VP1u region (K39Q), one in the VP2 domain (V151A), and 11 in VP3 (R447K, T450A, Q457M, S492A, E499D, F533Y, G546D, E548G, R585S, R588T, and A593T). Notably, all of the unique amino acid residues within VP3 are located within or near the variable region VR-IV to VR-VIII.

[0120] The VP3 region of AAVv66 was compared to that of other modern AAV serotypes (AAV1–AAV9). The most significant differences occur at four highly conserved positions (499, 533, 585, and 588) among AAV serotypes (Figure 8). At position 499, most serotypes possess asparagine, while AAVv66, AAV2, AAV4, and AAV9 have negatively charged aspartate or glutamate. The highly conserved phenylalanine at position 533 is tyrosine in AAVv66 (and T533 in AAV5). Finally, unlike AAV2, which possesses positively charged arginine residues at positions 585 and 58823 that define AAV2's ability to bind to heparan sulfate proteoglycan (HSPG), AAVv66 contains S585 and T588 (identical to AAV1, AAV3, AAV5, and AAV6).

[0121] The generation of the AAVv66 vector and its cell-infectivity differ from those of AAV2. The strong affinity of AAV2 for heparin and the resulting strong cell surface association have been proposed to lead to the relatively low packaging titer of the virus. The limited vector yield by AAV2 is thought to be due to unproductive binding and reinfection of packaging cells by vector particles during production. The vector production and cell infectivity of AAVv66 were compared with those of AAV2 and AAV3b. Notably, AAV3b is a distinct homolog (89% sequence similarity) that is the closest to AAV2, but it binds weakly to heparin using a different electrostatic surface charge of its three-turn protrusions. This difference between AAV3b and AAV2 likely explains the increased packaging titer of AAV3b resulting from transduction of HEK-producing cells.

[0122] The packaging profile of AAVv66 was compared with that of AAV2 and AAV3b by measuring the yield of capsid-formed vector genomes in cell lysates. For this purpose, an ORF of the AAVv66 capsid was synthesized and cloned into a transplasmid (pAAV2 / v66) expressing AAV2 Rep under the AAV2 p5 promoter. Small vector preparations of AAVv66, AAV2, and AAV3b were used to package a single-stranded vector (AAV-CB6-Fluc) consisting of a firefly luciferase transgene driven by a ubiquitous chicken-beta-actin promoter. Quantification of viral vector yield by crude lysate qPCR29 revealed that the yield of capsid-formed DNase-resistant genomes from the AAVv66 vector was approximately 2.4 times higher than that of AAV2 and approximately 30% higher than that of AAV3b (Figure 9, "combination" sample).

[0123] Whether the high abundance of AAVv66 in the crude lysate was due to the unproductive binding of particles to packaging cells was revealed by the dominance of AAVv66 particles in the culture medium rather than the cell lysate fraction, and this was then investigated. PCR analysis revealed that the capsidized genome of AAVv66 in the culture medium was approximately three times more abundant than in the cell lysate (Figure 9). In contrast, AAV2 particles were hardly detected in the culture medium of the packaging cells. To test whether the ability of AAVv66 to generate more DNase-resistant genomes is related to the reduced reinfectivity of packaging cells due to poor HSPG binding, a heparin competition assay was performed (Figure 10). For this purpose, large AAVv66 and AAV2 vectors packaging CB6-Fluc again were generated using a standard cesium chloride purification protocol. Transduction of AAVv66 was unaffected by the presence of heparin, whereas 1.25 μg / well of heparin blocked 50% of AAV2 transduction, and 5 μg / well of heparin completely eliminated transduction. These results suggest that the improved production efficiency of AAVv66 is at least partially attributable to poor heparin binding.

