AAV composition with high brain expression levels

Abstract

Described herein are compositions and kits that contain recombinant adeno-associated viruses (rAAVs) that enhance viral transduction in the CNS. Described herein are rAAV compositions that encapsidate transgenes, such as therapeutic nucleic acids. Described herein are gene therapies using rAAVs. Described herein are methods for treating CNS-related diseases and conditions. The rAAVs undergo repeated rounds of selection in non-human primates (NHPs) to produce variants that have enhanced transduction as measured in the CNS, with engineered transduction entering into the capsid structure.

Description

[Background technology]

[0001] background Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications due to their ability to transduce both dividing and non-dividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These features make them attractive for use in therapeutic applications such as gene therapy. However, there is a need to greatly improve the performance of existing AAV serotypes to be selectively and efficiently expressed in different cell types immediately after systemic delivery to a subject. This need is especially urgent when AAVs must be expressed within the central nervous system (CNS). Summary of the Invention [Means for solving the problem]

[0002] Summary of the Invention Disclosed herein are rAAVs in which repeated rounds of selection in non-human primates (NHPs) result in engineered transduction variants that reside within a capsid structure and thus increase transduction as measured within the CNS.

[0003] The present invention provides rAAV with broad transduction capabilities to the CNS.

[0004] The present invention provides, in one aspect, a peptide insert sequence comprising or consisting of an amino acid sequence set out in any one of Table 1, Figures 1-3, and / or formula I.

[0005] Another aspect of the present invention is an AAV capsid protein having a peptide insert comprising or consisting of an amino acid sequence set forth in any one of Table 1, Figures 1-3, and / or Formula I, characterized by increased CNS transduction in a subject.

[0006] The disclosure further includes a pharmaceutical composition comprising an rAAV having a peptide insert comprising or consisting of an amino acid sequence set forth in any one of Table 1, Figures 1-3, and / or Formula I, and a pharma- ceutical composition comprising a pharma- ceutical acceptable excipient.

[0007] The embodiments disclosed herein provide a method for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of a pharmaceutical preparation comprising the AAV capsid protein or AAV capsid of the present disclosure.In some embodiments, the disease or condition is a disease or condition of the CNS and brain of the subject.Relatedly, the present invention includes the use of rAAV in the manufacture of a medicament for treating or preventing a disease or medical condition.

[0008] Other aspects of the invention will become apparent from the following detailed description and claims.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0010] [Figure 1-1] 1 shows AAV capsid protein inserts and replacement amino acid sequences that encode amino acid sequences found in the non-human primate CNS after two rounds of evaluation of the engineered AAV library. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. [Figure 1-10] Same as above. [Figure 1-11] Same as above. [Figure 1-12] Same as above. [Figure 1-13] Same as above. [Figure 1-14] Same as above.

[0011] [Figure 2-1] The DNA sequences encoding the AAV capsid protein insert and replacement amino acid sequences, as well as the amino acid sequences found in certain non-human primate CNS, are shown. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0012] [Figure 3-1] The DNA sequences encoding the AAV capsid protein insert and replacement amino acid sequences, as well as amino acid sequences found in other non-human primate CNSs, are shown. [Figure 3-2] Same as above. [Figure 3-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Detailed Description of the Disclosure In one aspect, the present disclosure provides an rAAV that has high expression levels in the CNS.

[0014] In one aspect, the disclosure provides an rAAV having a peptide insertion and / or substitution comprising or consisting of an amino acid sequence set forth in any one of Table 1, Figures 1-3, and / or Formula I.

[0015] The embodiments disclosed herein include an amino acid sequence of formula I X 1 -X 2 -X 3 -NTTX 4 -X 5 -X 6 -X 7 -X8 (I) (SEQ ID NO: 72) (In the formula, X 1 is an amino acid selected from A, E, Q, T, and V; X 2 is an amino acid selected from Q, I, M, A, P, and V; X 3 is an amino acid selected from L, S, Q, M, and T; X 4 is an amino acid selected from K and R; X 5 is an amino acid selected from P, I, N, A, Q, H, I, V, S, and L; X 6 is an amino acid selected from T, I, V, A, Q, S, L, M, G, H, and R; X 7 is an amino acid selected from A, D, N, S, T, M, P, Q, E, G, V, I, and W; X 8 is an amino acid selected from Q, G, F, A, S, D, E, M, P, R, T, and Y.

[0016] In some embodiments, the AAV capsid protein is X 1 is A and X 2 is Q.

[0017] In some embodiments, the AAV capsid protein is X 3 is L.

[0018] In some embodiments, the AAV capsid protein is X 3 is N.

[0019] In some embodiments, the AAV capsid protein is X 4 is K.

[0020] In some embodiments, the AAV capsid protein is X 5 is P or S.

[0021] In some embodiments, the AAV capsid protein is X 6 is V or T.

[0022] In some embodiments, the sequence set forth in Table 1 is selected from LNTTKSV (SEQ ID NO:41), LNTTKPT (SEQ ID NO:42), LNTTKPS (SEQ ID NO:43), LNTTKPV (SEQ ID NO:44), LNTTKNI (SEQ ID NO:45), LNTTKPI (SEQ ID NO:46), NNTTKPI (SEQ ID NO:47), LNTTKNM (SEQ ID NO:48), LNTTKPR (SEQ ID NO:49), LNTTKNV (SEQ ID NO:50), LNTTKQM (SEQ ID NO:51), LNTTKPQ (SEQ ID NO:52), LNTTKIT (SEQ ID NO:53), LNTTKLH (SEQ ID NO:54), LNTTKNG (SEQ ID NO:55), VNTTKPI (SEQ ID NO:56), MNTTKPI (SEQ ID NO:57), LNTTKPL (SEQ ID NO:58), TNTTKPI (SEQ ID NO:59), LNTTKSF (SEQ ID NO:60), and LALPKPI (SEQ ID NO:114).

[0023] In some embodiments, the sequences set forth in Table 1 are AQLNTTKSVMQ (SEQ ID NO:2), AQLNTTKPTDR (SEQ ID NO:3), AQLNTTKPTVG (SEQ ID NO:4), AQLNTTKPSPG (SEQ ID NO:5), AQLNTTKPTGP (SEQ ID NO:6), AQLNTTKPTAG (SEQ ID NO:7), AQLNTTKPVAG (SEQ ID NO:8), AQLNTTKNITQ (SEQ ID NO:9), AQLNTTKPTSF (SEQ ID NO:10), AQLNTTKPTPS (SEQ ID NO:11), AQLNTTKPTGP (SEQ ID NO:12), AQLNTTKPTAG (SEQ ID NO:13), AQLNTTKPVAG (SEQ ID NO:14), AQLNTTKNITQ (SEQ ID NO:15), AQLNTTKPTSF (SEQ ID NO:16), AQLNTTKPTPS (SEQ ID NO:17), AQLNTTKPTGP (SEQ ID NO:18), AQLNTTKPTGP (SEQ ID NO:19), AQLNTTKPTGP (SEQ ID NO:20), AQLNTTKPTGP (SEQ ID NO:21), AQLNTTKPTGP (SEQ ID NO:22), AQLNTTKPTGP (SEQ ID NO:23), AQLNTTKPTGP (SEQ ID NO:24), AQLNTTKPTGP (SEQ ID NO:25), AQLNTTKPTGP (SEQ ID NO:26), AQLNTTKPTGP (SEQ ID NO:27), AQLNTTKPTGP (SEQ ID NO:28), AQLNTTKPTGP (SEQ ID NO:29), AQLNTTKPTGP (SEQ ID NO:30), AQLNTTKPTGP (SEQ No. 11), MANNTTKPIAQ (SEQ ID NO: 12), AQLNTTKPIAQ (SEQ ID NO: 13), AQLNTTKPTSY (SEQ ID NO: 14), AQLNTTKNMTQ (SEQ ID NO: 15), AQLNTTKPRDE (SEQ ID NO: 16), AQLNTTKPTAQ (SEQ ID NO: 17), AQLNTTKNVTQ (SEQ ID NO: 18), QTNNTTKPIAQ (SEQ ID NO:), AQLNTTKQMNQ (SEQ ID NO: 19), AQLNTTKHVDQ (SEQ ID NO: 20), AQLNTTKQI DQ (SEQ ID NO: 21), VALNTTKPIAQ (SEQ ID NO: 22), AMTNTTKPIAQ (SEQ ID NO: 23), AQLNTTKPANQ (SEQ ID NO: 24), AQLNTTKNVNQ (SEQ ID NO: 25), AQLNTTKPVVA (SEQ ID NO: 26), AQLNTTKPQNQ (SEQ ID NO: 27), AQLNTTKPQLQ (SEQ ID NO: 28), AQLNTTKITDQ (SEQ ID NO: 29), AQLNTTKLHDQ (SEQ ID NO: 30), AQLNTTKNGYQ (SEQ ID NO: 31), ANVNTTKPIAQ (SEQ ID NO: 32), EQMNTTKPIAQ (SEQ ID NO: 33), AQLNTTKPLQF (SEQ ID NO: 34), AQLNTTKPSSG (SEQ ID NO: 35), AQLNTTKPVMQ (SEQ ID NO: 36), AQLNTTKPLAQ (SEQ ID NO: 37), TITNTTKPIAQ (SEQ ID NO: 38), AQLNTTKSFGQ (SEQ ID NO: 39), AQLNTTKPTAQ (SEQ ID NO: 116), and AQLNTTKPTTS (SEQ ID NO: 117).

[0024] In some embodiments, the insert sequence is represented by a peptide sequence listed in Table 1. [Table 1-1] [Table 1-2]

[0025] In some embodiments, the inserted amino acid sequence is at least 71.4% identical to the amino acid sequence set forth in Table 1, Figures 1-3, and / or Formula I. In some embodiments, the inserted amino acid sequence is at least 86.7% identical to the amino acid sequence set forth in Table 1, Figures 1-3, and / or Formula I.

[0026] Also disclosed herein are methods and kits for generating therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising the rAAV particles, for treating diseases or conditions that affect the CNS.

[0027] Disclosed herein is an engineered AAV capsid that enhances viral transduction in CNS.AAV capsid can encapsidate viral vector with heterologous nucleic acid, for example, encoding therapeutic gene expression product.Transduction of heterologous nucleic acid in CNS can be achieved immediately after systemic delivery of the disclosed AAV capsid that encapsidates heterologous nucleic acid to a subject.The AAV capsid disclosed herein is useful for many applications of gene therapy for treating human diseases, including but not limited to disorders of the central nervous system.

[0028] Also provided herein is a recombinant AAV vector comprising the nucleic acid sequence encoding the AAV capsid protein of the present disclosure.For example, the viral vector of the present disclosure comprises the nucleic acid sequence encoding VP1, VP2 and VP3, and at least one of them is modified to generate the AAV capsid protein of the present disclosure.The recombinant AAV vector provided can be derived from an AAV serotype (e.g., AAV9) or a variant AAV serotype comprising the insert of the present invention. AAV Capsid

[0029] Provided herein are modified adeno-associated (AAV) viral capsid compositions useful for incorporating a transgene into a target cell or target environment (in a subject when administered systemically to the subject).

