Adeno-associated virus compositions with preferred brain enrichment and low liver enrichment

Abstract

Described herein are compositions and kits comprising recombinant adeno-associated viruses (rAAVs) that have increased transduction enrichment in the brain and, in some cases, decreased transduction in the liver. The rAAV compositions described herein encoat transgenes, such as therapeutic nucleic acids. Gene therapies using these rAAVs are described. Methods of treating brain-related diseases and conditions are also described.

Description

Background Art

[0001] Recombinant adeno-associated virus (rAAV) has been widely used as a vector for gene delivery in therapeutic applications because it can transduce both dividing and non-dividing cells, it can exist in infected cells in the form of episomal DNA for a long time, and it has low immunogenicity. These properties make it attractive for applications in therapeutic areas such as gene therapy. However, there is a need to significantly improve the performance of existing AAV serotypes to selectively and efficiently express in different cell types when systemically delivered to a subject. This need is particularly urgent when AAV has to be expressed in the brain.

[0002] Sequence Listing

[0003] This application contains a sequence listing that has been electronically submitted in XML format, and the sequence listing is hereby incorporated by reference in its entirety. The XML file, created on October 11, 2023, is named CAPS-027-01WO.xml and is 560 KB in size. Summary of the Invention

[0004] Disclosed herein are rAAVs that have peptide insertions and substitutions and have been engineered into the capsid structure by performing iterative rounds of selection in non-human primates (NHPs) to generate variants that have increased transduction when measured in the brain and / or decreased expression in the liver relative to the wild-type rAAV from which the variants are based.

[0005] The present invention provides rAAVs that provide extensive transduction of the brain but reduced transduction of the liver. After IV injection, unmodified rAAVs (such as rAAV derived from AAV9 (SEQ ID NO: 1)) may not have sufficient tissue enrichment to treat many human diseases by delivering the AAV cargo. Directed evolution of AAV9 as described herein has provided modified rAAVs that exhibit increased viral tissue enrichment in the brain. Thus, the engineered rAAVs described herein are particularly useful for delivering DNA cargo to the brain tissue. In addition, when current rAAVs are administered to patients in high enough amounts to provide effective disease treatment, off-target enrichment in certain tissues (such as the liver) may cause immune response problems. Thus, in certain embodiments, the modified rAAVs disclosed herein s have been selected to not only increase brain transduction but also reduce liver transduction.

[0006] In certain aspects, the present invention provides an AAV capsid protein comprising, consisting of, or consisting essentially of the amino acid sequence shown in any of Tables 1-3, Figure 1 and / or Formula I.

[0007] Certain aspects of the present invention include modified capsid proteins, wherein the AAV capsid protein comprises a peptide insertion / substitution that comprises any of the amino acid sequences shown in Tables 1-3, Figure 1 and / or consists of the amino acid sequences shown in Formula I. The modified capsid proteins of the present invention are characterized by increased brain transduction in a subject. In certain aspects, a modified capsid protein can be provided, wherein the AAV capsid protein comprises a peptide insertion / substitution that comprises any of the amino acid sequences shown in Tables 1-3, Figure 1 and / or consists of the amino acid sequences shown in Formula I, and is characterized by decreased liver transduction in a subject. When the transduction of a particular tissue discussed herein is increased or decreased, it may be related to an unmodified or wild-type AAV capsid protein.

[0008] In addition, the present disclosure also includes pharmaceutical compositions that comprise rAAV having a peptide insertion / substitution that comprises any of the amino acid sequences shown in Tables 1-3, Figure 1 and / or consists of the amino acid sequences shown in Formula I and a pharmaceutically acceptable excipient.

[0009] Aspects disclosed herein provide methods of treating a disease or condition in a subject that comprise administering a therapeutically effective amount of a pharmaceutical formulation 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 brain and the brain of the subject. Relatedly, the present invention includes the use of rAAV in the preparation of a medicament for treating or preventing the disease or medical condition.

[0010] Other aspects of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] 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 in which:

[0012] Figure 1 show AAV capsid protein insertion and substitution amino acid sequences that are found to have increased brain enrichment and / or decreased liver enrichment in the brain of non-human primates relative to the parental capsid. DETAILED DESCRIPTION

[0013] In certain aspects, the present invention provides modified rAAV that have increased expression levels in the brain and decreased expression levels in the liver when compared to parental AAV (e.g., AAV9).

[0014] In certain aspects, the present disclosure provides rAAV having a peptide insertion / substitution that comprises any of the amino acid sequences shown in Tables 1-3,Figure 1 rAAV in which the amino acid sequence shown in any one of the formula I or a peptide composed of the same is inserted and / or substituted.

[0015] Aspects disclosed herein provide an AAV capsid comprising an AAV capsid protein, the AAV capsid protein comprising the amino acid sequence of formula I

[0016] X 1 -X 2 -G-H-I-X 3 -I(I)(SEQ ID NO:2)

[0017] wherein X 1 is an amino acid selected from R, A, and F; X 2 is an amino acid selected from D, N, and A; and X 3 is an amino acid selected from L and F.

[0018] In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, wherein X 1 is R.

[0019] In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, wherein X 1 is A.

[0020] In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, wherein X 2 is N.

[0021] In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula I, wherein X 3 is L.

[0022] In some embodiments, the peptide insertion and / or substitution sequences are selected from AQRDGHILIAK (SEQ ID NO:3), AQANGHILIAK (SEQ ID NO:4), AQANGHILIAR (SEQ ID NO:5), AQFNGHILIAK (SEQ ID NO:6), AQRAGHILIAP (SEQ ID NO:7), AQRNGHIFIAH (SEQ ID NO:8), AQRNGHIFIAK (SEQ ID NO:9), AQRNGHIFIAR (SEQ ID NO:10), AQRNGHILIAK (SEQ ID NO:11), AQRNGHILIAQ (SEQ ID NO:12), AQRNGNILIAK (SEQ ID NO:13), and AQRNGQILIAK (SEQ ID NO:14).

[0023] In some embodiments, the insert and / or flanking replacement sequences are represented by the peptide sequences listed in Table 1.

[0024] Table 1.

[0025] Sequence SEQ ID NO Sequence SEQ ID NO RDGHILI 623 AQRDGHILIAK 3 ANGHILI 624 AQANGHILIAK 4 ANGHILI 624 AQANGHILIAR 5 FNGHILI 625 AQFNGHILIAK 6 RAGHILI 626 AQRAGHILIAP 7 RNGHIFI 627 AQRNGHIFIAH 8 RNGHIFI 627 AQRNGHIFIAK 9 RNGHIFI 627 AQRNGHIFIAR 10 RNGHILI 628 AQRNGHILIAK 11 RNGHILI 628 AQRNGHILIAQ 12 RNGNILI 629 AQRNGNILIAK 13 RNGQILI 630 AQRNGQILIAK 14

[0026] In certain embodiments, the parental AAV is AAV9. In some embodiments, the parental AAV contains SEQ ID NO:1. In various embodiments, the AAV capsid protein contains a heptamer insert inserted between amino acids 588 and 589 of the parental AAV, wherein the positions 587 - 597 (including the inserted AA position numbers) of the AAV capsid protein are selected from the sequences provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3 - 378.

[0027] The AAV capsid protein may contain amino acid substitutions relative to the parental AAV, which parental AAV contains one or more of the following: A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H, wherein the position numbers refer to the amino acid positions in the parental AAV before any insertions.

[0028] In some embodiments, the 450 - 460 or 461 (depending on the presence of an optional insert) sequences are represented by the peptide sequences listed in Table 2 or by peptide sequences selected from the group consisting of SEQ ID Nos: 379 - 622.

[0029] Table 2.

[0030] Sequence SEQ TD NO TIAGSGQNQQT 379 TIAPDAIRTQT 380 TIATENEKKQT 381 TICGSGQNQQT 382 TICTENEKKQT 383 TIDGALQRTQT 384 TIDGSGQNQQT 385 TIDTENEKKQT 386 TIEGSGQNQQT 387 TIENNQRSQQT 388 TIETENEKKQT 389 TIFGSGQNQQQT 390 TIFGSGQNQQT 391 TIFTENEKKQT 392 TIGGSGQNQQQT 393 TIGGSGQNQQT 394 TIGIDNVKFQT 395 TIGTENEKKQT 396

[0031] Aspects of the present invention may include an AAV capsid protein comprising a sequence provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3 - 378; and a sequence provided in Table 2 or selected from the group consisting of SEQ ID NOs: 379 - 622.

[0032] Typically, five, six, seven, or eight amino acid sequences (pentamers, hexamers, heptamers, or octamers, respectively) inserted or substituted at the 588 loop of the parental AAV capsid protein are inserted. Aspects provided herein provide amino acid insertions that include seven or eight amino acid polymers (heptamers or octamers) inserted at AA588 - 589 and may additionally include substitutions of one or two amino acids at amino acid positions flanking the heptamer sequence (e.g., AA587 - 588 and / or AA589 - 590 using the parental amino acid position numbering) to create an eleven amino acid polymer (11 - mer) at the 588 loop of the parental AAV capsid protein. Figure 1 The fourth column in Figure 1 lists the heptamer or octamer AA insertion sequences at 588 - 589, and two flanking amino acids on each side of the insertion (corresponding to AA positions 587, 588, 589, and 590 of the parental capsid). The flanking amino acids may include substitutions relative to the parental AAV capsid protein. Figure 1 The first column in Figure 1 lists the AA sequences at positions 450 - 460 relative to the parental capsid (or position 461 in the case of an optional insertion relative to the parental capsid).

[0033] In some aspects, the inserted amino acid sequence is at least 71.4% identical to the amino acid sequences provided in Tables 1 - 3, Figure 1 and / or Formula I. In some aspects, the inserted amino acid sequence is at least 86.7% identical to the amino acid sequences provided in Tables 1 - 3, Figure 1 and / or Formula I.