[0124] To determine whether the low affinity of AAVv66 for heparin corresponds to reduced cell transduction compared to AAV2, HEK293 cells were infected with purified AAVv66, AAV2, and AAV3b vectors. The data show that AAV2 exhibits higher transduction than AAVv66 (approximately 65-fold) and AAV3b (approximately 7.5-fold) (Figure 11). While vectorized AAVv66 proviral capsid sequences can efficiently transduce cells in vitro, their vectorization and cell infectivity characteristics are distinct from those of its closest serotype relative, AAV2.

[0125] AAVv66 exhibits a CNS transduction that is distinct from that of AAV2. We tested AAVv66's ability to transform selected target tissues via different administration routes. For this purpose, the in vivo distribution of AAVv66 was evaluated in mice via multiple delivery routes (Figures 12A–13D). Of all the routes tested, the most prominent was the transduction profile of AAVv66 after intracranial delivery to target cells in the central nervous system (CNS) (Figures 2A–2D). To determine whether AAVv66 has increased tropism in the CNS compared to AAV2, we packaged Egfp transgenes driven by a ubiquitous chicken-beta-actin promoter into AAVv66 and AAV2 capsids. The vectors were injected unilaterally into the right hemisphere of the hippocampus at a dose of 3.6E9vg / animal. Four weeks after injection, processed frozen sections of brain tissue showed that AAV2 tended to remain localized at the injection site, while AAVv66 transduced approximately 13 times more CNS cells than AAV2, as evidenced by enhanced diffusion throughout the tissue (Figures 2A-2B). High-magnification imaging of the contralateral region relative to the injection site showed that all subanatomical regions of the brain (Ammon's horns [CA1, CA2, CA3, and CA4], dentate gyrus, and corpus callosum, Figure 2C) exhibited detectable levels of EGFP expression (Figure 2D), suggesting that AAVv66 may efficiently spread throughout the hippocampal hemisphere.

[0126] We investigated specific cell types transduced by AAVv66. Antibody staining was performed using cell type-specific markers: anti-NEUN (neurons), anti-GFAP (astrocytes), anti-IBA1 (microglia), and anti-OLIG2 (oligodendritic cells) (Figures 3A, 3E, 3I, and 3M). 3D volume reconstructions of dissected CNS regions demonstrated co-localization of EGFP expression with each cell type investigated (Figures 3B, 3J, 3F, and 3N). Neurons were the dominant cell type, found in the cortex and CA1 region (Figure 3C). Interestingly, the CA2-4 region and the dentate gyrus showed the highest transduction (approximately 20-40%). Astrocytes and microglia shared similar distribution patterns, showing the highest enrichment in the dentate gyrus (Figures 3G and 3K). Astrocytes showed approximately 1–7% transduction across all regions (Figure 3H), while microglia exhibited a slightly higher transduction efficiency (2–12%) (Figure 3L). Oligodendrocytes were enriched in the corpus callosum (Figure 3O) and underwent approximately 1–7% transduction by AAVv66 across all regions (Figure 3P). These data suggest that AAVv66 can transform all major cell types in the CNS after intrahippocampal injection.

[0127] AAVv66 is serologically distinct from AAV2. Neutralization of AAV by the host immune system is a major limiting factor in the transduction efficiency of AAV vectors. Individuals who possess pre-existing antibodies against AAV serotypes used as capsids in therapeutic vectors are at increased risk of adverse effects and ineffective treatment. Furthermore, patients requiring repeated administration of AAV gene therapy are at risk of lower transduction efficiency, stronger immune responses, and the need for alternative vectors.

[0128] We investigated the question of whether AAVv66 transduction can be blocked by prior immunization with AAV2. To create existing anti-AAV2 antibodies in circulation, AAV2-Egfp vectors (1E11vg / mouse) were delivered intramuscularly to mice. Serum was collected after 4 weeks, and neutralizing antibody (NAb) titers were evaluated in vitro (Figures 13A-13D and 14A-14B). Low NAb titers (1 / 1,280 to 1 / 2,560) were required to achieve 50% neutralization (NAb50) of AAV2 infection in Huh-7.5 cells, indicating that antibodies produced from prior immunization with AAV2 were sufficient to inhibit AAV2 transduction. In contrast, NAb50 for AAVv66 infection with serum from AAV2-treated mice was 1 / 20 to 1 / 40, indicating that AAVv66 can infect cells despite the presence of NAb produced against AAV2.