[0030] rAAV contains an AAV capsid that can be engineered to encapsidate heterologous nucleic acids (e.g., therapeutic nucleic acids, gene editing machinery). The AAV capsid is composed of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid. VP1 covers the entirety of the VP2 protein in addition to an N-terminal region of about 137 amino acids (VP1u), and VP2 covers the entirety of VP3 in addition to an N-terminal region of about 65 amino acids (VP1 / 2 common region). These three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0031] Without wishing to be bound by theory, it is understood that the parent AAV capsid sequence comprises VP1 region.In certain embodiments, the parent AAV capsid sequence comprises VP1, VP2 and / or VP3 region, or any combination thereof.The parent VP1 sequence can be considered as synonymous with the parent AAV capsid sequence.

[0032] The AAV VP3 structure contains a highly conserved region common to all serotypes, a core eight-stranded β-barrel motif (βB-βI), and a small α-helix (αA). The loop regions inserted between the β-strands consist of the unique HI loop between β-strands H and I, the DE loop between β-strands D and E, and nine variable regions (VRs) that form the top of the loops. These VRs, such as the AA588 loop, are found on the capsid surface and may be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity.

[0033] In some embodiments, the rAAV variants of the invention comprise an AAV capsid protein having a peptide insertion at residues corresponding to amino acids 588-589 of the AAV9 native sequence of SEQ ID NO:1.

[0034] AAV capsids include AAV capsid proteins (e.g., VP1, VP2, and VP3), each of which has an insertion such as in the 588 loop of the parent AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains the heparan sulfate binding site of AAV2 and is suitable for peptide display. The only known receptor for AAV9 is the N-linked terminal galactose and AAV receptor (AAVR), but many indications indicate that there are others. Modifications to the AAV9 588 loop that confer increased transgene transduction in the target in vivo environment are presented herein.

[0035] The present invention, in one aspect, provides a peptide insert in the AAV 588 loop comprising or consisting of an amino acid sequence set forth in any one of Table 1, Figures 1-3, and / or Formula I.

[0036] Disclosed herein is an AAV capsid comprising an AAV capsid protein with an insert at the 588 loop, which confers higher transduction in CNS cell types (e.g., brain endothelial cells, neurons, astrocytes).In particular, the AAV capsid protein disclosed herein allows rAAV-mediated transduction of heterologous nucleic acid (e.g., transgene) in the CNS of a subject.The AAV capsid of the present disclosure can be formulated as a pharmaceutical composition.In addition, the AAV capsid can be isolated and purified to be used for various applications.

[0037] In some embodiments, the rAAV capsid of the present disclosure is produced using the method disclosed herein.In some cases, the rAAV capsid is chimeric.In some cases, the rAAV or variant AAV protein is contained within the rAAV capsid, and compared to the parent AAV capsid or capsid protein, rAAV localization is increased in target tissue. AAV Capsid Protein

[0038] Disclosed herein is a recombinant AAV (rAAV) capsid comprising an engineered AAV capsid protein having modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid protein of the present disclosure is produced using the methods disclosed herein. In some embodiments, the AAV capsid protein is used in a method for delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some cases, the rAAV capsid protein has a desired AAV expression, making it particularly suitable for a particular therapeutic use, such as the treatment of a disease or disorder in a subject, such as those disclosed herein.

[0039] The rAAV capsid protein is engineered for optimized expression in the CNS, for example, the brain, of a subject immediately after systemic administration of rAAV to the subject. The rAAV capsid protein is engineered to contain the inserts described in Table 1 and Figure 1. The rAAV capsid protein containing the inserts described in Table 1 and Figure 1 is engineered to achieve efficient transduction of encapsidated transgenes. In particular, the rAAV capsid protein is enhanced in the brain of the subject.

[0040] The engineered AAV capsid proteins described herein optionally have an insertion of an amino acid heterologous to the parent AAV capsid protein at an amino acid position within the 588 loop. In some embodiments, the amino acid is not endogenous to the parent AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid at the same or equivalent amino acid position as the replacement insertion in the different AAV capsid protein.

[0041] Generally, the inserts comprise 5, 6, or 7 amino acid sequences (5-mer, 6-mer, or 7-mer, respectively) that are inserted or substituted at the 588 loop in the parent AAV capsid protein. The embodiments provided herein provide amino acid inserts that comprise a 7 amino acid polymer (7-mer) inserted at AA588-589, and may additionally comprise substitutions of one or two amino acids at the amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and / or AA589-590) to generate an 11 amino acid polymer (11-mer) at the 588 loop of the parent AAV capsid protein. The 7-mers described herein are advantageously generated using polymerase chain reaction (PCR) with degenerate primers, each of the 7 amino acids being encoded by the deoxyribose nucleic acid (DNA) sequence NNK. "N" is any of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method of generating random heptamer amino acid sequences allows for 1.28 billion possible combinations at the protein level.

[0042] The rAAV capsid proteins of the present disclosure contain an insertion of an amino acid within the amino acid sequence of the AAV capsid protein. The AAV capsid from which the engineered AAV capsid proteins of the present disclosure are generated is referred to as the "parent" AAV capsid. The complete genome of AAV-1 is provided under GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829; the AAV-5 genome is provided under GenBank Accession No. AF085716; the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862; AAV-7 and and at least a portion of the AAV-8 genome are provided in GenBank accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004); a portion of the AAV-12 genome is provided in Genbank accession number DQ813647; and a portion of the AAV-13 genome is provided in Genbank accession number EU285562.

[0043] In some cases, the parent AAV is derived from an AAV having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The AAV capsid protein "derived" from another AAV capsid protein may be a variant AAV capsid protein. The variant may, for example, include a heterologous amino acid within the amino acid sequence of the AAV capsid protein. The heterologous amino acid may be one that does not naturally occur within the AAV capsid protein. The heterologous amino acid may be one that naturally occurs within a different AAV capsid protein. In some cases, the parent AAV capsid is described in U.S. Patent Application Publication No. 2020 / 0165576 and U.S. Patent Application No. 62 / 832,826 and International Application No. PCT / US20 / 20778; the contents of each of which are incorporated herein.

[0044] In some cases, the parent AAV is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of the AAV9 VP1 capsid protein (>tr|Q6JC40|Q6JC40_9VIRU capsid protein VP1 OS=adeno-associated virus 9 OX=235455 GN=cap PE=1 SV=1) is set forth in SEQ ID NO: 1. [ka] [ka] In some cases, the parent AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:1.

[0045] AAV capsid proteins from natural AAV serotypes, such as AAV9, which have a tropism that includes the liver, activate the innate immune response, which in some cases can cause a severe inflammatory response in a subject, which can lead to multiple organ failure. By improving the transduction of natural AAV serotypes to target in vivo tissues (e.g., the brain), the rAAV particles of the present disclosure reduce the immunogenicity of AAV-mediated transgene delivery and prevent the activation of the innate immune response.

[0046] In some cases, the parent AAV capsid protein includes the entire VP1 region set forth in SEQ ID NO:1 (e.g., amino acids 1-736). In some cases, the parent AAV capsid protein includes amino acids 217-736 in SEQ ID NO:1, which is a common region found in the VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein includes amino acids 64-736 in SEQ ID NO:1, which is a common region found in VP1 and VP2. The parent AAV capsid protein sequences are 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200-736, 210-736, 220-736, 230-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, 450-736, 460-736, 470-736, 480-736, 490-736, 500-736, 510-736, 520-736, 530-736, 540-736, 550-736, 560-736 736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, and 450-736. In some embodiments, the rAAV variant comprises an AAV capsid protein comprising an amino acid sequence at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO:1. In some cases, the amino acid insertion is at the three-fold axis of symmetry of the corresponding parent AAV capsid protein.

[0047] Disclosed herein are insertions of amino acid sequences within AAV capsid proteins. Where the sequence numbering designation "588-589" is noted for AAV9, e.g., AAV VP1, the invention also includes insertions within similar positions in other AAV serotypes. As used herein, "AA588-589" indicates that an insertion of an amino acid (or amino acid sequence) occurs immediately after the amino acid (AA) at position 588 and immediately before the AA at position 589 within the amino acid sequence of the parent AAV VP capsid protein (VP1 numbering). Amino acids 587-591 contain the motif "AQAQA" as shown in SEQ ID NO:1. Exemplary AAV capsid protein sequences are shown in Table 2. For example, LNTTKPT (SEQ ID NO:42) is inserted at AA588-589 within the AAV9 capsid amino acid sequence to provide variant A (SEQ ID NO:66). It is contemplated that the sequences disclosed herein (Table 1, Figures 1-3, and / or Formula I) may be inserted at AA588-589 or AA587-590 (replacing amino acids AA587-590) within the amino acid sequence of a parent AAV9 capsid protein, a variant thereof, or the equivalent amino acid position of a parent AAV of a different serotype (e.g., AAV1, AAV2, AAV3, etc.). In any AAV capsid protein sequence disclosed herein, the amino acid at position 449 may be R or K. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0048] The inserts described herein may optionally include a 7-mer insert at AA 588-589. It is contemplated that any 7-mer insert disclosed herein may include an 11-mer in addition to any amino acid substitution at amino acid positions 587-590 [AQAQ].

[0049] Disclosed herein is an AAV capsid protein having the above-mentioned insert in parent AAV capsid protein, which confers increased transduction in CNS in a subject even when delivered systemically.One of the many advantages of the AAV capsid protein described herein is its ability to target tissues and cells in CNS.Tissue can be brain.Non-limiting examples of CNS cells include neurons and glial cells.Glial cells can be selected from oligodendrocytes, ependymal cells, astrocytes, and microglia.

[0050] In some cases, the AAV capsid protein comprises an insertion of at least or about 5, 6, or 7 amino acids of the amino acid sequence of Table 1, Figures 1-3, and Formula I at amino acid positions 588-589 or 587-590 in the parent AAV9 capsid protein (SEQ ID NO:1). In some cases, the AAV capsid protein has high viral transduction efficiency in the brain.

[0051] The rAAV capsid proteins of the present disclosure may also have an amino acid sequence substitution at amino acid positions 452-458 in the parent AAV9 capsid protein or variant thereof, as described in WO 2020 / 068990. In some embodiments, the amino acid sequence substitution comprises KDNTPGR (SEQ ID NO: 73) at amino acid positions 452-458 in the parent AAV9 capsid protein. In some embodiments, the amino acid sequence substitution comprises DGAATKN (SEQ ID NO: 74) at amino acid positions 452-458 in the parent AAV9 capsid protein.

[0052] The rAAV capsid protein described herein can be isolated and purified. AAV can be isolated and purified by standard methods in the art, such as column chromatography, iodixanol gradient, or cesium chloride gradient. Methods for purifying AAV from helper virus are known in the art, and can include, for example, the methods disclosed in Clark et al., Hum.Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol.Med., 69:427-443 (2002); U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0053] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, can be formulated into pharmaceutical preparations, optionally further comprising a pharma- ceutically acceptable carrier.