[0034] Also disclosed herein are methods and kits for producing therapeutic recombinant AAV (rAAV) particles, and methods and pharmaceutical compositions or formulations comprising such rAAV particles for treating diseases or conditions affecting the brain.

[0035] Disclosed herein are AAV capsids engineered to have increased viral transduction in the brain. The AAV capsids can encapsidate a viral vector having a heterologous nucleic acid encoding, for example, a therapeutic gene expression product. Transduction of the heterologous nucleic acid in the brain can be achieved by systemic delivery of the AAV capsids of the present disclosure encapsidating the heterologous nucleic acid to a subject. The AAV capsids disclosed herein are advantageous for many applications of gene therapy for treating human diseases, including but not limited to conditions of the central nervous system.

[0036] The present disclosure also provides recombinant AAV vectors comprising nucleic acid sequences encoding the AAV capsid proteins of the present disclosure. For example, the viral vectors of the present disclosure comprise nucleic acid sequences that comprise AAV virus Cap (capsid) encoding VP1, VP2, and VP3, wherein at least one is modified to produce the AAV capsid proteins of the present disclosure. The provided recombinant AAV vectors can be derived from AAV serotypes (e.g., AAV9) or variant AAV serotypes that include the insertions of the present invention.

[0037] AAV capsid

[0038] The present disclosure provides modified adeno-associated (AAV) virus capsid compositions that can be used to integrate a transgene into target cells or environments of a subject when administered systemically to the subject.

[0039] rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., a therapeutic nucleic acid, a gene editing machine). The AAV capsid is composed of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact with each other in a ratio of 1:1:10 to form the viral capsid. Except for an approximately 137 amino acid N-terminal region (VP1u), VP1 also covers the entire VP2 protein, and except for an approximately 65 amino acid N-terminal region (VP1 / 2 common region), VP2 also covers the entire VP3. These three capsid proteins share the conserved amino acid sequence of VP3, which in some cases is the region starting from amino acid position 138 (e.g., AA139 - 736).

[0040] While not wishing to be bound by theory, it should be understood that the parental AAV capsid sequence contains the VP1 region. In certain embodiments, the parental AAV capsid sequence contains the VP1, VP2, and / or VP3 regions or any combination thereof. The parental VP1 sequence can be considered synonymous with the parental AAV capsid sequence.

[0041] The AAV VP3 structure contains highly conserved regions 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 a unique HI loop between β-strands H and I, a DE loop between β-strands D and E, and nine variable regions (VRs) that form the top of the loop. These VRs, such as the AA588 loop, are present on the capsid surface and can be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity.

[0042] In some aspects, the rAAV variants of the present invention comprise an AAV capsid protein that has a peptide insertion at a residue corresponding to amino acids 588 - 589 of the AAV9 native sequence of SEQ ID NO:1.

[0043] An AAV capsid contains AAV capsid proteins (e.g., VP1, VP2, and VP3), each of which has an insertion, for example, in the 588 loop of the parental AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains a site for heparan sulfate that binds to AAV2 and is suitable for peptide display. The only known receptor for AAV9 is N-linked terminal galactose and the AAV receptor (AAVR), but many indications suggest that there are other receptors. Modifications to the AAV9 588 loop are shown herein to confer increased transgene transduction in the target in vivo environment.

[0044] In one aspect, the present invention provides a peptide insertion at the AAV 588 loop, which comprises the amino acid sequences shown in any of Tables 1-3, Figure 1 and / or consists of or is composed of the amino acid sequences shown in any of Formula I.

[0045] AAV capsids are disclosed herein that contain AAV capsid proteins having an insertion at the 588 loop, which confers higher transduction in brain cell types (e.g., brain endothelial cells, neurons, astrocytes). Specifically, the AAV capsid proteins disclosed herein enable the transduction of rAAV-mediated heterologous nucleic acids (e.g., transgenes) in the brain of a subject. The AAV capsids of the present disclosure can be formulated into pharmaceutical compositions. Additionally, the AAV capsids can be isolated and purified for various applications.

[0046] In some embodiments, the rAAV capsids of the present disclosure are produced using the methods disclosed herein. In some cases, the rAAV capsids are chimeric. In some cases, compared to the parental AAV capsid or capsid protein, the rAAV or the variant AAV protein contained therein increases the targeting of rAAV within the target tissue.

[0047] AAV capsid protein

[0048] Recombinant AAV (rAAV) capsids are disclosed herein that contain AAV capsid proteins that have been engineered to have modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are produced using the methods disclosed herein. In some embodiments, the AAV capsid proteins are used in methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some cases, the rAAV capsid proteins have a desired AAV expression such that these rAAV capsid proteins are particularly suitable for certain therapeutic applications, e.g., the treatment of diseases or disorders of a subject as disclosed herein.

[0049] The rAAV capsid proteins are engineered to achieve optimized expression in the CNS (e.g., in the brain) of a subject upon systemic administration of rAAV to the subject. The rAAV capsid proteins are engineered to include Tables 1-3,Figure 1 and / or the insertions provided in Formula I, including Tables 1-3, Figure 1 and / or the rAAV capsid proteins with the insertions provided in Formula I are engineered to effect efficient transduction of the transgene encapsulated. Specifically, the expression of the rAAV capsid protein in the brain of the subject is increased.

[0050] In some cases, the engineered AAV capsid proteins described herein have insertions of amino acids heterologous to the parental AAV capsid protein at amino acid positions in the 588 loop. In some embodiments, at the inserted amino acid positions, the amino acid is not endogenous to the parental AAV capsid protein. The amino acid can be a naturally occurring amino acid in an amino acid position that is the same or equivalent to the substituted insertions in different AAV capsid proteins.

[0051] The heptamers described herein are advantageously generated using polymerase chain reaction (PCR) with degenerate primers, wherein each of the seven amino acids is encoded by the deoxyribonucleic acid (DNA) sequence N-N-K. "N" is any one of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method of generating random heptamer amino acid sequences enables 1.28 billion possible combinations at the protein level. Certain heptamer sequences are then modified by single amino acid substitutions or insertions, which in some cases results in octamer sequences for insertion, as Figure 1 visible.

[0052] The rAAV capsid proteins of the present disclosure can include insertions of amino acids within the amino acid sequence of an AAV capsid protein. The AAV capsid that gives rise to the engineered AAV capsid proteins of the present disclosure is referred to as the "parent" AAV capsid. The complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-2 is provided in GenBank accession numbers NC_001401 and Srivastava et al., Journal of Virology, 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the AAV-5 genome is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Molecular Therapy, 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; a portion of the AAV-13 genome is provided in Genbank accession number EU285562.

[0053] 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. An AAV capsid protein "derived" from another serotype can be a variant AAV capsid protein. Variants can include, for example, heterologous amino acids within the amino acid sequence of an AAV capsid protein. Heterologous amino acids can be non-naturally occurring in an AAV capsid protein. Heterologous amino acids can be naturally occurring in a different AAV capsid protein. In some cases, the parent AAV capsid is described in U.S. Patent Publication 2020 / 0165576 and U.S. Patent Application Serial Numbers 62 / 832,826 and PCT / US20 / 20778; the content of each of these U.S. patents is incorporated herein by reference.

[0054] In some cases, the parental 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 provided in SEQ ID NO:1:

[0055] In some cases, the parental 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.

[0056] AAV capsid proteins with tropism (including liver) from natural AAV serotypes such as AAV9 activate the innate immune response, which in some cases causes a severe inflammatory response in the subject, potentially leading to multiple organ failure. By enhancing the transduction of natural AAV serotypes into target tissues in the body (e.g., brain) and potentially reducing transduction in off-target tissues (e.g., liver), the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent the activation of the innate immune response.

[0057] In some cases, the parental AAV capsid protein comprises the entire VP1 region provided in SEQ ID NO:1 (e.g., amino acids 1-736). In some cases, the parental AAV capsid protein comprises amino acids 217-736 in SEQ ID NO:1, which is the common region found in the VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein comprises amino acids 64-736 in SEQ ID NO:1, which is the common region found in VP1 and VP2. The parental AAV capsid protein sequence may comprise amino acids selected from the following from SEQ ID NO:1: 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, and 450-736. In some aspects, the rAAV variant comprises an AAV capsid protein that comprises an amino acid sequence that is at least 98% identical to amino acids 217 to 736 of SEQ ID NO:1. In some cases, the amino acid insertion is at the three (3)-fold symmetry axis of the corresponding parental AAV capsid protein.

[0058] The present disclosure relates to the insertion of amino acid sequences in AAV capsid proteins. In view of the sequence numbering designation "588 - 589" of AAV9 (e.g., AAV VP1), the invention also includes insertions at analogous positions in other AAV serotypes. As used herein, "AA588 - 589" indicates that the insertion of an amino acid (or amino acid sequence) is immediately after the amino acid (AA) at position 588 within the amino acid sequence of the parental AAV VP capsid protein (VP1 numbering) and immediately before the AA at position 589. Amino acids 587 - 591 include a motif containing "AQAQA" as shown in SEQ ID NO:1. Exemplary AAV capsid protein sequences are provided in Table 3. For example, RDGHILI (SEQ ID NO:623) is inserted at AA588 - 589 in the AAV9 capsid amino acid sequence together with the Q590K substitution to provide Variant A (SEQ ID NO:631). It is contemplated that the sequences disclosed herein (Table 1, Figure 1 and Formula I) can be inserted at AA588 - 589 in the amino acid sequence of the parental AAV9 capsid protein or at AA587 - 590 (in place of amino acids AA587 - 590), their variants, or equivalent amino acid positions of the parental AAV of different serotypes (e.g., AAV1, AAV2, AAV3, etc.). In certain embodiments, the foregoing "AQAQ" sequences flanking the insertion may include one or more substitutions. In any of the AAV capsid protein sequences disclosed herein, the amino acid at position 449 can be R or K. The sequence may include one or more substitutions and / or insertions between positions 450 - 460 (including the end values).