[0129] To test these findings regarding the products of therapeutic transgenes secreted in vivo, we readmitted AAV2-immunized mice with AAV2 or AAVv66 (AAV2-A1AT or AAVv66-A1AT) packaging the alpha-1 antitrypsin transgene. Serum was collected at weeks 5, 6, 7, and 8, and secreted A1AT levels were quantified by ELISA31 (Figure 14C). Low A1AT expression would suggest that NAb generated from the first vector administration interfered with transduction from the second vector administration. Naive mice were treated in the same manner to establish a baseline for "maximum" A1AT expression. At weeks 6 and 7, A1AT expression in mice treated with AAV2-Egfp and then AAVv66-A1AT reached approximately 90% of naive levels, while mice re-administered AAV2-A1AT only reached approximately 40% of naive levels (Figure 14C). These results are consistent with the in vitro observation of robust infectivity by AAVv66 in the presence of serum from mice pre-immunized with AAV2 capsid.

[0130] Furthermore, pre-immunity was also examined to investigate whether a wide range of AAV serotypes (AAV1, AAV2, AAV3b, AAV8, AAV9, AAV-DJ, AAVrh.8, and AAVrh.10) could impair transduction of the AAVv66 vector. Antisera from rabbits pre-immunized separately with eight different serotypes were screened for neutralization of the AAVv66 vector. AAV1, AAV3b, and AAV-DJ showed approximately one-order-of-magnitude differences in NAb50 titers compared to AAVv66, while AAV2, AAV8, AAVrh.8, and AAVrh.10 showed two-order-of-magnitude differences, and AAV9 showed a three-order-of-magnitude difference (Figure 14D). In summary, these data indicate that AAVv66 is serologically distinct from AAV2 and several other modern AAV capsids.

[0131] AAVv66 capsids have higher thermal stability than AAV2 across a certain pH range. Efficient capsid formation and structural stability are essential for the production, purification, and storage of viral vectors. Furthermore, for productive infection to occur, the vector particle must maintain stability throughout the entry process and uncoating only under conditions where the delivery of the genomic payload can lead to cellular transduction. While AAV vectors have been widely studied and their potent transduction profiles have been utilized in a range of tissues, the processes of intracellular transport, endosomal exodus, and capsid transport to the nucleus are not fully understood. Of the presumed intracellular checkpoints that influence AAV intracellular transport and transduction, which depend on capsid dynamics, endosomal exodus is the best understood. This process is thought to be triggered by pH-dependent structural changes in the capsid. Acidification of the endosomal lumen leads to conformational changes in the VP1 domain and exposure of the PLA2 domain within VP1, which induces exodus from the endosomal compartment. In principle, a vector capsid that can maintain stability throughout intracellular transport is desirable and can exhibit high transduction potential.

[0132] To determine the overall stability of the AAVv66 capsid, differential scanning fluorescence (DSF) analysis was used to measure the thermal stability of the AAVv66 capsid over a range of physiological pH values ​​(pH 7 to pH 4) (Figure 4). This range includes pH 4.5, which is observed in the lumen of late endosomes and lysosomes. In this assay, vector particles are suspended in a SYPRO Orange dye that fluoresces upon binding to hydrophobic residues of the protein. Therefore, the peak of the fluorescence signal is an indirect readout of the maximally bound hydrophobic region exposed during protein unfolding. The melting temperature of AAVv66 (maximum slope [Dsignal / Dtemp], Tm) was more than 5 degrees higher than that for AAV2 across all pH conditions tested. The most extreme difference was observed at pH 7, where the Tm of AAVv66 (75.29±0.34℃) was almost 10 degrees higher than that of AAV2 (65.85±0.18℃) (Figure 4A). Therefore, the AAVv66 capsid is more thermally stable and tolerant of pH than AAV2.