[0054] rAAV capsid protein can be conjugated to nanoparticle, second molecule, or viral capsid protein.In some cases, nanoparticle or viral capsid protein encapsidates therapeutic nucleic acid as described herein.In some cases, second molecule is therapeutic agent, such as small molecule, antibody, antigen-binding fragment, peptide, or protein, such as those described herein.

[0055] "Percent identity" is the percent of characters that actually match. Percent similarity is the percent of characters that are similar. Characters across a gap are ignored. Similarity is scored if the score matrix value for a pair of characters is equal to or greater than the similarity threshold of 0.50. The scoring matrix used in version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0056] The sequence identity / similarity values ​​provided herein may refer to values ​​obtained using the BLAST+ 2.5.0 suite of programs (blast.ncbi.nlm.nih.gov) using default settings (Camacho, C. et al. (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0057] As those skilled in the art will appreciate, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequences, which may be homopolymer tracts, short period repeats, or regions enriched with one or more amino acids. Such low-complexity regions can be aligned between unrelated proteins, even if other regions of the proteins are completely different. Several low-complexity filter programs can be used to reduce such low-complexity alignments. For example, SEG (Wooten and Federhen (1993) Comput. Chem. 17:149-63) and XNU (Ci-ayerie and States (1993) Comput. Chem. 17:191-201) low-complexity filters can be used alone or in combination.

[0058] The terms "substantial identity" and "substantially identical" refer to a polypeptide or nucleic acid comprising a sequence having 55-100% sequence identity to a reference sequence, at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity, or any percentage value within the range of 55-100% sequence identity to a reference sequence. The sequence identity percentage may occur over a specified comparison window. Optimal alignment may be confirmed or performed using the homology alignment algorithm of Needleman and Wunsch, supra.

[0059] For example, insertion sequences can include, but are not limited to, sequences that are not exactly the same as the sequences disclosed herein, but have additional substitutions of amino acid residues in addition to the substitutions explicitly described for the various sequences recited herein, which do not substantially impair the activity or properties of the sequences described herein, e.g., as predicted by homology software, e.g., the BLOSUM62 matrix. AAV particles

[0060] rAAV particles having an insertion sequence described herein have increased transduction in the CNS. In some cases, the increased transduction includes 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold or more. In some cases, the increased transduction is at least 2-fold. In some cases, the increased transduction is at least 4-fold. In some cases, the increased transduction is at least 8-fold.

[0061] The rAAV particles having the insert sequence described herein have increased expression in CNS.Detecting whether rAAV has more or less expression includes measuring the level of gene expression product (e.g., RNA or protein) expressed from the heterologous nucleic acid encapsidated by rAAV in tissue samples obtained from subject.Methods suitable for measuring the expression of gene expression product include next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0062] The increased expression within the CNS is represented by the cpm values ​​shown in FIG. heterologous nucleic acid

[0063] Disclosed herein are therapeutic nucleic acids useful for treating or preventing disease or symptoms, or symptoms of disease or symptoms. In some embodiments, the therapeutic nucleic acid encodes a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.), and gene editing components that are used to treat, prevent, and / or improve disease or disorder or symptoms of disease or disorder. In some cases, the therapeutic nucleic acid is placed in the subject's organism, cell, tissue, or organ by rAAV such as those disclosed herein.

[0064] Disclosed herein are rAAVs, each comprising a viral vector (e.g., a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector comprises two inverted terminal repeat (ITR) sequences, each of about 145 bases, flanking a transgene. In some embodiments, the transgene comprises a promoter in cis with a therapeutic nucleic acid, in some cases, a therapeutic nucleic acid in an open reading frame (ORF). The promoter can initiate transcription of the therapeutic nucleic acid in the nucleus of a target cell. The ITR sequence can be derived from any AAV serotype. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some cases, the ITR is derived from AAV2. In some cases, the ITR is derived from AAV9.

[0065] Disclosed herein are transgenes that may comprise any number of nucleotides. In some cases, the transgene may comprise less than about 100 nucleotides. In some cases, the transgene may comprise at least about 100 nucleotides. In some cases, the transgene may comprise at least about 200 nucleotides. In some cases, the transgene may comprise at least about 300 nucleotides. In some cases, the transgene may comprise at least about 400 nucleotides. In some cases, the transgene may comprise at least about 500 nucleotides. In some cases, the transgene may comprise at least about 1000 nucleotides. In some cases, the transgene may comprise at least about 5000 nucleotides. In some cases, the transgene may comprise more than 5,000 nucleotides. In some cases, the transgene may comprise from about 500 to about 5000 nucleotides. In some cases, the transgene comprises about 5000 nucleotides. In any of the cases disclosed herein, the transgene may comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene may be single stranded. In some cases, the transgene may be double-stranded.

[0066] Disclosed herein is a transgene that is useful for regulating the expression or activity of target gene or its gene expression product.In some cases, the transgene is encapsidated by the rAAV capsid protein of the rAAV particle described herein.In some cases, the rAAV particle is delivered to a subject to treat the disease or symptoms disclosed herein in the subject.In some cases, the delivery is systemic.

[0067] The transgenes disclosed herein are useful for expressing endogenous genes at levels similar to those of healthy or normal individuals. This is particularly useful for treating diseases or conditions associated with under- or lack of expression of gene expression products. In some embodiments, the transgenes disclosed herein are useful for over-expressing endogenous genes such that the expression level of the endogenous gene exceeds that of a healthy or normal individual. In addition, the transgenes can be used to express exogenous genes (e.g., active agents such as antibodies, peptides, nucleic acids, or gene editing components). In some embodiments, the therapeutic gene expression product can modify, enhance, increase, or induce the activity of one or more endogenous biological processes in the cell. In some embodiments, the transgenes disclosed herein are useful for reducing the expression of endogenous genes, such as dominant negative genes. In some embodiments, the therapeutic gene expression product can modify, inhibit, reduce, prevent, eliminate, or impair the activity of one or more endogenous biological processes in the cell. In some embodiments, an increase in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In one embodiment, the protein product of the target gene can be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In some embodiments, a decrease in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In one embodiment, the protein product of the target gene can be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%.

[0068] When an endogenous sequence (endogenous or part of a transgene) is expressed by a transgene, the endogenous sequence may be a full-length sequence (wild-type or mutant) or a partial sequence. The endogenous sequence may be functional. Non-limiting examples of the functions of these full-length or partial sequences include extending the serum half-life of the polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0069] A transgene can be inserted into an endogenous gene so that all or part of the endogenous gene is expressed, or none of the endogenous gene is expressed.For example, a transgene as described herein can be inserted into an endogenous locus so that part of the endogenous sequence (the N-terminus and / or C-terminus of the transgene) is expressed, or none of the endogenous sequence is expressed, for example as a fusion with the transgene.In other cases, a transgene (e.g., with or without additional coding sequences of the endogenous gene) is integrated into any endogenous locus, for example, a safe harbor locus.For example, a frataxin (FXN) transgene can be inserted into an endogenous FXN gene.A transgene can be inserted into any gene, for example, a gene as described herein.

[0070] At least one advantage of the present disclosure is that virtually any therapeutic nucleic acid can be used to express any therapeutic gene expression product. In some cases, the therapeutic gene expression product is a therapeutic protein or peptide (e.g., an antibody, an antigen-binding fragment, a peptide, or a protein). In one embodiment, the protein encoded by the therapeutic nucleic acid is 50-5000 amino acids in length. In some embodiments, the encoded protein is 50-2000 amino acids in length. In some embodiments, the encoded protein is 50-1000 amino acids in length. In some embodiments, the encoded protein is 50-1500 amino acids in length. In some embodiments, the encoded protein is 50-800 amino acids in length. In some embodiments, the encoded protein is 50-600 amino acids in length. In some embodiments, the encoded protein is 50-400 amino acids in length. In some embodiments, the encoded protein is 50-200 amino acids in length. In some embodiments, the encoded protein is 50-100 amino acids in length. In some embodiments, the encoded peptide is 4-50 amino acids in length. In some embodiments, the encoded protein is a tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In some embodiments, the encoded protein comprises a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. In some embodiments, the encoded protein comprises a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide of 50 amino acids or less, e.g., 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0071] Non-limiting examples of therapeutic proteins or peptides include adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietic agents, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartate antagonists, plexins, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphoring, semaphorin receptors, serotonin transporter proteins, serotonin uptake inhibitors, serotonin receptors, serpins, serpin receptors, and tumor suppressors. In certain embodiments, the therapeutic protein or peptide is selected from the group consisting of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropins, interferon-gamma (IFN), insulin-like growth factor 1 (IFG-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), transforming growth factor (TGF), transforming growth factor (TGF-1), and the like. The inhibitor is selected from transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, somatotropin, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10 viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.

[0072] The therapeutic gene expression product may include gene editing components. Non-limiting examples of gene editing components include those required for CRISPR / Cas, artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), and transcription factor-like effector nucleases (TALENs). In a non-limiting example, a subject suffering from Huntington's disease is identified. The subject is then systemically administered a first amount of rAAV that encapsidates a viral vector encoding a ZFN engineered to suppress transcription of the huntingtin (HTT) gene. The rAAV will include a modified AAV capsid protein that includes an amino acid sequence provided in any one of Table 1, Figures 1-3, and Formula I to allow proper targeting of the ZFN to the nervous system while reducing expression in extra-target organs such as the liver. If necessary, the subject is administered a second or third dose of rAAV until a therapeutically effective amount of the ZFN is expressed in the subject's nervous system.

[0073] Therapeutic nucleic acids may include sequences encoding non-protein-coding genes, such as antisense RNA, RNAi, shRNA, and microRNA (miRNA), miRNA sponges or decoys, recombinase delivery for conditional gene deletion, conditional (recombinase-dependent) expression, including those required for gene editing components described herein. Non-protein-coding genes may also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (IncRNA). In some cases, non-protein-coding genes can regulate the expression or activity of target genes or gene expression products. For example, the RNAs described herein can be used to inhibit gene expression in the CNS. In some cases, inhibition of gene expression refers to at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100% inhibition. In some cases, the protein product of the target gene can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. The gene can be either a wild-type gene or a gene having at least one mutation. The target protein can be either a wild-type protein or a protein having at least one mutation.

[0074] The therapeutic nucleic acid can regulate the expression or activity of the gene involved in CNS disease or disorder or the gene expression product expressed from the gene.For example, the therapeutic nucleic acid can be a gene or a modified version of the gene described herein.In some cases, the gene or gene expression product is inhibited.In some cases, the gene or gene expression product is enhanced.

[0075] In another example, the therapeutic nucleic acid comprises an effector gene expression product, e.g., a gene editing component specific for targeting a gene therein. Non-limiting examples of genes include ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEXl, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line derived neurotrophic factor (GDNF), motor neuron survival 1, STXBP1, telomere (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-AT and target genes or gene expression products selected from Pase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some cases, the gene or gene expression product is inhibited. In some cases, the gene or gene expression product is enhanced. AAV vectors

[0076] The embodiments disclosed herein include plasmid vectors that contain the nucleic acid sequence encoding AAV capsid and the AAV capsid protein described herein.The AAV vectors described herein are useful for the assembly of rAAV and the viral packaging of heterologous nucleic acid.In addition, AAV vectors can code for transgenes that contain heterologous nucleic acid.