[0059] Table 3. Exemplary AAV Capsid Protein Sequences

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] In some cases, the insertions described herein can include heptameric or octameric insertions at AA588 - 589. It is contemplated that any heptameric or octameric insertion disclosed herein can also include, in addition to being substituted with any amino acid at amino acid positions 587 - 590 [AQAQ], a selection fromFigure 1 and the 11-mer or 12-mer sequences of Table 1.

[0067] Disclosed herein are AAV capsid proteins having the insertions described above in the parental AAV capsid protein, which insertions confer increased transduction of the brain of a subject even upon systemic delivery. One of the many advantages of the AAV capsid proteins described herein is their ability to target tissues and cells within the brain. The tissue can be the brain. Non-limiting examples of brain cells include neurons and glial cells. Glial cells can be selected from oligodendrocytes, ependymal cells, astrocytes, and microglia. Another advantage of certain AAV capsid proteins described herein is their reduced off-targeting to liver tissue relative to the parental AAV capsid protein.

[0068] In some cases, the AAV capsid protein comprises an insertion / substitution of at least or about seven, eight, nine, ten, eleven, or twelve amino acids in amino acid positions 588 - 589 or at 587 - 590 in the parental AAV9 capsid protein (SEQ ID NO:1), in Tables 1 - 3, Figure 1 and the amino acid sequence of Formula I. In some cases, the AAV capsid protein has increased viral transduction enrichment in the brain. In some cases, the AAV capsid protein has reduced viral transduction enrichment in the liver.

[0069] The rAAV capsid proteins of the present disclosure may also have substitutions of the amino acid sequence at amino acid positions 452 - 458 in the parental AAV9 capsid protein or a variant thereof, as described in W02020 / 068990. Exemplary substitutions at amino acid positions 452 - 458 in the parental AAV9 capsid protein can be found in Figure 1 or the second column of Table 2.

[0070] The rAAV capsid proteins described herein can be isolated and purified. AAV can be isolated and purified by standard methods in the art, such as by column chromatography, iodixanol gradient, or cesium chloride gradient. Methods for purifying AAV from helper virus are known in the art and can include methods disclosed, for example, 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.

[0071] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, can be formulated into pharmaceutical formulations which, in some cases, further comprise a pharmaceutically acceptable carrier.

[0072] The rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In some cases, the nanoparticle or viral capsid protein will encapsidate the therapeutic nucleic acid described herein. In some cases, the second molecule is a therapeutic agent, e.g., a small molecule, an antibody, an antigen-binding fragment, a peptide, or a protein, such as those described herein.

[0073] "Percent identity" is the percentage of symbols that actually match. Percent similarity is the percentage of similar symbols. Symbols across gaps are ignored. Similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50 (similarity threshold). The scoring matrix used in the Wisconsin Genetics Software Package, version 10, is BLOSUM62 (see: Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0074] The sequence identity / similarity values provided herein can refer to those obtained using the BLAST+ 2.5.0 program suite with default settings (blast.ncbi.nlm.nih.gov) (Camacho, C. et al., (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0075] One of ordinary skill in the art will appreciate that BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequence, which may be homopolymer segments, short-period repeats, or regions rich in one or more amino acids. Such low-complexity regions can align between unrelated proteins even if other regions of the proteins are completely different. Many low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the 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 employed alone or in combination.

[0076] The terms "substantially identical" and "substantially the same" indicate that a polypeptide or nucleic acid comprises a sequence having a sequence identity between 55% and 100% with a reference sequence, having at least 55% sequence identity relative to the reference sequence, 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 of a value within the range of 55% - 100% sequence identity. The percent sequence identity may occur in a specified comparison window. The homology alignment algorithm of Needleman and Wunsch described above can be used to determine or perform an optimal alignment.

[0077] For example, an insert sequence may include, but is not limited to, a sequence that is not exactly the same as the sequences disclosed herein, but in addition to the substitutions specifically described for the various sequences listed herein, these insert sequences also have additional substitutions of amino acid residues that do not substantially impair the activity or properties of the sequences described herein, such as sequences predicted by homology software (e.g., the BLOSUM62 matrix).

[0078] AAV particle

[0079] rAAV particles having the insert sequences described herein have increased transduction enrichment in the brain. In some cases, the increased transduction enrichment comprises an increase of 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more. In some cases, the increased transduction enrichment is at least 1-fold. In some cases, the increased transduction enrichment is at least 2-fold. In some cases, the increased transduction enrichment is at least 4-fold.

[0080] rAAV particles having the insert sequences described herein have increased expression enrichment in the brain. Detecting whether an rAAV has more or less specificity for the target in vivo includes measuring the level of a gene expression product (e.g., RNA or protein) expressed by a heterologous nucleic acid encapsulated by the rAAV capsid in a tissue sample obtained from a subject. Suitable methods for measuring the expression of the gene expression product include next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0081] Heterologous nucleic acid

[0082] The present disclosure relates to therapeutic nucleic acids that can be used to treat or prevent a disease or condition or symptoms of the disease or condition. In some embodiments, the therapeutic nucleic acids encode 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 for treating, preventing, and / or ameliorating a disease or condition, or symptoms of a disease or condition. In some cases, the therapeutic nucleic acids are placed in an organism, a subject's cell, tissue, or organ by an rAAV as disclosed herein.

[0083] The present disclosure relates to rAAVs, each of which contains a viral vector (e.g., a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector contains two inverted terminal repeat (ITR) sequences of about 145 bases each flanking a transgene. In some embodiments, the transgene contains a therapeutic nucleic acid, and in some cases, a promoter that is arranged cis to the therapeutic nucleic acid in an open reading frame (ORF). The promoter is capable of initiating transcription of the therapeutic nucleic acid in the nucleus of a target cell. The ITR sequences can be 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 from AAV2. In some cases, the ITR is from AAV9.

[0084] The present disclosure relates to transgenes that can contain any number of nucleotides. In some cases, the transgene can contain fewer than about 100 nucleotides. In some cases, the transgene can contain at least about 100 nucleotides. In some cases, the transgene can contain at least about 200 nucleotides. In some cases, the transgene can contain at least about 300 nucleotides. In some cases, the transgene can contain at least about 400 nucleotides. In some cases, the transgene can contain at least about 500 nucleotides. In some cases, the transgene can contain at least about 1000 nucleotides. In some cases, the transgene can contain at least about 5000 nucleotides. In some cases, the transgene can contain more than 5,000 nucleotides. In some cases, the transgene can contain from about 500 to about 5000 nucleotides. In some cases, the transgene contains about 5000 nucleotides. In any case disclosed herein, the transgene can contain DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene can be single-stranded. In some cases, the transgene can be double-stranded.

[0085] The present disclosure relates to transgenes that can be used to modulate the expression or activity of a target gene or its gene expression product. In some cases, the transgene is encapsulated by the rAAV capsid protein of the rAAV particles described herein. In some cases, the rAAV particles are delivered to a subject to treat a disease or condition disclosed herein in the subject. In some cases, the delivery is systemic.

[0086] The transgenes disclosed herein can be used to express an endogenous gene at a level similar to that of a healthy or normal individual. This is particularly useful in treating diseases or conditions associated with low or insufficient expression of a gene expression product. In some embodiments, the transgenes disclosed herein can be used to overexpress an endogenous gene such that the expression level of the endogenous gene is higher than that of a healthy or normal individual. Additionally, the transgene can be used to express a foreign gene (e.g., an active agent such as an antibody, a peptide, a nucleic acid, or a gene editing component). In some embodiments, the therapeutic gene expression product is capable of altering, enhancing, increasing, or inducing the activity of one or more endogenous biological processes in a cell. In some embodiments, the transgenes disclosed herein can be used to reduce the expression of an endogenous gene, e.g., a dominant negative gene. In some embodiments, the therapeutic gene expression product is capable of altering, inhibiting, reducing, preventing, eliminating, or attenuating the activity of one or more endogenous biological processes in a cell. In some aspects, 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 certain aspects, the protein product of the targeted gene can be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In some aspects, 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 certain aspects, the protein product of the targeted gene can be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%.

[0087] When an endogenous sequence (endogenous or partial transgene) is expressed together with the transgene, the endogenous sequence can be a full-length sequence (wild-type or mutant) or a partial sequence. The endogenous sequence can be functional. Non-limiting examples of the functions of these full-length or partial sequences include increasing the serum half-life of a polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0088] Transgenes can be inserted into endogenous genes such that all, some, or none of the endogenous gene is expressed. For example, the transgenes described herein can be inserted into an endogenous locus such that some of the endogenous sequences (at the N-terminus and / or C-terminus of the transgene) are expressed or the endogenous sequences are not expressed, e.g., as a fusion with the transgene. In other cases, a transgene (e.g., with or without additional coding sequences of an endogenous gene) is integrated into any endogenous locus, such as a safe harbor locus. For example, a frataxin (FXN) transgene can be inserted into the endogenous FXN gene. A transgene can be inserted into any gene (e.g., the genes described herein).

[0089] 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, antigen-binding fragment, peptide, or protein). In one embodiment, the protein encoded by the therapeutic nucleic acid is between 50 and 5000 amino acids in length. In some embodiments, the encoded protein is between 50 and 2000 amino acids in length. In some embodiments, the encoded protein is between 50 and 1000 amino acids in length. In some embodiments, the encoded protein is between 50 and 1500 amino acids in length. In some embodiments, the encoded protein is between 50 and 800 amino acids in length. In some embodiments, the encoded protein is between 50 and 600 amino acids in length. In some embodiments, the encoded protein is between 50 and 400 amino acids in length. In some embodiments, the encoded protein is between 50 and 200 amino acids in length. In some embodiments, the encoded protein is between 50 and 100 amino acids in length. In some embodiments, the encoded peptide is between 4 and 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 no more than 50 amino acids (e.g., no more than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids).