[0133] The effect of AAVv66 capsid stability on vector genome release was investigated. Vector genome release as a function of temperature range was used as a proxy for DNA efflux driven by pressure exerted by the nucleolar environment. The temperature dependence of AAVv66 genome release was compared with that of AAV2 at different pH levels. For this purpose, DSF analysis with SYBR Gold dye, which fluoresces upon binding to DNA, was used. The fluorescence peak is an indirect measure of the maximum accessibility of the capsid-formed genome to the dye solution. Vector genome release at pH 7 was observed in association with capsid stability, with signal peaks at approximately 65°C for AAV2 and approximately 74°C for AAVv66. However, at lower pH levels, peak fluorescence of dye-accessible DNA was detected at lower temperatures than peak fluorescence of unfolded capsid protein (Figure 4B). Furthermore, DNA accessibility for AAVv66 was more pronounced than that for AAV2, with peaks in DNA accessibility for AAV2 occurring at approximately 53°C and 42°C, respectively, while AAVv66 showed a peak signal at 25°C. This surprising observation suggests that the DNA in the AAVv66 capsid is particularly accessible at low pH (4–5) even at room temperature.

[0134] Next, we investigated the question of whether AAVv66-specific amino acid residues contribute to the structural and functional differences observed between AAVv66 and AAV2. Thirteen amino acid residues defining AAVv66 were mutated to those of AAV2, and their effects on vector genome packaging during production in HEK293 cells were tested (Figure 4C). Furthermore, thermal capsid stability and vector genome release were evaluated for the mutant capsids (Figures 4D and 4E). All but four mutations (A151V, K447R, Y533F, and S585R) resulted in lower yields of DNase-resistant genomes, similar to or lower than those of AAV2 (Figure 4C). Surprisingly, the relatively conserved mutation D499E, which does not involve a change in charge, reduced the packaging yield to approximately 5% of the AAV2 yield. Modification D499E, along with the S585R and S585R / T588R double mutations, reduced Tm by 5.9°C, 3.8°C, and 5.4°C, respectively, thus affecting capsid stability (Figure 4D), while other mutations only affected Tm by 1–2°C. Vector genome accessibility with the same amino acid mutations exhibited reduced peak signal temperature, while other mutations had little or no change (Figure 4E). Notably, the overall titer of the purified vector was not dramatically affected (Table 3). Therefore, it is suggested that the packaging yield of AAVv66 depends only partially on capsid stability, and that partial capsid destabilization may be sufficient to promote genome release. Only residue D499 dramatically affects both packaging and capsid stability. [Table 3]

[0135] Cryo-EM structural analysis of capsid differences between AAVv66 and AAV2 To characterize the structural properties of AAVv66, the AAV2v66-Egfp vector was purified for cryo-EM analysis. 52,874 particle images were obtained, and cryo-EM maps with a resolution of 2.5 Å were acquired (Figures 5E and 16) to obtain a structural model with optimal real-space fit and stereochemical parameters (Table 2). Overall, the structure of AAVv66 is similar to that of AAV2 (mean squared deviation (RMSD) of atomic coordinates = 0.456 Å) (Figure 16). Thus, AAVv66 exhibits characteristic properties of an AAV capsid, including a 2-rotational recess, 3-rotational projections defining 3-fold symmetry, and 5-rotational pores composed of 5 monomers forming interfaces and pores for Rep bonding (Figure 5A). Notably, the VP1u and VP2 domains were represented by approximately 1 / 12th of the VP3 domain for each particle, and, as with other previous AAV structures, were not elucidated in our symmetrized cryo-EM maps. Therefore, only residues 219–736, which include 11 of the 13 residues defining AAVv66, are definitively identified within the cryo-EM map (Figure 5B).