[0077] The AAV vector may contain a transgene, optionally encoding a heterologous gene expression product (e.g., a therapeutic gene expression product, a recombinant capsid protein, etc.). The transgene is in cis with two inverted terminal repeats (ITRs) flanking the transgene. The transgene may contain a therapeutic nucleic acid encoding a therapeutic gene expression product. Due to the limited packaging capacity of rAAV (approximately 5 kB), in some cases, a longer transgene may be split into two AAV vectors, the first one with a 3' splice donor and the second one with a 5' splice acceptor. Shortly after co-infection of cells, concatemers are formed, which are spliced ​​together to express the full-length transgene.

[0078] A transgene is generally inserted such that its expression is driven by an endogenous promoter at the integration site, i.e., the promoter that drives the expression of the endogenous gene into which the transgene is inserted.In some cases, the transgene includes a promoter and / or an enhancer, such as a constitutive promoter or an inducible or tissue / cell-specific promoter.As a non-limiting example, the promoter can be a CMV promoter, a CMV-β-actin-intron-β-globin hybrid promoter (CAG), a CBA promoter, a FRDA or FXN promoter, a UBC promoter, a GUSB promoter, an NSE promoter, a synapsin promoter, a MeCP2 promoter, a GFAP promoter, a H1 promoter, a U6 promoter, a NFL promoter, a NFH promoter, a SCN8A promoter, or a PGK promoter. As non-limiting examples, the promoter may be a tissue-specific expression element, including, but not limited to, human elongation factor 1 alpha-subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC). The transgene may include tissue-specific expression elements for neurons, such as, but not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk, and EAAT2 promoters. The transgene may include tissue-specific expression elements for astrocytes, such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may include tissue-specific expression elements for oligodendrocytes, such as, but not limited to, the myelin basic protein (MBP) promoter.

[0079] In some embodiments, the promoter is less than 1 kb. , 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, or more than 800. The promoter may be 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800 in length. The promoter may effect expression of a therapeutic gene expression product for a period of time in a target tissue, such as, but not limited to, the CNS.Expression of therapeutic gene products was measured at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 2 The period may be 3 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years. Expression of the payload can be 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, 5-10 years, 10-15 years, 15-20 years, 20-25 years, 25-30 years, 30-35 years, 35-40 years, 40-45 years, 45-50 years, 50-55 years, 55-60 years, or 60-65 years.

[0080] AAV vectors can contain the genome of helper virus. Helper virus proteins are required for the assembly of recombinant AAV (rAAV) and the packaging of transgenes containing heterologous nucleic acid into rAAV. Helper virus genes are adenovirus genes E4, E2a, and VA, which support AAV replication when expressed in cells. In some embodiments, AAV vectors contain E2. In some embodiments, AAV vectors contain E4. In some embodiments, AAV vectors contain VA. In some cases, AAV vectors contain one or any combination of helper virus proteins.

[0081] The target genes or gene expression products used for transgenesis were ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEXl, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line derived neurotrophic factor (GDNF), motor neuron survival 1, STXBP1, telomere (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE6 5), sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0082] The AAV vector may comprise a viral genome comprising a nucleic acid encoding a recombinant AAV (rAAV) capsid protein described herein. The viral genome may comprise a replication (Rep) gene encoding a Rep protein, and a capsid (Cap) gene encoding an AAP protein in a first open reading frame (ORF1) or a Cap protein in a second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some cases, the Cap gene is modified to encode a modified AAV capsid protein described herein. The wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1-VP3 are modified. The AAV vector may comprise a nucleic acid encoding a wild-type Rep78, Rep68, Rep52, Rep40, and AAP protein.

[0083] In some cases, the AAV9 VP1 gene set forth in SEQ ID NO: 71 shown in Table 3 is modified to include any one of SEQ ID NOs: 75-113 in Table 4. The AAV vectors described herein can be used to generate variant AAV capsids by the methods described herein. [Table 3-1] [Table 3-2] [Table 3-3] [Table 4-1] [Table 4-2] How to generate rAAV

[0084] Disclosed herein is a method for producing AAV capsids, including AAV capsid proteins and viral vectors encoding therapeutic nucleic acids. AAV capsid proteins are produced by introducing into cells (e.g., immortalized stem cells) a first vector containing a transgene cassette (the transgene cassette has a promoter sequence that drives the transcription of heterologous nucleic acid in the nucleus of target cells) flanked by inverted terminal repeat (ITR) sequences from parent AAV virus, a second vector (encoding AAV Rep genes and modified Cap genes for the variant to be produced) that encodes an AAV genome with AAV capsid proteins, and a third vector that encodes helper virus proteins required for the assembly of AAV capsid structures and the packaging of transgenes within the modified AAV capsid structures. The assembled AAV capsids can be isolated and purified from cells using suitable methods known in the art. Table 1 provides the DNA sequences for use in the methods described herein.

[0085] Also provided herein is a transgene that is contained in a recombinant AAV (rAAV) vector and is encapsidated by the AAV capsid protein of the present disclosure.The transgene disclosed herein is delivered to a subject for various purposes, such as to treat a disease or condition in the subject.The transgene can be a gene editing component that regulates the activity or expression of a target gene or gene expression product.In addition, the transgene is a gene that codes for a therapeutic gene expression product that is effective for regulating the activity or expression of itself or another target gene or gene expression product.

[0086] The embodiments disclosed herein provide a method of producing a rAAV virus or virus particle, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector containing a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parent AAV virus, the transgene cassette having a promoter sequence driving the transcription of a heterologous nucleic acid in the nucleus of a target cell; (ii) a second vector encoding an AAV genome having an AAV capsid protein of the present invention; and (iii) a vector encoding a helper virus protein required for the assembly of an AAV capsid structure and packaging of the transgene within the modified AAV capsid structure; (b) expressing in the cell the AAV capsid protein described herein; (c) assembling an AAV particle comprising the AAV capsid protein disclosed herein; and (d) packaging the AAV particle. In some cases, the cell is a mammalian cell. In some cases, the cell is immortalized. In some cases, the immortalized cell is an embryonic stem cell. Optionally, the embryonic stem cell is a human embryonic stem cell. Optionally, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. Optionally, the Cap gene is derived from the deoxyribose nucleic acid (DNA) set forth in SEQ ID NO: 71. Optionally, the 5'ITR and 3'ITR are derived from an AAV2 serotype. Optionally, the 5'ITR and 3'ITR are derived from an AAV5 serotype. Optionally, the 5'ITR and 3'ITR are derived from an AAV9 serotype. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are in trans. Optionally, the first nucleic acid sequence and the second nucleic acid sequence are in cis. Optionally, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in trans.

[0087] The Cap genes disclosed herein include any one of SEQ ID NOs: 75-113 in Table 4, which are DNA sequences that encode the modified AAV capsid protein portion of the present disclosure.

[0088] In some cases, the method includes packaging a first nucleic acid sequence encoding a therapeutic gene expression product such that it is encapsidated by a modified AAV capsid protein. In some embodiments, the rAAV particles are isolated, concentrated, and purified using a suitable virus purification method, such as those described herein.

[0089] In some cases, the rAAV of the present disclosure is produced using the method described in Challis, RC et al., Nat. Protoc. 14, 379 (2019). Briefly, triple transfection of HEK293T cells (ATCC) using polyethyleneimine (PEI) is performed, and virus is collected from both cell lysate and medium after 120 hours and purified with iodixanol. In a non-limiting example, rAAV is produced by triple transfection of progenitor cells (e.g., HEK293T) using a standard transfection protocol (e.g., PEI). Viral particles are collected from the medium after a period of time (e.g., 72 hours after transfection), and from the cells and medium at a later time point (e.g., 120 hours after transfection). Virus present in the medium is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500 mM sodium chloride, and the precipitated virus is added to a lysate prepared from the collected cells. Virus is purified by iodixanol (Optiprep, Sigma) step gradient (15%, 25%, 40%, and 60%). Virus is concentrated and formulated in PBS. Viral titers are determined by measuring the number of DNaseI-resistant vector genome copies (VG) using qPCR and a linearized genome plasmid as a control.

[0090] The cell can be selected from human, primate, murine, feline, canine, porcine, ovine, bovine, equine, epinephrine, caprine, and wolf host cells. In some cases, the cell is a progenitor or precursor cell, such as a stem cell. In some cases, the stem cell is a mesenchymal cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a fibroblast, or other tissue-specific stem cell. The cell can be immortalized. In some cases, the immortalized cell is a HEK293 cell. In some cases, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in combination with any transgenic line that expresses a recombinase in a target cell type of interest to develop an AAV capsid that more efficiently transduces that target cell population. Treatment

[0091] Disclosed herein is a method of treating a disease or condition in a subject, or a symptom of a disease or condition, comprising administering to the subject a therapeutically effective amount of one or more compositions (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) disclosed herein. In some embodiments, the composition is a rAAV capsid protein described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein described herein. In some embodiments, the rAAV particles encapsidate an AAV vector comprising a transgene (e.g., a therapeutic nucleic acid). In some embodiments, the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising rAAV particles and a pharma- ceutical acceptable carrier. In some embodiments, one or more compositions are administered alone to the subject (e.g., a stand-alone treatment). In some embodiments, the composition is a first-line treatment for a disease or condition. In some embodiments, the composition is a second, third, or fourth line treatment for a disease or condition.

[0092] Recombinant adeno-associated virus (rAAV)-mediated gene delivery employs the AAV mechanism of viral transduction for nuclear expression of episomal heterologous nucleic acids (e.g., transgenes, therapeutic nucleic acids). Upon delivery to the host in vivo environment, rAAV (1) binds or attaches to cell surface receptors on the target cell, (2) is internalized by endocytosis, (3) is transported to the nucleus, (4) uncoats the virus to release the encapsidated heterologous nucleic acid, (5) converts the heterologous nucleic acid from single-stranded DNA to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribes the episomal heterologous nucleic acid in the nucleus of the host cell ("transduction"). rAAV engineered to increase transduction (transcription of episomal heterologous nucleic acid in the host cell) is desirable for gene therapy applications.

[0093] Aspects disclosed herein provide a method of treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of an rAAV of the disclosure, or a pharmaceutical formulation of the disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, administration is intracranially, intracerebroventricularly, intraventricularly, intravenously, intraarterially, intranasally, intrathecally, intracisternae magna, or subcutaneously.

[0094] Provided herein is a method for treating a disease or condition associated with abnormal expression or activity of a target gene or its gene expression product, comprising regulating the expression or activity of the target gene or gene expression product in a subject by administering a rAAV that encapsidates a heterologous nucleic acid of the present disclosure. In some cases, the expression or activity of the target gene or gene expression product is reduced compared to that in a normal (undiseased) individual; administering the rAAV to the subject is sufficient to increase the expression or activity of the target gene or gene expression product. In some cases, the expression or activity of the gene or gene expression product is increased compared to that in a normal individual; administering the rAAV to the subject is sufficient to reduce the expression or activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer's disease, in some cases caused by a gain-of-function of presenilin 1 and / or presenilin 2 (encoded by the genes PSEN1 and PSEN2, respectively), is administered an rAAV disclosed herein encapsidated with a therapeutic nucleic acid that is a silencing RNA (siRNA) or other RNAi that has a loss-of-function effect on PSEN1 mRNA.