[0090] Non-limiting examples of therapeutic proteins or peptides include adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietin, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuropeptides, neuropeptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartic acid antagonists, plexins, proteases, protease inhibitors, protein decarboxylase, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphorins, semaphorin receptors, serotonin transporters, serotonin uptake inhibitors, serotonin receptors, serine protease inhibitors, serine protease inhibitor receptors, and tumor inhibitors. In certain embodiments, the therapeutic protein or peptide is selected from the following: brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropin, 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-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, growth hormone, 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.

[0091] The therapeutic gene expression product can comprise 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 activator-like effector nucleases (TALENs). In a non-limiting example, a subject with Huntington's disease is identified. Then, a first amount of rAAV is administered systemically to the subject, the rAAV encapsidating a viral vector encoding a ZFN that is engineered to inhibit the transcription of the huntingtin (HTT) gene. The rAAV will comprise a modified AAV capsid protein, the modified AAV capsid protein comprising Table 1-3,Figure 1 The amino acid sequence provided in any of Formulas I, thereby allowing the ZFN to correctly target the nervous system while reducing expression in off-target organs such as the liver. If desired, a second or third dose of rAAV is administered to the subject until a therapeutically effective amount of ZFN is expressed in the nervous system of the subject.

[0092] The therapeutic nucleic acid can comprise a non-protein-coding gene, e.g., a sequence encoding an antisense RNA, RNAi, shRNA, and microRNA (miRNA), miRNA sponge, or decoy, for conditional gene deletion, delivery of recombinases, conditional (recombinase-related) expression including those required for the gene editing components described herein. The non-protein-coding gene can also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (lncRNA). In some cases, the non-protein-coding gene can regulate the expression or activity of a target gene or gene expression product. For example, the RNAs described herein can be used to inhibit gene expression in the brain. 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 targeted gene can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. The gene can be a wild-type gene or a gene having at least one mutation. The targeted protein can be a wild-type protein or a protein having at least one mutation.

[0093] The therapeutic nucleic acid can regulate the expression or activity of a gene associated with a disease or disorder of the brain or a gene expression product expressed by the gene. For example, in some cases, the therapeutic nucleic acid is the gene described herein or a modified version of the gene. In some cases, the gene or gene expression product is inhibited. In some cases, the gene or gene expression product is enhanced.

[0094] In another example, the therapeutic nucleic acid comprises an effector gene expression product, such as a gene editing component that is specific for a gene targeted therein. Non-limiting examples of genes include target genes or gene expression products selected from the following: ATP1A2, CACNA1A, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamate decarboxylase 65 (GAD65), glutamate decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), survival motor neuron 1, STXBP1, telomeric (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarcoplasmic / 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), antimicrotubule agent, copper-zinc superoxide dismutase (SOD1), iduronate-2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the following: PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX110, 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.

[0095] AAV vector

[0096] Aspects disclosed herein include plasmid vectors that comprise nucleic acid sequences encoding the AAV capsids and AAV capsid proteins described herein. The AAV vectors described herein can be used for the assembly of rAAV and the viral packaging of heterologous nucleic acids. Additionally, the AAV vectors can encode a transgene comprising a heterologous nucleic acid.

[0097] An AAV vector can contain a transgene that, in some cases, encodes 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 can contain a therapeutic nucleic acid encoding a therapeutic gene expression product. Due to the limited packaging capacity of rAAV (about 5 kB), in some cases, a longer transgene can be split between two AAV vectors, with the first having a 3' splice donor and the second having a 5' splice acceptor. After co-infection of cells, concatemers are formed and these concatemers are spliced together to express the full-length transgene.

[0098] The transgene is typically inserted such that its expression is driven by an endogenous promoter at the integration site (i.e., the promoter driving the expression of the endogenous gene into which the transgene is inserted). In some cases, the transgene contains a promoter and / or enhancer, such as a constitutive promoter or an inducible or tissue / cell-specific promoter. As non-limiting examples, the promoter can be the CMV promoter, the CMV-β-actin-intron-β-globin hybrid promoter (CAG), the CBA promoter, the FRDA or FXN promoter, the UBC promoter, the GUSB promoter, the NSE promoter, the synapsin promoter, the MeCP2 promoter, the GFAP promoter, the H1 promoter, the U6 promoter, the NFL promoter, the NFH promoter, the SCN8A promoter, or the PGK promoter. As non-limiting examples, the promoter can be a tissue-specific expression element, including but not limited to human elongation factor 1α-subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC). The transgene can 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 can contain tissue-specific expression elements for astrocytes, such as but not limited to glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene can contain tissue-specific expression elements for oligodendrocytes, such as but not limited to the myelin basic protein (MBP) promoter.

[0099] In some embodiments, the promoter is less than 1 kb. The length of the promoter can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 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 greater than 800. The length of the promoter can be between 200 - 300, between 200 - 400, between 200 - 500, between 200 - 600, between 200 - 700, between 200 - 800, between 300 - 400, between 300 - 500, between 300 - 600, between 300 - 700, between 300 - 800, between 400 - 500, between 400 - 600, between 400 - 700, between 400 - 800, between 500 - 600, between 500 - 700, between 500 - 800, between 600 - 700, between 600 - 800 or between 700 - 800. The promoter can provide expression of a therapeutic gene product in a targeted tissue (such as but not limited to the brain) for a period of time.Expression of the therapeutic gene product can persist for the following time periods: 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, 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, 23 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 persist for 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, or 5 - 10 years, or 10 - 15 years, or 15 - 20 years, or 20 - 25 years, or 25 - 30 years, or 30 - 35 years, or 35 - 40 years, or 40 - 45 years, or 45 - 50 years, or 50 - 55 years, or 55 - 60 years or 60 - 65 years.

[0100] The AAV vector can contain the genome of a helper virus. Assembly of recombinant AAV (rAAV) and packaging of a transgene containing heterologous nucleic acid into rAAV require helper virus proteins. The helper virus genes are the adenovirus genes E4, E2a, and VA that assist AAV replication when expressed in cells. In some embodiments, the AAV vector contains E2. In some embodiments, the AAV vector contains E4. In some embodiments, the AAV vector contains VA. In some cases, the AAV vector contains one or any combination of the helper virus proteins.

[0101] Target genes or gene expression products for gene transfer may be selected from the following: ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAH, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamate decarboxylase 65 (GAD65), glutamate decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), survival motor neuron 1, STXBP1, telomere (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarcoplasmic reticulum / 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), antimicrotubule agent, copper-zinc superoxide dismutase (SOD1), iduronate-2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the following: PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0102] An AAV vector can contain a viral genome that includes nucleic acids encoding the recombinant AAV (rAAV) capsid proteins described herein. The viral genome can contain 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 contain the 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 the modified AAV capsid proteins described herein. The wild-type Cap gene encodes three proteins, namely 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 of VP1-VP3 are modified. The AAV vector can contain nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40, and an AAP protein.

[0103] In some cases, the AAV9 VP1 gene provided in SEQ ID NO: 641 shown in Table 4 can be modified to encode Figure 1 any insertions and / or substitutions found therein. The AAV vectors described herein can be used to generate variant AAV capsids by the methods described herein.

[0104] Table 4. Nucleic Acid Sequences of VP1 Capsid Proteins

[0105]

[0106]

[0107]

[0108] Methods for Producing rAAV

[0109] The present disclosure relates to methods for generating AAV capsids that contain AAV capsid proteins and viral vectors encoding therapeutic nucleic acids. The AAV capsid proteins are generated by introducing a first vector, a second vector, and a third vector into a cell (e.g., an immortalized stem cell), where the first vector contains a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette having a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell), the second vector encodes an AAV genome having an AAV capsid protein (a modified Cap gene encoding the AAV Rep gene and the resulting variants), and the third vector encodes helper viral proteins that are required for assembly of the AAV capsid structure and packaging of the transgene in the modified AAV capsid structure. The assembled AAV capsids can be isolated and purified from the cells using suitable methods known in the art.

[0110] The present disclosure also provides transgenes contained in recombinant AAV (rAAV) vectors and capsidated by the AAV capsid proteins of the present disclosure. The transgenes disclosed herein are delivered to a subject for various purposes, such as to treat a disease or condition of the subject. The transgene can be a gene editing component that modulates the activity or expression of a target gene or gene expression product. Alternatively, the transgene is a gene encoding a therapeutic gene expression product that effectively modulates the activity or expression of itself or another target gene or gene expression product.

[0111] Aspects disclosed herein provide methods for preparing rAAV viruses or virus particles, the methods comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector that contains a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette having a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell); (ii) a second vector that encodes an AAV genome having an AAV capsid protein of the invention; and (iii) a vector encoding helper viral proteins that are required for assembly of the AAV capsid structure and packaging of the transgene in the modified AAV capsid structure; (b) expressing in the cell the AAV capsid protein described herein; (c) assembling AAV particles comprising the AAV capsid protein disclosed herein; and (d) packaging the AAV particles. In some cases, the cell is mammalian. In some cases, the cell is immortalized. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In some cases, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In some cases, the Cap gene is derived from the deoxyribonucleic acid (DNA) provided in SEQ ID NO:6. In some cases, the 5' ITR and 3' ITR are derived from the AAV2 serotype. In some cases, the 5' ITR and 3' ITR are derived from the AAV5 serotype. In some cases, the 5' ITR and 3' ITR are derived from the AAV9 serotype. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are in trans. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are in cis. In some cases, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are in trans.