[0136] By comparing the structure of AAVv66 with that of AAV2, several structural differences that may contribute to improved DNA packaging and / or capsid stability are revealed. The key difference lies in the VP3 monomer-interface of the protrusion around the three rotational axes. Mutations to longer glutamate residues resulted in dramatic defects in vector genome packaging (Figure 4C). D499 forms electrostatic interactions and / or hydrogen bonds with S501 (Figure 5D). This region is densely packed with adjacent VP3 monomers (Figure 5D). Here, the skeletal atoms of D499 and S501 interact with the symmetry-related side chains N449 and T448, respectively, while the hydroxyl group of S501 hydrogen bonds with the symmetry-related carbonyl of the S446 skeleton. Therefore, the strong effect of the D499 mutation likely stems from the disruption of the VP3 monomer-interface, leading to capsid destabilization. In the same region, the adjacent monomer residues K447 and A450 rule out the possibility of electrostatic interactions between the corresponding AAV2-R447 and T450 side chains (Figure 6D). The amino acid M457 is located in the three-turn projection of AAVv66 in variable region IV, and the side chain is in equilibrium with respect to the solvent (Figure 6E). Interestingly, this methionine is a unique feature among other serotypes (Figure 8), suggesting the possibility of unique capsid interactions with cell receptors, host factors, or antibodies. The polar hydroxyl group of AAVv66-Y533 (AAV2-F533) may stabilize the polar environment between the R487 and K532 side chains, potentially contributing to interactions with the symmetry-related monomer L583 (Figure 6A). AAVv66-D546 and G548 redistribute the surface charge imparted by AAV2-G546 and E548 (Figure 6B), and are yet another feature that defines AAVv66.

[0137] The critical functional region of AAV2 contains positively charged arginine residues at positions 585 and 588. These residues are located on the surface of the tri-turn projection and govern the interaction between the HSPG receptor and the capsid, which is essential for adhesion and entry in many cell types. In contrast, S585 and T588 of the AAVv66 capsid are neutrally charged residues (Figures 5D and 6), similar to S586 and T589 of AAV3b (Figure 8). The physical and functional interaction of AAV3b with HSPG relies on electrostatic interactions provided by residues R447 and R594 (R447 and R593 in AAV2), whereas AAVv66 also lacks these arginine residues (K447 and T593). These differences from AAV2 and AAV3b are consistent with our finding that AAVv66 lacks heparin binding, suggesting that it associates differently from the standard cell surface receptors typically utilized by AAV clades B and C capsids.

[0138] AAV2 and AAVv66 show differences in surface charge. Since the electrostatic properties of viruses are important for capsid-receptor interactions,7,43 we investigated how the net loss of positive charge in the AAV2 and related AAVv66 capsids affects the electrostatic properties of the capsids. First, we compared the calculated electrostatic potential values ​​for the structures of AAV2 and AAVv66 (Figure 7A). The distribution of electrostatic potential on the surface of AAVv66 differs from that of AAV2. The most significant difference is at the three-turn projection, where the positive charge imparted by R585 and R588 in AAV2 is dramatically reduced by S585 and T588 in AAVv66 (Figure 7B).

[0139] Next, we investigated whether the distinct structure and surface electrostatics of AAVv66 affect the charge-dependent particle migration (zeta potential) of the capsid (Figure 7C). The zeta potential of AAVv66 (-10mV) differs significantly from that of AAV2 (-3.5mV), which is consistent with the difference in electrostatic potential between capsids. To test the contribution of individual substitutions, we measured the particle migration of AAVv66 possessing single amino acid substitutions that convert residues to corresponding residues in AAV2 (Figure 7C). Single mutations S585R and T588R resulted in the most dramatic changes in zeta potential (only about 3mV each), bringing the zeta potential closer to that of AAV2 (Figure 7C). These observations indicate that the electrostatic properties of AAVv66 differ from those of AAV2, and that this difference is mainly due to substitutions at positions 585 and 588. Therefore, the interactions of the AAVv66 capsid with receptors, antibodies, and other proteins are likely substantially different from those of other closely related capsids.

[0140] [ka] [ka]

Claims

[Claim 1] The invention described in the specification.

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