[0095] Also provided is a method for preventing a disease or condition disclosed herein in a subject, comprising administering to the subject a therapeutically effective amount of a rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product as described herein.The rAAV vector can be encapsidated in a modified capsid protein or rAAV viral particle as described herein.In some cases, the therapeutic gene expression product is effective for regulating the activity or expression of a target gene or gene expression product.

[0096] Disclosed herein is a method for treating a disease or condition in a subject by administering a composition comprising the rAAV disclosed herein.The advantage of the rAAV disclosed herein is that it can be used to treat virtually any disease or condition that benefits from transgene therapy, including but not limited to spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Pompe's disease, mucopolysaccharidosis type II, fragile X syndrome, STXBP1 encephalopathy, Krabbe's disease, Huntington's chorea, Alzheimer's disease, Batten's disease, lysosomal storage disorder, glioblastoma multiforme, Rett's syndrome, Leber's congenital amaurosis, late infantile neuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinal cord injury, traumatic brain injury, and lysosomal storage disorder.

[0097] In some cases, disease or condition is localized in a specific in vivo environment in a subject, such as CNS.The composition of the present disclosure is particularly useful for treating the disease or condition described herein, since it specifically or more effectively targets in vivo environment and delivers the therapeutic nucleic acid that is engineered to regulate the activity or expression of the target gene expression product that is involved in the etiology or pathology of disease or condition.

[0098] Provided herein are methods of treating a disease or condition, or a symptom of a disease or condition, in a subject, comprising: (a) diagnosing a subject suffering from a disease or condition that affects a target in vivo environment; and (b) treating the disease or condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions), where the composition has been engineered to have increased expression.

[0099] Disclosed herein are methods of treating a disease or condition, or a symptom of a disease or condition, in a subject to afflict a target, comprising: (a) administering a composition (e.g., an rAAV particle, an AAV vector, a pharmaceutical composition) to the subject; and (b) increasing transduction and expression of a therapeutic nucleic acid in a target in vivo environment in the subject.

[0100] In some embodiments, the method further comprises reducing or eliminating delivery of the heterologous nucleic acid in off-target in vivo environments, such as the liver, In some embodiments, the delivery is characterized by increased transduction (e.g., of the heterologous nucleic acid) within the CNS.

[0101] In some embodiments, a method of treating a disease or condition that affects the CNS comprises administering rAAV particles to the CNS in a subject, the rAAV particles comprising a rAAV capsid protein comprising an insertion of about 5, 6, or 7 amino acids of an amino acid sequence set forth in Table 1, Figures 1-3, and Formula I at amino acid positions 588-589 in the parent AAV capsid protein. In some embodiments, a method of treating a disease or condition that affects the CNS comprises administering rAAV particles to the CNS in a subject, the rAAV particles comprising a rAAV capsid protein comprising an insertion of about 5, 6, or 7 amino acids of an amino acid sequence set forth in Table 1, Figures 1-3, and Formula I, for example, and one or more substitutions at amino acids found at amino acid positions 587-590[AQAQ]. In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein (e.g., as set forth in SEQ ID NO:1).

[0102] Also provided are methods of modulating a target gene expression product, comprising administering to a subject in need thereof a composition (e.g., an rAAV particle, an AAV vector, a pharmaceutical composition) disclosed herein. For example, the methods provided herein include administering to a subject an rAAV having an rAAV capsid protein that encapsidates a viral vector that includes a heterologous nucleic acid that modulates the expression or activity of a target gene expression product.

[0103] The term "normal individual" refers to an individual not afflicted with a disease or condition characterized by altered expression or activity of a gene or its gene expression product.

[0104] In some embodiments, the disease or condition of the CNS is selected from the group consisting of absence of septum pellucidum, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie pupil, Adie syndrome, adrenoleukodystrophy, corpus callosum hypoplasia, agnosia, Aicardi syndrome, Aicardi-Gutierrez syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangiomatosis, anoxia, anti-inflammatory, anti-inflammatory drugs ... Phospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger syndrome ataxia, ataxia-telangiectasia, ataxia and cerebellar or spinocerebellar degeneration, atrial fibrillation and stroke, attention deficit hyperactivity disorder, autism spectrum disorder, autonomic dysfunction, back pain, Barth syndrome, Batten disease, Becker myotonia, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhard-Ross syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, Brachial plexus birth injury, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal tumors, cerebral aneurysm, brain injury, Brown-Séquard syndrome, bulbospinal muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, causalgia, cavernoma, cavernous hemangioma, cavernous malformation, central cervical cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine demyelination syndrome, craniopathy, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous hemangioma, cerebral gigantism, cerebral hypoxemia encephalomyelitis, cerebral palsy, cerebro-oculofacial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Charcot-Marie-Tooth syndrome, classic rhizomelic chondrodysplasia punctata (RCDP), Chiari malformation, cholesterol ester storage disease, chorea, acanthocytic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, coma, complex regional pain syndrome, congenital facial paralysis, congenital myasthenia, congenital muscle disease, congenital cavernous vascular malformation,Corticobasal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalic inclusion disease, cytomegalovirus infection, Dancing Eyes Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson's Disease, Hearing Loss, Domorsia Syndrome, Degerine-Klumpke Palsy, Dementia, Multi-infarct Dementia, Semantic Dementia, Subcortical Dementia, Dementia with Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne Muscular Dystrophy, Autonomic Neuropathy, Dysgraphia, Dyslexia, Dysphagia, Coordination Disorders, Cerebellar Atrophy, Progressive Cerebellar Dyssynchrony, Dystonia, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephalopathy, Encephalopathy (Family Infant), Trigeminal Region Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb-Duchenne and Degerines-Klumpke palsy, essential tremor, extrapontine myelinolysis, Fabry disease, Fahr syndrome, syncope, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, floppy infant syndrome, foot drop, fragile X syndrome, Friedreich's ataxia, frontotemporal dementia (FTD), Gaucher disease, systemic gangliosidosis Gerstmann syndrome, Gerstmann-Sträussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, glioblastoma, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head trauma, headache, persistent hemihyperglycemic headache, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary tonic paraplegia, hereditary polyneuropathic ataxia, herpes zoster, herpes zoster oticus, Hirayama syndrome, Holmes-Addie syndrome group, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's chorea, hydroanencephaly, hydrocephalus, normal pressure hydrocephalus, hydromyelopathy, hyperadrenocorticism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease (IRD), infantile spasms, inflammatory myopathy, foramen occipitalis, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaac's syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Klein-Lewin syndrome, Klippel-Feille syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, learning disability, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, Levi syndrome N. Critschli syndrome, Dementia with Lewy bodies, Lipid storage diseases, Lipid proteinopathies, Lissencephaly, Locked-in syndrome, Lou Gehrig's disease, Lupus - neurological sequelae, Lyme disease - neurological complications, Machado-Joseph disease, Megaencephaly, Maple syrup urine disease, Megaencephalopathy, Melkersson-Rosenthal syndrome, Meningitis, Meningitis and encephalopathy, Menkes disease, Menkes syndrome, Lateral femoral cutaneous neuralgia, Metachromatic leukodystrophy, Microcephaly, Migraine, Miller-Fisher syndrome, Mild stroke, Mitochondrial myopathy, Moebius syndrome, Single Limb muscular atrophy, Motor neuron disease, Moyamoya disease, Mucolipidosis, Mucopolysaccharidosis, Mucopolysaccharidosis type II, Multi-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis, Multiple system atrophy, Multiple system atrophy with orthostatic hypotension, Muscular dystrophy, Congenital myasthenia, Myasthenia gravis, Myelinating diffuse sclerosis, Infantile myoclonus encephalopathy, Myoclonus, Muscle disease, Congenital myopathy, Muscle disease thyrotoxicity, Myotonia, Congenital myotonia, Myotonic dystrophy, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with cerebral iron accumulation, Neurofibromatosis , neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, hereditary neuropathies, neurosarcoidosis, cerebral syphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-MacLeod syndrome, occipital neuralgia, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndrome, Chronic pain, Pantothenate kinase-related neurodegeneration, Paraneoplastic syndromes, Paresthesia, Parkinson's disease, Paroxysmal choreoathetosis, Paroxysmal migraine, Parry-Romberg, Pelizaeus-Merzbach disease, Pena-Choqueir syndrome type II, Perineural cyst, Periodic paralysis, Peripheral neuropathy, Periventricular leukomalacia, Persistent vegetative state, Pervasive developmental disorder, Phenylketonuria, Phytanic acid storage disease, Pick's disease, Nerve compression, Piriformis syndrome, Pituitary tumor, Polymyositis, Pompe disease, Porencephaly, Post-polio syndrome, Postherpetic neuralgia, Post-infectious encephalomyelitis, Post-orthostatic hypotension, Post-orthostatic tachycardia syndrome , postural orthostatic tachycardia syndrome, Prader-Willi syndrome, primary dentate atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive gait ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliodystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II, Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum's disease, infantile Refsum's disease, repetitive movement disorder, repetitive stress injury, restless legs syndrome syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye syndrome, rheumatic encephalitis, Riley-Day syndrome, sacral root cyst, chorea, salivary gland disease, Sandhoff disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic nerve dysplasia, severe myoclonic epilepsy in infancy (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar ataxia ... Leslie-Lechardson-Olszewski syndrome, generalized tonic syndrome, striatonigral degeneration, stroke, Sturge-Weber syndrome, STXBP1 encephalopathy, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short-lasting unilateral neuralgiform headache (SUNCT), dysphagia, Sydenham chorea, syncope, syphilitic spinal sclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, Tangier disease, tardive facial palsy, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen myotonia, thoracic outlet syndrome, thyrotoxic myopathy, trigeminal neuralgic tics,Todd's palsy, Tourette's syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer's syndrome, tuberous sclerosis, vascular erectile tumors, vasculitis syndromes of the central nervous system, von Echonomo disease, von Hippel-Lindau disease (VHL), von Hippel-Lindau syndrome, von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash syndrome, Whipple's disease, Williams syndrome, Wilson's disease, Wolman disease, X-linked spinal-bulbar muscular atrophy, and Zellwerger syndrome.

[0105] In some embodiments, the pharmaceutical formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some cases, the therapeutic gene expression product is selected from the group consisting of ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line derived neurotrophic factor (GDNF), motor neuron survival 1, STXBP1, telomere (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase. The present invention is effective in modulating the activity or expression of a target gene or gene expression product selected from: (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0106] In some embodiments, other examples of genes involved in CNS diseases or disorders include MAPT, IDUA, SNCA, ATXN2, Ube3a, GNS, HGSNAT, NAGLU, SGSH, CLN1, CLN3, CLN4, CLN5, CLN6, CLN7, CLN8, CTSD, ABCD1, HEXA, HEXB, ASM, ASPA, GLB1, AADC, MFN2, GNAO1, SYNGAP1, GRIN2A, GRIN2B, KCNQ2, EPM2A, NHLRC1, SLC6A1, SLC13A5, SURF1, GBE1, ATXN1, ATXN3, and ATXN7.