[0112] In some cases, the methods comprise packaging a first nucleic acid sequence encoding a therapeutic gene expression product such that it is encapsulated by the modified AAV capsid protein. In some embodiments, the rAAV particles are isolated, concentrated, and purified using suitable virus purification methods (such as the methods described herein).

[0113] In some cases, the rAAV of the present disclosure is produced using the methods described in: Challis, R.C. et al., Nat. Protoc. 14, 379 (2019). Briefly, triple transfection of HEK293T cells (ATCC) is performed using polyethyleneimine (PEI), and the virus is collected from both the cell lysate and the culture medium after 120 hours and purified by iodixanol. In a non-limiting example, rAAV is produced by triple transfection of precursor cells (e.g., HEK293T) using a standard transfection protocol (e.g., PEI). Viral particles are harvested from the culture medium after a certain period of time (e.g., 72 hours post-transfection), and viral particles are harvested from the cells and the culture medium at a later time point (e.g., 120 hours post-transfection). The virus present in the culture medium is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500 mM sodium chloride, and the precipitated virus is added to the lysate prepared from the collected cells. The virus is purified by step gradient (15%, 25%, 40%, and 60%) of iodixanol (Optiprep, Sigma). The virus is concentrated and formulated in PBS. The virus titer is determined by measuring the number of DNase I-resistant vector genomes (VG) using qPCR and a linearized genomic plasmid as a control.

[0114] The cells can be selected from human, primate, murine, feline, canine, porcine, ovine, bovine, equine, epine, caprine, and lupine host cells. In some cases, the cells are progenitor or precursor cells, such as stem cells. In some cases, the stem cells are mesenchymal cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), fibroblasts, or other tissue-specific stem cells. The cells can be immortalized. In some cases, the immortalized cells are HEK293 cells. In some cases, the cells are differentiated cells. Based on the provided disclosure, it is expected that this system can be used in combination with any transgenic strain that expresses a recombinase in the target cell type of interest to develop AAV capsids that more efficiently transduce that target cell population.

[0115] Therapeutic methods

[0116] The present disclosure provides methods for treating a disease or condition or a symptom of the disease or condition in a subject, the methods comprising administering to the subject a therapeutically effective amount of one or more of the compositions disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions). In some embodiments, the composition is an rAAV capsid protein as described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein as 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 an rAAV capsid protein conjugated to a therapeutic agent as disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising rAAV particles and a pharmaceutically acceptable carrier. In some embodiments, one or more of the compositions are administered to the subject alone (e.g., as a monotherapy). In some embodiments, the composition is a first-line therapy for a disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy for a disease or condition.

[0117] Recombinant adeno-associated virus (rAAV)-mediated gene delivery exploits the AAV mechanism of viral transduction for nuclear expression of episomal heterologous nucleic acids (e.g., transgenes, therapeutic nucleic acids). After delivery into the host in vivo environment, rAAV will (1) bind or attach to a cell surface receptor on a target cell, (2) be endocytosed, (3) travel to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid, (5) convert the heterologous nucleic acid from single-stranded DNA to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe the episomal heterologous nucleic acid in the nucleus of the host cell (“transduction”). rAAV engineered to have increased transduction enrichment (transcription of episomal heterologous nucleic acid in the host cell) is desirable for gene therapy applications.

[0118] Aspects disclosed herein provide methods for treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the disclosed rAAV or the disclosed pharmaceutical formulation, wherein the gene product is a therapeutic gene product. In some embodiments, the administration is by intracranial, intraventricular, intrathecal, intravenous, intraarterial, intranasal, intrathecal, intracisternal, or subcutaneous.

[0119] Methods are provided herein for treating a disease or condition associated with abnormal expression or activity of a target gene or its gene expression product, the method comprising modulating the expression or activity of the target gene or gene expression product in a subject by administering rAAV that encapsidates the disclosed heterologous nucleic acid. In some cases, relative to the expression or activity in a normal (non-diseased) individual, the target gene or gene expression product

[0120] The expression or activity is reduced; and administering rAAV to a subject is sufficient to increase the expression of the 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 relative to the expression or activity in a normal individual; and administering rAAV to a subject is sufficient to reduce the expression or activity of the target gene or gene expression product. In a non-limiting example, rAAV disclosed herein is administered to 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)), and the rAAV encapsidates a therapeutic nucleic acid, which is a silencing RNA (siRNA) or other RNAi having a loss-of-function effect on PSEN1 mRNA.

[0121] Also provided are methods of preventing the diseases or conditions disclosed herein in a subject, the methods comprising administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The rAAV vector can be encapsidated in a modified capsid protein or rAAV viral particle described herein. In some cases, the therapeutic gene expression product effectively regulates the activity or expression of a target gene or gene expression product.

[0122] Disclosed herein are methods of treating a disease or condition in a subject by administering a composition comprising rAAV disclosed herein. The advantage of the rAAV disclosed herein is that rAAV can be used to treat almost any disease or condition that would benefit from gene therapy, including but not limited to spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Pompe disease, mucopolysaccharidosis type II, fragile X syndrome, STXBP1 encephalopathy, Krabbe disease, Huntington's disease, Alzheimer's disease, Batten's disease, lysosomal storage disorders, glioblastoma multiforme, Rett syndrome, Leber's congenital amaurosis, late infantile neuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinal cord injury, traumatic brain injury, and lysosomal storage disorders.

[0123] In some cases, the disease or condition is confined to a specific in vivo environment of the subject, e.g., the brain. The compositions of the present disclosure are particularly useful in treating the diseases or conditions described herein because these compositions specifically or more effectively target the in vivo environment and deliver therapeutic nucleic acids engineered to modulate the activity or expression of a target gene expression product associated with the pathogenesis or pathology of the disease or condition.

[0124] Provided herein are methods of treating a disease or condition of a subject or a symptom of the disease or condition, the methods comprising: (a) diagnosing a subject having 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., an rAAV particle, an AAV vector, a pharmaceutical composition), wherein the composition is engineered to have an increased specificity for the target in vivo environment.

[0125] Disclosed herein are methods of treating a disease or condition of a subject that afflicts a target or a symptom of the disease or condition, the methods comprising: (a) administering to the subject a composition (e.g., an rAAV particle, an AAV vector, a pharmaceutical composition); and (b) expressing a therapeutic nucleic acid in a target in vivo environment of the subject with an increased transduction enrichment.

[0126] In some embodiments, the methods further comprise reducing or eliminating delivery of the heterologous nucleic acid to an off-target in vivo environment, such as the liver. In some embodiments, the delivery is characterized by an increased transduction enrichment in the brain (e.g., of the heterologous nucleic acid).

[0127] In some embodiments, a method of treating a disease or condition that affects the brain comprises administering to the brain of a subject an rAAV particle comprising an rAAV capsid protein that comprises an insertion of about five, six, seven, or eight amino acids of the amino acid sequences provided in Table 1-3, Figure 1 and Formula I at amino acid positions 588-589 of the parental AAV capsid protein. In some embodiments, a method of treating a disease or condition that affects the brain comprises administering to the brain of a subject an rAAV particle comprising an rAAV capsid protein that comprises an insertion of about five, six, seven, or eight amino acids of the amino acid sequence and one or more substitutions of amino acids found at amino acid positions 587-590 [AQAQ] as provided in Table 1-3, Figure 1 and Formula I. In some embodiments, the parental AAV capsid protein is an AAV9 capsid protein (e.g., as provided in SEQ ID NO:1).

[0128] Methods for modulating the expression product of a target gene are also provided, which methods comprise administering to a subject in need thereof a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions). For example, the methods provided herein comprise administering to a subject an rAAV having an rAAV capsid protein that encapsidates a viral vector comprising a heterologous nucleic acid that modulates the expression or activity of the expression product of a target gene.

[0129] The term "normal individual" refers to an individual who does not have a disease or condition characterized by an alteration in the expression or activity of a gene or its gene expression product.