[0107] In some cases, the therapeutic gene expression product comprises a gene editing component. In some cases, the gene editing component is selected from an artificial site-specific RNA endonuclease (ASRE), a zinc finger endonuclease (ZFN), a transcription factor-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRlSPR) / Cas enzyme, and a CRlSPR) / Cas guide RNA.

[0108] In some cases, expression of the gene or expression or activity of the gene expression product is inhibited by administration of the composition to a subject. In some cases, expression of the gene or expression or activity of the gene expression product is enhanced by administration of the composition to a subject. Formulations, dosages, and routes of administration

[0109] Disclosed herein are methods comprising delivering rAAV particles that encapsidate a heterologous nucleic acid to the CNS in a subject, the rAAV particles comprising (i) increased transduction of the heterologous nucleic acid within the CNS, the rAAV particles having an rAAV capsid protein comprising an insertion of 5, 6, or 7 amino acids of the amino acid sequence set forth in Table 1, Figures 1-3, and Formula I at amino acid positions 588-589 in the parent AAV capsid protein, as well as one or more substitutions at amino acids found at amino acid positions 587-590[AQAQ], e.g., as set forth in Table 1, Figures 1-3, and Formula I.

[0110] In general, the methods disclosed herein include administering the therapeutic rAAV composition by systemic administration. In some cases, the methods include administering the therapeutic rAAV composition by intravenous ("iv") administration. The therapeutic rAAV composition can be administered by additional routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, percutaneous administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intraventricular administration, intrathecal administration, intracisternal administration, or any other suitable parenteral administration. The route, dosage, time point, and duration of administration of the therapeutic agent can be adjusted. In some embodiments, administration of the therapeutic agent is before or after the onset of either or both acute and chronic symptoms of the disease or condition. Other routes of delivery to the CNS include, but are not limited to, intracranial administration, lateral ventricular administration, and intravascular administration.

[0111] Effective doses and dosages of pharmaceutical compositions for preventing or treating a disease or condition disclosed herein are defined by the observed beneficial response associated with the disease or condition, or the symptoms of the disease or condition. A beneficial response includes preventing, alleviating, arresting, or curing the disease or condition, or the symptoms of the disease or condition. In some embodiments, a beneficial response can be measured by detecting a measurable improvement in the presence, level, or activity of a biomarker, transcriptome risk profile, or gut microbiota in a subject. As used herein, "improvement" refers to a shift in the presence, level, or activity relative to that observed in a normal individual (e.g., an individual not suffering from a disease or condition). If a therapeutic rAAV composition is not therapeutically effective or does not provide sufficient relief of the disease or condition, or the symptoms of the disease or condition, the dosage and / or route of administration may be altered or additional agents may be administered to the subject together with the therapeutic rAAV composition. In some embodiments, once a patient is initiated on a regimen of a therapeutic rAAV composition, the patient also discontinues the second treatment regimen (e.g., dose tapering).

[0112] In some cases, the dose of the pharmaceutical composition is at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 In some cases, the concentration of infectious particles may be greater than or equal to 2×10 7 , 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16, or 2 × 10 17 In some cases, the concentration of infectious particles is as high as 3 × 10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , or 3 × 10 17 In some cases, the concentration of infectious particles is as high as 4 × 10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 11 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 , or 4 × 10 17 In some cases, the concentration of infectious particles is as high as 5 × 10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 , or 5 × 10 17 In some cases, the concentration of infectious particles is as high as 6 × 10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11 , 6×10 12 , 6×10 13 , 6×10 14 , 6×10 15 , 6×10 16 , or 6 × 10 17 In some cases, the concentration of infectious particles is as high as 7 × 10 7 , 7×10 8 , 7×10 9 , 7×1010 , 7×10 11 , 7×10 12 , 7×10 13 , 7×10 14 , 7×10 15 , 7×10 16 , or 7 × 10 17 In some cases, the concentration of infectious particles is as high as 8 × 10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , or 8 × 10 17 In some cases, the concentration of infectious particles is as high as 9 × 10 7 , 9×10 8 , 9×10 9 , 9×10 10 , 9×10 11 , 9×10 12 , 9×10 13 , 9×10 14 , 9×10 15 , 9×10 16 , or 9×10 17 There are 10.

[0113] Disclosed herein, in some embodiments, are formulations of pharma- ceutically acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as administration and treatment regimens suitable for using the particular compositions described herein in various treatment regimens.In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition can be prepared so that an appropriate dosage is obtained in any given unit dose of compound.Various dosages and treatment regimens may be desirable, since factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are taken into account by those skilled in the art of preparing such pharmaceutical formulations.

[0114] In some embodiments, pharmaceutical forms of rAAV-based virus compositions suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride.

[0115] In some cases, for administration of injectable aqueous solutions, the solution may be appropriately buffered as necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose.These particular aqueous solutions are suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration, among others.Some variation in dosage will necessarily occur depending on the condition of the subject to be treated.The person responsible for administration will in any case determine the appropriate dose for each individual subject.In addition, for administration to humans, the preparation should meet the sterility, pyrogenicity, and general safety and purity standards required by FDA Office of Biologics standards.

[0116] Disclosed herein is a sterile injectable solution comprising the rAAV composition disclosed herein, which is prepared by incorporating the rAAV composition disclosed herein in the required amount in a suitable solvent with some other components as listed above, if necessary, and then filter sterilization.In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other required components from those listed above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains a powder of active ingredient plus any additional desired ingredients from its previously sterile filtered solution.Injectable solutions can be useful for systemic administration, for example, by intravenous or intrathecal administration.

[0117] Suitable doses and dosages to be administered to a subject will be determined by factors including, but not limited to, the particular therapeutic rAAV composition, the disease state and its severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined according to the particular circumstances surrounding the case, including, for example, the particular agent to be administered, the route of administration, the condition to be treated, and the subject or host to be treated.

[0118] The amount of rAAV composition and the administration time of such composition will be within the scope of a person skilled in the art who receives the benefit of the present teachings.However, it is highly likely that the administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, such as a single injection, of a sufficient number of infectious particles to provide therapeutic benefit to the patient undergoing such treatment.This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, eliminating the need for multiple administrations or chronic administration.

[0119] In certain embodiments, data obtained from cell culture assays and animal studies are used in formulating therapeutically effective daily dosage ranges and / or therapeutically effective unit dosages for use in mammals, including humans. In certain embodiments, the dosage ranges and / or unit dosages vary within this range depending on the dosage form used and the route of administration utilized. Combination therapy

[0120] Therapeutic rAAV can be used alone or in combination with additional therapeutic agents ("therapeutic agents"). In some cases, therapeutic rAAV as used herein is administered alone. Therapeutic agents can be administered together in combination therapy or sequentially. The combination therapy can be administered within the same day, or one or more days, weeks, months, or years apart.

[0121] The additional therapeutic agent may include a small molecule. The additional therapeutic agent may include an antibody or antigen-binding fragment. The additional therapeutic agent may include lipid nanoparticle-based therapies, antisense oligonucleotide therapies, and other viral therapies.

[0122] The additional therapeutic agent may include cell-based therapy. Exemplary cell-based therapy includes, but is not limited to, immune effector cell therapy, chimeric antigen receptor T cell (CAR-T) therapy, natural killer cell therapy, and chimeric antigen receptor natural killer (NK) cell therapy. NK cells, or CAR-NK cells, or a combination of both NK cells and CAR-NK cells may be used in combination with the methods disclosed herein. In some embodiments, the NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSC), umbilical cord blood, or cell lines. The NK cells and CAR-NK cells may include cytokine receptors and suicide genes. The cell-based therapy may include stem cell therapy. The stem cell therapy may be embryonic or somatic stem cells. The stem cells may be isolated from a donor (allogeneic) or from a subject (autologous). The stem cells can be expanded adipose-derived stem cells (eASCs), hematopoietic stem cells (HSCs), mesenchymal stem (stromal) cells (MSCs), or induced pluripotent stem cells (iPSCs) derived from the subject's cells. kit

[0123] Disclosed herein is a kit that comprises the composition disclosed herein.Disclosed herein is also a kit for treating or preventing CNS disease or condition.In some cases, the disease or condition is cancer, pathogen infection, pulmonary disease or condition, neurological disease, muscular disease, or immune disorder, such as those described herein.

[0124] In one embodiment, the kit may include a therapeutic or prophylactic composition that contains an effective amount of a composition of recombinant AAV (rAAV) capsid protein of the present disclosure and rAAV particles that encapsidate a recombinant AAV vector (e.g., a therapeutic nucleic acid) encoding a therapeutic nucleic acid. In another embodiment, the kit may include a therapeutic or prophylactic composition that contains an effective amount of cells ("modified cells") modified by rAAV as described herein in a unit dosage form that expresses a therapeutic nucleic acid. In some embodiments, the kit includes a sterile container that may contain a therapeutic composition. Such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a pharmaceutical agent.

[0125] Optionally, the kit further comprises a cell. Optionally, the cell is a mammalian cell. Optionally, the cell is immortalized. Optionally, the immortalized cell is an embryonic stem cell. Optionally, the embryonic stem cell is a human embryonic stem cell. Optionally, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. Optionally, the kit further comprises an AAV vector comprising a heterologous nucleic acid encoding a therapeutic gene expression product. Optionally, the AAV vector is an episome.

[0126] In some cases, the rAAV is provided with instructions for administering the rAAV to a subject suffering from or at risk of developing a disease or condition (e.g., a CNS disease). The instructions may generally include information regarding the use of the composition for the treatment or prevention of the disease or condition.

[0127] In some cases, the instructions include at least one of the following: description of the therapeutic rAAV composition; dosage schedule and administration for treating or preventing the disease or condition disclosed herein; cautions; warnings; instructions; contraindications; overdosing information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (if present), or as a label attached to the container, or as a separate sheet, pamphlet, card, or folder provided in or with the container. In some cases, the instructions provide instructions for administering the rAAV to the subject alone. In some cases, the instructions describe that the rAAV is formulated for systemic delivery. definition

[0128] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be as inclusive as the term "comprising."

[0129] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to convention for a given value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be considered to mean an acceptable error range for the particular value.

[0130] As used herein, "consisting essentially of" when used to define compositions and methods shall mean excluding any non-essential elements of the combination for the purpose described. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure, such as compositions for treating skin disorders such as acne, eczema, psoriasis, and rosacea.

[0131] The terms "homology," "homology," or "percent homology" are used herein to generally refer to an amino acid sequence or a nucleic acid sequence that has the same or similar sequence as a reference sequence. Percent sequence homology can be determined using the latest version of BLAST as of the filing date of this application.

[0132] The term "increased" or "increasing" is generally used herein to mean an increase of a statistically significant amount. In some embodiments, the term "increased" or "increasing" means an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or up to 100% compared to a reference level, standard, or control, or any increase between 10-100%. Other examples of "increasing" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more compared to a reference level.

[0133] The term "reduced" or "reducing" is used herein generally to mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reducing" means a reduction of at least 10% compared to a reference level, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or up to 100% reduction compared to a reference level (e.g., nonexistent or undetectable levels compared to a reference level), or any reduction between 10-100%. In the context of a marker or indication, the term means a statistically significant reduction in such level. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to a level that is accepted as within the normal range for an individual not suffering from a given disease.

[0134] The term "subject" is any living organism. In some cases, the organism is a mammal. Non-limiting examples of mammals include any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; domestic animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. In one aspect, the mammal is a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, such as a rodent, such as a rat or mouse. In one embodiment, a "non-human primate" is a mammal, such as a monkey. In some instances, the subject is a patient, which as used herein may refer to a subject diagnosed as suffering from a particular disease or disorder.

[0135] As used herein, the term "gene" refers to a segment of nucleic acid that encodes a particular protein or RNA (also referred to as a "coding sequence" or "coding region"), along with associated regulatory regions, such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence as appropriate.

[0136] The term "adeno-associated virus" or "AAV" used herein refers to adeno-associated virus or its derivatives.Non-limiting examples of AAV include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.In some cases, AAV is described as "primate AAV", which refers to the AAV that infects primates. Similarly, the AAV can infect bovine animals (e.g., "bovine AAV"). In some cases, the AAV is wild-type or naturally occurring. In some cases, the AAV is recombinant.

[0137] The term "AAV capsid" as used herein refers to the capsid protein or peptide of adeno-associated virus.In some cases, the AAV capsid protein is configured to encapsidate genetic information (e.g., transgene, therapeutic nucleic acid, viral genome).In some cases, the AAV capsid of the present disclosure is a modified AAV capsid compared to the corresponding parent AAV capsid protein.

[0138] The term "tropism" as used herein refers to a quality or characteristic of an AAV capsid, which may include the specificity of expressing encapsidated genetic information in an in vivo environment and / or the increased or decreased efficiency of doing so, compared to a second in vivo environment. The in vivo environment is optionally a cell type. The in vivo environment is optionally an organ or organ system.

[0139] The term "AAV vector" as used herein refers to a nucleic acid polymer that encodes genetic information related to a virus.AAV vector can be a recombinant AAV vector (rAAV), which refers to an AAV vector that is produced using recombinant genetics methods.In some cases, rAAV vector comprises at least one heterologous polynucleotide (e.g., a polynucleotide other than wild-type AAV genome or naturally occurring AAV genome, such as a transgene).

[0140] As used herein, the term "AAV particle" refers to an AAV virus, virion, AAV capsid protein, or a component thereof. In some cases, the AAV particle is modified relative to the parent AAV particle.

[0141] The term "gene product" of "gene expression product" refers to the product of expression of a polynucleotide sequence, such as, for example, a polypeptide, peptide, protein, or RNA, including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA).

[0142] As used herein, the term "heterologous" refers to genetic elements (e.g., coding regions) or gene expression products (e.g., RNA, proteins) derived from an entity that is genotypically distinct from the rest of the entity to which it is being compared.

[0143] As used herein, the term "endogenous" refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is naturally occurring in or associated with an organism or a particular cell within an organism.

[0144] As used herein, the terms "treat", "treating" and "treatment" refer to the alleviation or elimination of a disorder, disease or condition; or one or more symptoms associated with a disorder, disease or condition; or the alleviation or eradication of the cause of the disorder, disease or condition itself. The desired effects of treatment may include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis.

[0145] The term "therapeutically effective amount" refers to an amount of a compound or treatment that, when administered, is sufficient to prevent the onset of, or alleviate to some extent, one or more symptoms of a disorder, disease, or symptom of a disease, or an amount of a compound sufficient to elicit the biological or medical response in a cell, tissue, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician.

[0146] The terms "pharmaceutical acceptable carrier", "pharmaceutical acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component may be "pharmaceutically acceptable" in the sense of being compatible with other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / loss ratio. Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams and Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th ed.; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004).

[0147] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein with other chemical components, such as a diluent or carrier. A pharmaceutical composition can facilitate administration of a compound to an organism. There are multiple techniques in the art for administering a compound, including but not limited to systemic administration.

[0148] Non-limiting examples of "samples" include any material from which nucleic acids and / or proteins can be obtained. Non-limiting examples include whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph, fecal extracts, buccal swabs, cells, or other bodily fluids or tissues, including but not limited to tissues obtained by surgical biopsy or surgical resection. Alternatively, samples can be obtained via primary patient-derived cell lines, or patient samples stored in the form of archived samples, or fresh frozen samples.

[0149] The term "in vivo" is used to describe events that take place inside the body of a subject.

[0150] The term "in vitro" is used to describe events that occur within a container for holding laboratory reagents such that the material is separate from the biological source from which it is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0151] The term "CNS" or "central nervous system" refers to tissues selected from the brain, thalamus, cortex, dura, lateral ventricles, medulla oblongata, pons, amygdala, motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brainstem, and spinal cord. The brain includes various cortical and subcortical regions, including the frontal, temporal, occipital, and parietal lobes.

[0152] The term "systemic delivery" is defined as a route of administration of a drug or other substance into the circulatory system such that the entire body is affected. Administration can be via enteral administration (absorption of the drug through the digestive tract) or parenteral administration (generally via injection, infusion, or implantation). "Circulatory system" includes both the blood system and the cerebrospinal fluid circulatory system. Examples of systemic administration to the CNS include intra-arterial, intravenous, or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location within the spine (i.e., including but not limited to, the lumbar spine) or brain (i.e., including but not limited to, the cisterna magna). The terms "systemic administration" and "systemic delivery" are used interchangeably.

[0153] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0154] Example 1 Methods for identifying modified capsid proteins in African green monkeys A major concern for the therapeutic applicability of engineered adeno-associated viruses (AAVs) is the extent to which their transduction profiles translate to human use. Previous engineering efforts have focused on in vitro or in vivo rodent screening platforms due to their ease of use and flexibility, but direct non-human primate (NHP) screening efforts are much more likely to identify viruses that translate. For the engineering efforts that helped develop the present invention, the African green monkey, an Old World NHP, was selected. The engineering efforts were primarily directed at a region of the AAV9 capsid surface located at amino acid position 588, which is a variable region between natural AAV serotypes and one of the most exposed loops on the capsid surface that has a role in receptor binding. Insertion of peptides between positions 588 and 589 has been previously investigated by the present inventors and resulted in novel receptor binding (AAV-PHP.B / AAV-PHP.eB binding to Ly6a on rodent brain endothelium to facilitate blood-brain barrier crossing and high transduction of the brain) and dramatically altered capsid tropism. A library of viral capsids was generated by making random 7 amino acid insertions at this site in AAV9 in hopes of achieving novel tropism for the NHP CNS.

[0155] A first-round viral DNA library with all possible mutations was created by amplifying a portion of the AAV9 capsid genome between amino acids 450 and 599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert a random amino acid between amino acids 588 and 589. The resulting library inserts were then introduced into the rAAV-ΔCap-in-rev-RNA plasmid via Gibson assembly, as previously described. The resulting capsid DNA library, rAAV-Cap-Cag-GFP11, contained a diversity of approximately 1.28 billion variants at the amino acid level. A second-round viral DNA library was created similarly to the first round, but instead of the NNK degenerate primer inserted at 588, a synthetic oligo pool (Twist Biosicence) was used to generate only variants selected in the UBC-Cap-DNA and CAG-Cap-DNA constructs with CAP. This second-round DNA library contained a diversity of 3,000-15,000 variants at the amino acid level, with 2-6 barcoded replicates per variant.

[0156] The AAV2 / 9 REP-AAP-ΔCAP plasmid, which is transfected into HEK293T cells to provide the Rep genes for library virus generation, prevents the generation of wild-type AAV9 capsids during viral library generation after plausible recombination events between the plasmids, which are co-transfected with the library plasmids at each stage containing the library inserts.

[0157] Virus production. Recombinant AAV was produced according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were transfected four times with four vectors using polyethylenimine (PEI). The first vector was the rAAV-Cap-Cap-in-cis-Lox library flanked by inverted terminal repeat (ITR) sequences from the parent AAV virus. The second vector was the AAV2 / 9 REP-AAP-ΔCAP plasmid. The third vector contains nucleic acids encoding helper virus proteins required for virus assembly and packaging of heterologous nucleic acid into a modified capsid structure. The fourth is a pUC-18 plasmid included to achieve an appropriate PEI / DNA ratio for optimal transfection efficiency. Only 10 ng of rAAV-Cap-in-cis-Lox library DNA was transfected (per 150 mm plate) to reduce the possibility of multiple library DNAs entering the same cell. 60 hours after transfection, viral particles are harvested from the cells and media. The virus present in the medium is concentrated by precipitation with 8% polyethylene glycol and 500 mM sodium chloride, and the precipitated virus is added to the lysate prepared from the harvested cells. The virus is purified by iodixanol (Optiprep, Sigma) step gradient (15%, 25%, 40%, and 60%). The virus is concentrated and formulated in PBS. The virus titer is determined by measuring the number of DNaseI-resistant vector genome copies (VG) using qPCR and linearized genome plasmid as a control.

[0158] Animals. African Green Monkey procedures were approved by the Virscio, Inc. IACUC Committee. African Green Monkeys were born and raised in the Virscio, Inc. colony and housed under standard conditions. They were fed ad libitum and were enriched as part of the primate enrichment program for NHPs at Virscio, Inc. For AAV injections, animals were screened for the presence of endogenous neutralizing antibodies (nAbs). None of the screened animals showed a detectable blocking response at a 1:5 dilution of serum. The day before injection, animals were deprived of food. Test articles were administered via intravenous or lumbar intrathecal administration. Activity and behavior were monitored daily for the duration of their lives.

[0159] DNA / RNA recovery and sequencing. Round 1 and round 2 viral libraries were cultured in African green monkeys at 5 × 10 12 ~1×10 13rAAV genomes were harvested 4 weeks after injection, with the animals being euthanized and the brain (both round 1 and round 2), spinal cord (round 1 and round 2), and liver (round 1 and round 2) harvested, snap frozen, and stored long term at -80°C (as were other peripheral tissues such as heart, spleen, adrenal gland, kidney, and abdominal quad). For round 1, the brain was separated into 11 brain regions of 20 mg each, and for round 2, into 11 brain regions. Each brain slice, spinal cord, and liver, 20-300 mg each, were homogenized in buffer using MagMAX DNA ULTRA (A25597) and Bead Ruptor 96 (OMNI, INC.), and viral DNA was isolated according to the manufacturer's recommended protocol. The recovered viral DNA was treated with RNase and purified with Zymo DNA Clean and Concentrator kit (D4033). Using 50% of the extracted total viral DNA as template, viral genomes were enriched by 25 cycles of PCR amplification with primers flanking the 588-589 insertion site in the capsid genome. After Zymo DNA purification, samples were diluted 1:10 to 1:1000 depending on tissue type and each dilution was further amplified around the library variable region by 10 cycles of PCR. Samples were then further amplified for >10 cycles using custom primers with Illumina Indices. Amplification products were run on a 2% low melting agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 210bp band.