[0130] In some embodiments, the diseases or conditions of the brain are selected from agenesis of the septum pellucidum, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's Pupil, Adie's Syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS - neurological complications, Alexander Disease, Alpers' Disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), congenital anencephaly, aneurysm, Angelman Syndrome, angiomatosis, anoxia, antiphospholipid 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's Myotonia, Behcet's Disease, Bell's Palsy, benign essential blepharospasm, benign focal amyotrophy, benign intracranial hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, brachial plexus injury, Bradbury-Eggleston Syndrome, brain and spinal tumors, brain aneurysm, brain injury, Brown-Sequard Syndrome, bulbospinal muscularAtrophy), Autosomal Dominant Cerebral Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernoma, Cavernous Hemangioma, Cavernous Venous Malformation, Central Cervical Myelopathy, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Head Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Brain Aneurysm, Cerebral Arteriosclerosis, Brain Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Brain Hypoxia, Cerebral Palsy, Cerebro-oculofacial Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth syndrome, Recessive Congenital Dissecting Dwarfism (RCDP), Chiari Malformation, Cholesteryl Ester Storage Disease, Chorea, Choreoacanthocytosis, 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 Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformation, Cortical Basal Ganglionic Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalovirus Infection, Dancing Eyes and Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Multi-infarct Dementia, Semantic Dementia, Subcortical Vascular Dementia, Dementia with Lewy Bodies, Dentatorubral-Pallidoluysian Atrophy, Dental Muscular Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne musculardystrophy), familial dysautonomia, dysgraphia, dyslexia, dysphagia, dyskinesia, myoclonic cerebellar ataxia, progressive cerebellar ataxia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, epidemic encephalitis, encephalocele, brain lesion, brain lesion (familial infantile), cerebrospinal cavernous angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Duchenne and Dejerine-Klumpke palsies, essential tremor, extrapontine myelinolysis, Fabry disease, Fahr's syndrome, fainting, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber's disease, febrile convulsion, fibromuscular dysplasia, Fisher syndrome, infantile hypotonia syndrome, foot drop, fragile X syndrome, Friedreich's ataxia, frontotemporal dementia (FTD), Gaucher disease, generalized gangliosidosis, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, cytomegalic inclusion disease, glioblastoma, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barre syndrome, Hallervorden-Spatz disease, head injury, headache, chronic paroxysmal hemicrania, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, polyneuropathic hereditary ataxia, herpes zoster, Ramsay Hunt syndrome, Hirayama syndrome, Holmes-Adie syndrome, holoprosencephaly, HTLV-1 associated myelopathy, Hughes syndrome, Huntington's disease, hydranencephaly, hydrocephalus, normal pressure hydrocephalus, syringomyelia, Cushing's syndrome, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic storage disease, infantile RefsumDisease, IRD), infantile spasms, inflammatory myopathy, occipital encephalocele, intestinal lipodystrophy, intracranial cysts, intracranial hypertension, Isaac's Syndrome, Joubert syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome, KTS, Klüver-Bucy Syndrome, Korsakoff's Amnesic Syndrome, Krabbe disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, lateral femoral cutaneous nerve entrapment, lateral medullary syndrome, learning disorders, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, leukodystrophy, chorea associated with acanthocytosis, Levine-Critchley Syndrome, Lewy body dementia, lipid storage diseases, lipoid proteinosis, lissencephaly, locked-in syndrome, Lou Gehrig's Disease, lupus - neurological sequelae, Lyme Disease - neurological complications, Machado-Joseph Disease, megalencephaly, maple syrup urine disease, macrocephaly, Melkersson-Rosenthal Syndrome, meningitis, meningitis and encephalitis, Menkes Disease, Menkes syndrome, meralgia paresthetica, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher Syndrome, minor stroke, mitochondrial myopathy, MoebiusSyndrome), Unilateral Muscle Atrophy, Motor Neuron Disease, Moyamoya Disease, Mucolipidosis, Mucopolysaccharidosis, Mucopolysaccharidosis II, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Congenital Myasthenia, Myasthenia Gravis, Myelodyscytogenic Diffuse Sclerosis, Infantile Myoclonic Encephalopathy, Myoclonus, Myopathy, Congenital Myopathy, Thyroid Myopathy, Myotonia, Congenital Myotonia, Myotonic Dystrophy, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain 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 Abnormalities, Hereditary Neuropathy, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Cavernous Hemangioma, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Myoclonic Ataxia, Orthostatic Hypotension, Overuse Syndrome, Chronic Pain, Pantothenate Kinase-Associated Neurodegeneration, Paraneoplastic Syndrome, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Migraine, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, 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, Poliomyelitis Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Prader-Willi Syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Disease, Progressive Hemifacial Atrophy, Progressive Motor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliomyelitis, Progressive Supranuclear Palsy, Prosopagnosia, Pseudotorch Syndrome, Pseudotoxoplasmosissyndrome), pseudotumor cerebri, psychogenic movement disorders, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, reflex sympathetic dystrophy syndrome, Refsum Disease, Refsum Disease - infantile type, repetitive movement disorders, repetitive stress injuries, restless legs syndrome, retrovirus - associated myelopathy, Rett syndrome, Reye's Syndrome, rheumatic encephalitis, Riley - Day Syndrome, sacral nerve root cysts, Saint Vitus Dance, salivary gland diseases, Sandhoff Disease, Schilder's Disease, schizencephaly, Seitelberger Disease, seizures, semantic dementia, optic septum dysplasia, severe myoclonic epilepsy in infancy (SMEI), shaken baby syndrome, shingles, Shy - Drager Syndrome, Sjogren's Syndrome, sleep apnea, sleep disorders, Sotos Syndrome, spasticity, spina bifida, spinal infarction, spinal cord injury, spinal cord tumors, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele - Richardson - Olszewski Syndrome, stiff - person syndrome, striatonigral degeneration, stroke, Sturge - Weber Syndrome, STXBP1 encephalopathy, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, short - lasting unilateral neuralgiform (SUNCT) headache, dysphagia, Sydenham Chorea, syncope, syphilitic myelosclerosis, syringohydromyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, Tangier disease, tardive dyskinesia, Tarlov Cysts, Tay - Sachs Disease, temporal arteritis, tethered cord syndrome, Thomsen's Myotonia, thoracic outlet syndrome, thyrotoxic myopathy, Tic Douloureux, Todd's Paralysis, TouretteSyndrome), transient ischemic attack, Creutzfeldt-Jakob disease, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, tuberous sclerosis, angiofibroma, central nervous system vasculitis syndrome, Von Economo's disease, Von Hippel-Lindau disease (VHL), Von Hippel-Lindau syndrome, Von Recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash, Whipple's disease, Williams syndrome, Wilson disease, Wolman's disease, X-linked spinal and bulbar muscular atrophy, and Zellweger syndrome.

[0131] 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 effectively regulates the activity or expression of a target gene or gene expression product selected from the following: ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamate decarboxylase 65 (GAD65), glutamate decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), survival motor neuron 1, STXBP1, telomeric (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarcoplasmic / 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), antimicrotubule agent, copper-zinc superoxide dismutase (SOD1), iduronate-2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the following: PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0132] In some aspects, other examples of genes involved in neurological or brain 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.

[0133] In some cases, the therapeutic gene expression product comprises a gene editing component. In some cases, the gene editing component is selected from: artificial site-specific RNA endonuclease (ASRE), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) / Cas enzyme, and CRISPR / Cas guide RNA.

[0134] In some cases, the expression or activity of a gene or gene expression product is inhibited by administering a composition to a subject. In some cases, the expression or activity of a gene or gene expression product is enhanced by administering a composition to a subject.

[0135] Formulations, Dosages and Routes of Administration

[0136] Disclosed herein are methods for delivering rAAV particles that will encapsidate a heterologous nucleic acid to the brain of a subject, the rAAV particles comprising (i) increased transduction of the heterologous nucleic acid in the brain, wherein the rAAV particle has an rAAV capsid protein that contains an insertion of five, six, seven, or eight amino acids from the amino acid sequences provided in Tables 1-3, Figure 1 and Formula I, and one or more substitutions of amino acids found at amino acid positions 587-590 [AQAQ] as provided in Tables 1-3, Figure 1 and Formula I. In various embodiments, the rAAV capsid protein may contain one or more substitutions at amino acid positions 452-458, either alone or in combination with the above-described modifications.

[0137] Generally, the methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some cases, the methods comprise administering a therapeutic rAAV composition by intravenous (“i.v.”) administration. The therapeutic rAAV composition may be administered by additional routes, such as subcutaneous injection, intramuscular injection, intradermal injection, percutaneous injection, percutaneous administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intracerebroventricular administration, intrathecal administration, intracisternal administration, or any other suitable parenteral administration. The route, dose, time point, and duration of administration of the therapeutic agent may be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of either or both acute and chronic symptoms of a disease or condition. Other routes of delivery to the brain include, but are not limited to, intracranial administration, lateral ventricle administration, and intravascular administration.

[0138] The effective dose and dosage of the pharmaceutical composition for preventing or treating the diseases or conditions disclosed herein are defined by the observed beneficial responses related to the disease or condition or the symptoms of the disease or condition. Beneficial responses include preventing, alleviating, stopping or curing the disease or condition or the symptoms of the disease or condition. In some embodiments, the beneficial response can be measured by detecting the measurable improvement of the presence, level or activity of biomarkers, transcriptome risk profiles or intestinal microbiome in the subject. As used herein, "improvement" refers to the presence, level or activity to the presence, level or activity observed in normal individuals (e.g., individuals without diseases or conditions). In the case where the therapeutic rAAV composition is ineffective or does not fully alleviate the disease or condition or the symptoms of the disease or condition, the dosage and / or route of administration can be changed, or another agent can be administered to the subject together with the therapeutic rAAV composition. In some embodiments, when the patient begins the regimen of the therapeutic rAAV composition, the patient also gradually stops (e.g., the dose is gradually reduced) the second treatment regimen.

[0139] In some cases, a dose of a pharmaceutical composition can contain infectious particles at a concentration of 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 was 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 was 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 , 3×1016 or 3×10 17 。In some cases, the concentration of infectious particles is 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 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 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 7×10 7 、7×10 8 、7×10 9 、7×10 10 、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 8×10 7 、8×10 8 、8×10 9 、8×10 10 、8×10 11 、8×1012 , 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 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 .

[0140] In some embodiments, there are disclosed herein formulations of pharmaceutically acceptable excipients and carrier solutions suitable for delivering the rAAV compositions described herein, as well as suitable dosages and treatment regimens for using the specific compositions described herein in a variety of treatment regimens. In some embodiments, the amount of the therapeutic gene expression product in each therapeutically useful composition can be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological factors, and as such, a variety of dosages and treatment regimens may be desirable.

[0141] In some embodiments, the pharmaceutical forms of rAAV-based viral compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of the action of microorganisms can be provided by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, the inclusion of isotonic agents (such as sugars or sodium chloride) will be preferred.

[0142] In some cases, for the administration of injectable aqueous solutions, if desired, the solution can be suitably buffered and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for the individual subject in any case. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA's Office of Biologics Standards.

[0143] Disclosed herein are sterile injectable solutions containing the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amounts, as needed, together with several other ingredients enumerated above into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredients from their previously sterile-filtered solutions. Injectable solutions can be advantageous for systemic administration (e.g., by intravenous or intrathecal administration).

[0144] The appropriate dosage and dose to be administered to a subject are determined by factors including but not limited to the following: the particular therapeutic rAAV composition, the disease condition and its severity, the identity of the subject to be treated (e.g., weight, gender, age), and can be determined according to the specific circumstances surrounding the case (including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated).

[0145] The amount of the rAAV composition and the time of administration of such composition will be within the capabilities of those skilled in the art benefiting from this teaching. However, it is likely that the administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, e.g., a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to a patient receiving such treatment. This is made possible at least in part by the fact that certain target cells (e.g., neurons) do not divide, thus eliminating the need for multiple or chronic dosing.

[0146] In certain embodiments, data obtained from cell culture assays and animal studies are used to formulate a therapeutically effective daily dose range and / or a therapeutically effective unit dose for mammals (including humans). In certain embodiments, the dose range and / or unit dose vary within this range, which depends on the dosage form employed and the route of administration utilized.

[0147] Combination therapy

[0148] The therapeutic rAAV can be used alone or in combination with additional therapeutic agents (collectively referred to as "therapeutic agents"). In some cases, the therapeutic rAAV as used herein is administered alone. The therapeutic agents can be administered together or sequentially in a combination therapy. The combination therapy can be administered on the same day, or can be administered one or more days, weeks, months, or years apart.

[0149] The additional therapeutic agents can include small molecules. The additional therapeutic agents can include antibodies or antigen-binding fragments. The additional therapeutic agents can include lipid nanoparticle-based therapies, antisense oligonucleotide therapies, and other viral therapies.

[0150] The additional therapeutic agents can include cell-based therapies. Exemplary cell-based therapies include, but are not limited to, immune effector cell therapies, chimeric antigen receptor T cell (CAR-T) therapies, natural killer cell therapies, and chimeric antigen receptor natural killer (NK) cell therapies. Either NK cells or CAR-NK cells or a combination of both NK cells and CAR-NK cells can 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 (iPSCs), umbilical cord blood, or cell lines. The NK cells and CAR-NK cells can include cytokine receptors and suicide genes. The cell-based therapies can include stem cell therapies. The stem cell therapies can be embryonic stem cells or somatic stem cells. The stem cells can be isolated from a donor (allogeneic) or from the 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 cells of the subject.

[0151] Kit

[0152] Kits containing the compositions disclosed herein are disclosed herein. Kits for treating or preventing diseases or conditions of the brain are also disclosed herein. In some cases, the disease or condition is cancer, pathogen infection, lung disease or condition, neurological disease, muscle disease, or immune disorder, such as the diseases or conditions described herein.

[0153] In one embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of rAAV particles and a composition of a recombinant AAV (rAAV) capsid protein of the present disclosure, wherein the rAAV particles encapsidate a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., a therapeutic nucleic acid). In another embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of cells modified by rAAV as described herein in unit dosage form that express a therapeutic nucleic acid ("modified cells"). In some embodiments, the kit contains a sterile container that can contain the therapeutic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, sachets, blister packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs.

[0154] In some cases, the kit further contains cells. In some cases, the cells are mammalian. In some cases, the cells are immortalized. In some cases, the immortalized cells are embryonic stem cells. In some cases, the embryonic stem cells are human embryonic stem cells. In some cases, the human embryonic stem cells are human embryonic kidney 293 (HEK293) cells. In some cases, the kit further contains an AAV vector that contains a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the AAV vector is an episome.

[0155] In some cases, rAAV is provided together with instructions for administering rAAV to a subject having or at risk of having a disease or condition (e.g., a brain disease). The instructions can generally include information regarding the use of the composition for treating or preventing a disease or condition.

[0156] In some cases, the instructions include at least one of the following: a description of the therapeutic rAAV composition; a dosage schedule and administration for treating or preventing the diseases or conditions disclosed herein; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions can be printed directly on the container (if present), or as a label applied to the container or as a separate sheet, booklet, card, or folder provided with or applied in the container. In some cases, the instructions provide a procedure for administering rAAV to a subject alone. In some cases, these instructions provide that rAAV is formulated for systemic delivery.

[0157] Definitions

[0158] The terms used herein are for the purpose of describing particular situations only and are not intended to be limiting. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" are intended to include the plural forms as well. Additionally, where the terms "including", "having", or variants thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0159] The term "about" or "approximately" means within an acceptable error range of 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., the limitations of the measuring system. For example, depending on the practice with a given value, "about" can mean within 1 or more standard deviations. Where a particular value is described in this application and the claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable error range for that particular value.

[0160] When used to define compositions and methods, "consisting essentially of" as used herein shall mean excluding other elements that are of any significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein will 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 conditions such as acne, eczema, psoriasis, and rosacea.

[0161] The terms "homologous", "homology", or "percent homology" as used herein generally mean an amino acid sequence or nucleic acid sequence having the same or similar sequence as a reference sequence. As of the filing date of this application, the percent homology of a sequence can be determined using the most recent version of BLAST.

[0162] The term "increased" is used herein to generally mean an increase in a statistically significant amount. In some embodiments, the term "increased" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% compared to a reference level, standard, or control, or any increase between 10% - 100%. Other examples of "increased" 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 compared to a reference level.

[0163] The term "decreased / decrease" is generally used herein to mean a decrease in a statistically significant amount. In some embodiments, "decreased / decrease" means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% decrease (e.g., a level that is absent or undetectable compared to the reference level) or any decrease between 10-100%. In the context of a biomarker or symptom, these terms mean a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably to a level that is accepted within the normal range for an individual not suffering from a given disease.

[0164] 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; farm animals such as cows, horses, sheep, goats, pigs, etc.; domestic animals such as rabbits, dogs, and cats, etc.; laboratory animals including rodents such as rats, mice, and guinea pigs, etc. In certain aspects, the mammal is a human. As used herein, the term "animal" encompasses humans and non-human animals. In one embodiment, the "non-human animal" is a mammal, e.g., a rodent (such as a rat or a mouse). In one embodiment, the "non-human primate" is a mammal, e.g., a monkey. In some cases, the subject is a patient, as used herein, which can refer to a subject diagnosed with a specific disease or disorder.

[0165] As used herein, the term "gene" refers to a nucleic acid fragment (also referred to as a "coding sequence" or "coding region") that optionally encodes a single protein or RNA together with associated regulatory regions (such as promoters, operators, terminators, etc.), and the associated regulatory regions can be located upstream or downstream of the coding sequence.

[0166] As used herein, the term "adeno-associated virus" or "AAV" refers to adeno-associated virus or derivatives thereof. 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 AAV that infects primates. Similarly, AAV can infect bovines (e.g., "bovine AAV," etc.). In some cases, AAV is wild-type or naturally occurring. In some cases, AAV is recombinant.

[0167] As used herein, the term "AAV capsid" 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 capsids of the present disclosure are modified AAV capsids relative to the corresponding parental AAV capsid proteins.

[0168] As used herein, the term "tropism" refers to a quality or property of an AAV capsid that can include specificity for expressing encapsidated genetic information into a body environment relative to a second body environment, and / or an increase or decrease in enrichment for expressing encapsidated genetic information into a body environment relative to a second body environment. In some cases, the body environment is a cell type. In some cases, the body environment is an organ or organ system.

[0169] As used herein, the term "AAV vector" refers to a nucleic acid polymer encoding virus-related genetic information. An AAV vector can be a recombinant AAV vector (rAAV), which refers to an AAV vector produced using recombinant genetic methods. In some cases, the rAAV vector contains at least one heterologous polynucleotide (e.g., a polynucleotide other than the wild-type or naturally occurring AAV genome, such as a transgene).

[0170] As used herein, the term "AAV particle" refers to an AAV virus, virion, AAV capsid protein, or components thereof. In some cases, the AAV particle is modified relative to the parental AAV particle.

[0171] The "gene product" of the term "gene expression product" refers to the expression product of a polynucleotide sequence, for example, a polypeptide, a peptide, a protein, or an RNA (including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA)).

[0172] As used herein, the term "heterologous" refers to a genetic element (e.g., a coding region) or a gene expression product (e.g., an RNA, a protein) that is derived from an entity that is genetically distinct from the remainder of the entity with which it is being compared.

[0173] As used herein, the term "endogenous" refers to a genetic element (e.g., a coding region) or a gene expression product (e.g., an RNA, a protein) that is naturally present in or associated with an organism or a particular cell within an organism.

[0174] As used herein, the term "treat / treating / treatment" refers to alleviating or eliminating a disorder, disease, or medical condition; or one or more symptoms associated with the disorder, disease, or medical condition; or alleviating or eliminating the cause of the disorder, disease, or medical condition itself. Desirable treatment effects can include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and alleviating or improving the prognosis.

[0175] The term "therapeutically effective amount" refers to an amount of a compound or therapy that, when administered, is sufficient to prevent the development or to alleviate to some extent one or more symptoms of a disorder, disease, or medical condition of a disease; or an amount of a compound that is sufficient to elicit a biological or medical response in a cell, tissue, system, animal, or human that a researcher, veterinarian, physician, or clinician is seeking.

[0176] The terms "pharmaceutically acceptable carrier", "pharmaceutically 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 can be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It can also be suitable for contact with the tissues or organs of humans and animals in a reasonable benefit / risk ratio without producing excessive toxicity, irritation, allergic responses, immunogenicity, or other problems or complications. See Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Edited by Rowe et al., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Edited by Ash and Ash, Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Edited by Gibson, CRC Press LLC: Boca Raton, FL, 2004.

[0177] 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 the administration of the compound to an organism. There are various techniques for administering a compound in the art, and these techniques include, but are not limited to, systemic administration.

[0178] Non-limiting examples of a “sample” include any material from which nucleic acids and / or proteins can be obtained. As non-limiting examples, this includes whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph fluid, fecal extracts, buccal swabs, cells or other body fluids or tissues, which tissues include but are not limited to tissues obtained by surgical biopsy or surgical resection. Alternatively, a sample can be obtained from a primary patient-derived cell line or can be an archived patient sample in the form of a stored sample or a fresh frozen sample.

[0179] The term “in vivo” is used to describe an event that occurs within a subject.