[0160] For the second round library only, the packaged viral library DNA was isolated from the injected viral library by digestion of the viral capsid and purification of the contained ssDNA. These viral genomes, like the viral DNA extracted from tissues, were amplified by two PCR amplification steps to add adapters and indexes for Illumina next-generation sequencing, and purified after gel electrophoresis. This viral library DNA, together with the viral DNA extracted from tissues, was sent for deep sequencing using the Illumina NextSeq 2000 system.

[0161] NGS data alignment and processing. Raw fastq files from the NGS runs were processed by custom scripts (Capsida CapSeq Tools). For the first round libraries, the pipeline for processing these datasets involved filtering to remove low quality reads, utilizing a quality score per sequence, and eliminating bias from PCR-induced mutations or high GC content. The filtered datasets were then aligned by a perfect string match algorithm and trimmed to improve alignment quality. Read counts per sequence were extracted and displayed per tissue, at which point all sequences found in the brain were compiled for the formation of the second round libraries.

[0162] For the second round libraries, read counts per tissue were similarly tabulated. Then, all brain regions per sequence were summed together, adding a read count of 1 to each sequence and removing values ​​of 0, and the read sequences per codon duplication of a given 7-mer amino acid sequence were summed together to obtain a single value per peptide insert. Finally, data were normalized by log 10 counts per million (Cpm). Enrichment values ​​were calculated by normalized cpm of brain relative to cpm of virus and transformed to log 10.

[0163] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of the claims, and their equivalents, be covered by the invention.

[0164] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An AAV capsid protein comprising a sequence set forth in Table 1 or Figures 1-3.

2. Insert sequence of formula I: X １ -X ２ -X ３ -N-T-T-X ４ -X ５ -X ６ -X ７ -X ８ (I) (Allocation number 72) (In the formula, X １ is an amino acid selected from A, E, Q, T, and V; X ２ is an amino acid selected from Q, I, M, A, P, and V; X ３ is an amino acid selected from L, S, Q, M, and T; ４ is an amino acid selected from K and R; X ５ is an amino acid selected from P, I, N, A, Q, H, I, V, S, and L; X ６ is an amino acid selected from T, I, V, A, Q, S, L, M, G, H, and R; X ７ is an amino acid selected from A, D, N, S, T, M, P, Q, E, G, V, I, and W; X ８ is an amino acid selected from Q, G, F, A, S, D, E, M, P, R, T, and Y.

3. X １ is A; X ２ 3. The AAV capsid protein of claim 2, wherein X is Q, X 3 is L or N, X 4 is K, X 5 is S, and / or X 6 is V.

4. The sequences are AQLNTTKSVMQ (SEQ ID NO: 2), AQLNTTKPTDR (SEQ ID NO: 3), AQLNTTKPTVG (SEQ ID NO: 4), AQLNTTKPSPG (SEQ ID NO: 5), AQLNTTKPTGP (SEQ ID NO: 6), AQLNTTKPTAG (SEQ ID NO: 7), AQLNTTKPVAG (SEQ ID NO: 8), AQLNTTKNITQ (SEQ ID NO: 9), AQLNTTKPTSF (SEQ ID NO: 10), AQLNTTKPTPS (SEQ ID NO: 11), MANTTKPIAQ (SEQ ID NO: 12), AQLNTTKPTGP (SEQ ID NO: 13), AQLNTTKPTAG (SEQ ID NO: 14), AQLNTTKPVAG (SEQ ID NO: 15), AQLNTTKNITQ (SEQ ID NO: 16), AQLNTTKPTSF (SEQ ID NO: 17), AQLNTTKPTPS (SEQ ID NO: 18), AQLNTTKPIAQ (SEQ ID NO: 19), AQLNTTKPTGP (SEQ ID NO: 20), AQLNTTKPTAG (SEQ ID NO: 21), AQLNTTKPVAG (SEQ ID NO: 22), AQLNTTKNITQ (SEQ ID NO: 23), AQLNTTKPTSF (SEQ ID NO: 24), AQLNTTKPTPS (SEQ ID NO: 25), AQLNTTKPIAQ (SEQ ID NO: 26), AQLNTTKPTGP (SEQ ID NO: 27), AQLNTTKPTAG (SEQ ID NO: 28), AQLNTTKPVAG (SEQ ID NO: 29), AQLNTTKNIT No. 12), AQLNTTKPIAQ (SEQ ID NO: 13), AQLNTTKPTSY (SEQ ID NO: 14), AQLNTTKNMTQ (SEQ ID NO: 15), AQLNTTKPRDE (SEQ ID NO: 16), AQLNTTKPTAQ (SEQ ID NO: 17), AQLNTTKNVTQ (SEQ ID NO: 18), QTNNTTKPIAQ (SEQ ID NO:), AQLNTTKQMNQ (SEQ ID NO: 19), AQLNTTKHVDQ (SEQ ID NO: 20), AQLNTTKQIDQ (SEQ ID NO: 21), VALNTTKP IAQ (SEQ ID NO: 22), AMTNTTKPIAQ (SEQ ID NO: 23), AQLNTTKPANQ (SEQ ID NO: 24), AQLNTTKNVNQ (SEQ ID NO: 25), AQLNTTKPVVA (SEQ ID NO: 26), AQLNTTKPQNQ (SEQ ID NO: 27), AQLNTTKPQLQ (SEQ ID NO: 28), AQLNTTKITDQ (SEQ ID NO: 29), AQLNTTKLHDQ (SEQ ID NO: 30), AQLNTTKNGYQ (SEQ ID NO: 31), ANVNTTKPIAQ (SEQ ID NO: 32), 2. The AAV capsid protein of claim 1, selected from EQMNTTKPIAQ (SEQ ID NO: 33), AQLNTTKPLQF (SEQ ID NO: 34), AQLNTTKPSSG (SEQ ID NO: 35), AQLNTTKPVMQ (SEQ ID NO: 36), AQLNTTKPLAQ (SEQ ID NO: 37), TITNTTKPIAQ (SEQ ID NO: 38), AQLNTTKSFGQ (SEQ ID NO: 39), AQLNTTKPTAQ (SEQ ID NO: 116), and AQLNTTKPTTS (SEQ ID NO: 117).

5. LNTTKSV (SEQ ID NO: 41), LNTTKPT (SEQ ID NO: 42), LNTTKPS (SEQ ID NO: 43), LNTTKPV (SEQ ID NO: 44), LNTTKNI (SEQ ID NO: 45), LNTTKPI (SEQ ID NO: 46), NNTTKPI (SEQ ID NO: 47), LNTTKNM (SEQ ID NO: 48), LNTTKPR (SEQ ID NO: 49), LNTTKNV (SEQ ID NO: 50), LNTTKQM (SEQ ID NO: 51), LNTTKP and AAV capsid proteins comprising an AAV capsid protein comprising an insertion sequence selected from Q (SEQ ID NO:52), LNTTKIT (SEQ ID NO:53), LNTTKLH (SEQ ID NO:54), LNTTKNG (SEQ ID NO:55), VNTTKPI (SEQ ID NO:56), MNTTKPI (SEQ ID NO:57), LNTTKPL (SEQ ID NO:58), TNTTKPI (SEQ ID NO:59), and LNTTKSF (SEQ ID NO:60).

6. The AAV capsid protein of any of claims 1, 2, 4 or 5, wherein the AAV is AAV9, optionally as set forth in SEQ ID NO:

1.

7. 6. The AAV capsid of any one of claims 2, 4 or 5, wherein the insertion is between amino acid 588 and amino acid 589.

8. The AAV capsid protein of any one of claims 1, 2, 4 or 5, wherein 60 copies of the AAV capsid protein are assembled into an AAV capsid.

9. The AAV capsid protein of claim 1, 2, 4 or 5, wherein the AAV capsid protein is present within VP1, VP2, and VP3 of the AAV capsid.

10. The AAV capsid protein of any of claims 1, 2, 4 or 5, characterized by at least one of increased transduction efficiency as measured within the CNS in a subject when delivered systemically to the subject.

11. The AAV capsid protein of any one of claims 1, 2, 4 or 5, further comprising an amino acid substitution including A589N and / or Q590P.

12. 10. An AAV capsid comprising an AAV capsid protein according to any one of claims 1, 2, 4 or 5, optionally wherein the AAV capsid is chimeric.

13. The AAV capsid of claim 12, formulated as a pharmaceutical preparation for systemic administration to treat a disease or condition of the CNS, said pharmaceutical preparation further comprising a pharma- ceutical acceptable carrier.

14. The AAV capsid protein of any one of claims 1, 2, 4 or 5, wherein the capsid protein is expressed in the CNS.

15. The AAV capsid protein of claim 14, wherein the CNS comprises tissues selected from the brain, thalamus, cortex, dura, lateral ventricles, medulla oblongata, pons, amygdala, motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, thalamus, cerebrum, cerebellum, brainstem, and spinal cord.

16. A nucleic acid sequence encoding a peptide according to any one of claims 1, 2, 4 or 5, selected from the group consisting of SEQ ID NOs: 75 to 113.

17. Nucleic acid sequences encoding the peptide sequences set forth in Table 4.

18. A recombinant vector comprising a nucleic acid encoding an AAV capsid protein according to any one of claims 1, 2, 4 or 5.

19. a) a first vector comprising the recombinant vector of claim 18; b) a second vector encoding helper virus proteins; and c) a third vector comprising a therapeutic nucleic acid encoding a therapeutic gene expression product; Kit including:

20. A method for producing recombinant AAV particles from the AAV capsid of claim 12, comprising: a. i. a first nucleic acid sequence encoding a therapeutic gene expression product; ii. a second nucleic acid sequence encoding a recombinant viral genome comprising a capsid (Cap) gene modified to express the AAV capsid of claim 12; and iii. a third nucleic acid sequence encoding an AAV helper virus genome; introducing into a cell a nucleic acid comprising b. assembling the recombinant AAV particles, the recombinant AAV particles comprising the AAV capsid that encapsidates the first nucleic acid; The method includes:

21. LNTTKSV (SEQ ID NO: 41), LNTTKPT (SEQ ID NO: 42), LNTTKPS (SEQ ID NO: 43), LNTTKPV (SEQ ID NO: 44), LNTTKNI (SEQ ID NO: 45), LNTTKPI (SEQ ID NO: 46), NNTTKPI (SEQ ID NO: 47), LNTTKNM (SEQ ID NO: 48), LNTTKPR (SEQ ID NO: 49), LNTTKNV (SEQ ID NO: 50), LNTTKQM (SEQ ID NO: 51), LNT A peptide comprising an amino acid sequence selected from KPQ (SEQ ID NO:52), LNTTKIT (SEQ ID NO:53), LNTTKLH (SEQ ID NO:54), LNTTKNG (SEQ ID NO:55), VNTTKPI (SEQ ID NO:56), MNTTKPI (SEQ ID NO:57), LNTTKPL (SEQ ID NO:58), TNTTKPI (SEQ ID NO:59), LNTTKSF (SEQ ID NO:60), and LALPKPI (SEQ ID NO:114).

22. A recombinant adeno-associated virus (rAAV) comprising a capsid containing any one of the sequences described in claim 4 or 5.