[0180] The term “in vitro” is used to describe an event that occurs in a container used to hold laboratory reagents such that the material is separated from the biological source from which it was obtained. In vitro assays can encompass cell-based assays in which live or dead cells are employed. In vitro assays can encompass cell-free assays in which intact cells are not employed.

[0181] The term “brain” means tissue selected from the following: brain, thalamus, cortex, dura mater, lateral ventricle, medulla, pons, amygdala, motor cortex, caudate nucleus, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brain stem, and spinal cord. The brain includes various cortical and subcortical regions, including the frontal, temporal, occipital, and parietal lobes.

[0182] The term “systemic delivery” is defined as the route of administration by which a drug or other substance enters the circulatory system such that the entire body is affected. Administration can be by enteral administration (where the drug is absorbed through the gastrointestinal tract) or parenteral administration (usually by injection, infusion, or implantation). The “circulatory system” includes both the blood circulatory system or the cerebrospinal fluid circulatory system. Examples of systemic administration to the brain include intra-arterial, intravenous, or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location in the spinal cord (i.e., but not limited to the lumbar region) or the brain (i.e., but not limited to the cisterna magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0183] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0184] Examples

[0185] Example 1

[0186] Method for Identifying Modified Capsid Proteins in Cynomolgus Macaques

[0187] The therapeutic applicability of engineered adeno-associated virus (AAV) is mainly concerned with how its transduction profile translates to human applications. Although previous engineering efforts have focused on in vitro or in vivo rodent screening platforms due to their ease of use and flexibility, screening efforts directly in non-human primates (NHPs) are more likely to identify viruses for translation. Cynomolgus monkeys (an Old World NHP) were selected for the engineering effort. The effort was focused on the region on the surface of the AAV9 capsid located at amino acid position 588, which is one of the most exposed loops on the capsid surface, a variable region between native AAV serotypes, and plays a role in receptor binding. Insertions of peptides between positions 588 and 589 have been studied in the past and have achieved novel receptor binding (AAV-PHP.B / AAV-PHP.eB binds to Ly6a on rodent brain endothelium to facilitate blood-brain barrier crossing and high transduction of the brain) and significantly altered capsid tropism. A viral capsid library was established by randomly inserting 7 amino acids at this site within AAV9, with the hope of having a novel tropism for the NHP brain. Through two rounds of screening of this process, an rAAV was identified that had a specific 7-amino acid peptide insertion that conferred high brain tropism, and this rAAV was used as the parental capsid for mutagenic substitutions and insertions on this parental capsid in rounds 3 and 4 of screening.

[0188] Plasmid。The first-round viral DNA library was generated by amplifying the segment of the AAV9 capsid genome between amino acids 450 - 599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 588 and 589 with all possible variations. The resulting library inserts were then introduced into the rAAV-ΔCap-in-rev-RNA plasmid by 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. The second-round viral DNA library was generated in a similar manner to the first round, but instead of inserting the NNK degenerate primer at 588, a synthetic oligomer pool (Twist Bioscience) was used to generate only the selected variants in the UBC-Cap-DNA and CAG-Cap-DNA constructs with CAP. The third- and fourth-round viral DNA libraries were generated similarly to the second round, but synthetic oligomer pools of the selected variants were ordered from IDT and used to generate capsids with amino acid insertions and substitutions in the 588 loop and / or 452 loop. The third-round library contained a diversity of 10,000 variants at the amino acid level, and the fourth round contained approximately 1,000 variants; in both cases, 2 barcode replicates were used for each variant.

[0189] The AAV2 / 9REP-AAP-ACAP plasmid transfected into HEK293T cells to provide the Rep gene for library virus production prevented the generation of wild-type AAV9 capsids during virus library production after a plausible recombination event occurred between this plasmid and the library plasmids containing the library inserts at each stage.

[0190] Virus ProductionRecombinant AAVs are generated according to the established protocol. Briefly, immortalized HEK293T cells (ATCC) are quadruple transfected with four vectors using polyethyleneimine (PEI). The first vector is an rAAV-Cap-in-cis-Lox library flanked with inverted terminal repeat (ITR) sequences from the parental AAV virus. The second vector is an AAV2 / 9REP-AAP-ACAP plasmid. The third vector contains nucleic acids encoding the helper viral proteins required for viral assembly and packaging of heterologous nucleic acids into the modified capsid structure. The fourth is a pUC-18 plasmid, and including this plasmid achieves the correct PEI / DNA ratio for optimal transfection enrichment. Only 10 ng of the rAAV-Cap-in-cis-Lox library DNA (per 150 mm plate) is transfected to reduce the likelihood of multiple library DNAs entering the same cell. Sixty hours after transfection, viral particles are harvested from the cells and the medium. 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 collected cells. The virus is purified by a step gradient (15%, 25%, 40%, and 60%) of iodixanol (Optiprep, Sigma). The virus is concentrated and formulated in PBS. The virus titer is determined by measuring the number of DNase I-resistant vector genomes (VGs) using qPCR and a linearized genomic plasmid as a control.

[0191] Animals The cynomolgus monkey procedures are approved by the IACUC committee of Envol Biomedical. Cynomolgus monkeys are born and raised in the Envol Biomedical colony and are housed in family groups under standard conditions. They are fed ad libitum and receive enrichment as part of the primate enrichment program for NHPs at Envol Biomedical. For AAV infusion, animals are screened for endogenous neutralizing antibodies (Nabs). None of the screened animals showed any detectable blocking response at a 1:10 diluted serum. The animals are then group-housed for several days and acclimated to the new room before injection. The animals are restrained, and the test article is administered by intravenous infusion for 10 minutes. Two juvenile monkeys are used in each of the first and second rounds of screening, and three juvenile monkeys are used in the third and fourth rounds of screening. Activity and behavior are monitored daily throughout life.

[0192] DNA / RNA Recovery and Sequencing The viral library is at 1 - 3×10 13Doses of vg / kg animals were injected into cynomolgus monkeys, and rAAV genomes were recovered two weeks after injection. The animals were euthanized and the brain, spinal cord, heart, and liver, as well as other peripheral tissues such as the heart, spleen, adrenal glands, kidneys, and limbs, were recovered, snap-frozen, and stored long-term at -80 °C. The brain was divided into 11 - 13 brain regions, and 100 mg of each brain region was homogenized in buffer using MagMAX DNA ULTRA (A25597) and Bead Ruptor96 (OMNI, INC), and viral DNA was isolated according to the manufacturer's recommended protocol. The recovered viral DNA was treated with RNase and purified using the Zymo DNA Clean and Concentrator Kit (D4033). Using 50% of the total extracted viral DNA as a template, the viral genome was enriched by 25 cycles of PCR amplification with primers flanking the AA 452 - 588 region in the capsid genome. After Zymo DNA purification, the samples were diluted 1:10 to 1:1000 according to tissue type, and each dilution was further amplified by 10 cycles of PCR around the variable region of the library. Subsequently, the samples were further amplified by 10 cycles using custom primers with Illumina indices. The amplification products were run on a 2% low melting point agarose gel (ThermoFisher Scientific, 16520050) to better separate and recover the 600 bp band.

[0193] For the second, third, and fourth round libraries, the packaged viral library DNA was isolated from the injected viral library by digesting the viral capsid and purifying the contained ssDNA. These viral genomes were amplified through two PCR amplification steps, as was the viral DNA extracted from tissues, to add adapters and indices for Illumina next-generation sequencing and were purified after gel electrophoresis. This viral library DNA, along with the viral DNA extracted from tissues, was deep-sequenced using the Illumina NextSeq 2000 system.

[0194] NGS Data Alignment and Processing . The raw fastq files from the NGS runs were processed with custom scripts (Capsida CapSeq tool). For the first round library, the pipeline for processing these datasets involved filtering to remove low-quality reads, leveraging the quality scores of each sequence and eliminating biases caused by PCR-induced mutations or high GC content. Then, the filtered datasets were aligned by a perfect string matching algorithm and adjusted to improve alignment quality. The read counts for each sequence were extracted and presented by tissue, at which point all sequences found in the brain were compiled to form the second round library.

[0195] For the second, third, and fourth round libraries, the read counts organized by tissue were similarly tabulated. Then, a read count of 1 was added to each sequence to remove zero values, all brain regions for each sequence were summed, and the read sequences for each codon duplication of a given capsid amino acid sequence were summed to obtain a single value for each variant. Finally, the read count data was normalized relative to counts per million (Cpm). The enrichment value for each capsid variant was calculated as the normalized cpm from [tissue of interest] / cpm from the viral library.

[0196] While the preferred embodiments of the 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. Many variations, changes, and substitutions will now 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 may be employed in practicing the invention. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0197] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the 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 the sequences provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378.

2. The AAV capsid protein according to claim 1, comprising an insertion sequence of Formula I X 1 -X 2 -G-H-I-X 3 -I(I)(SEQ ID NO:2) wherein X 1 is an amino acid selected from R, A, and F; X 2 is an amino acid selected from D, N, and A; and X 3 is an amino acid selected from L and F.

3. The AAV capsid protein according to claim 2, wherein X 1 is R.

4. The AAV capsid protein according to claim 2, wherein X 1 is A.

5. The AAV capsid protein according to claim 2, wherein X 2 is N.

6. The AAV capsid protein according to claim 2, wherein X 3 is L.

7. The AAV capsid protein according to claim 1, wherein the sequence is selected from the group consisting of: AQRDGHILIAK (SEQ ID NO: 3), AQANGHILIAK (SEQ ID NO: 4), AQANGHILIAR (SEQ ID NO: 5), AQFNGHILIAK (SEQ ID NO: 6), AQRAGHILIAP (SEQ ID NO: 7), AQRNGHIFIAH (SEQ ID NO: 8), AQRNGHIFIAK (SEQ ID NO: 9), AQRNGHIFIAR (SEQ ID NO: 10), AQRNGHILIAK (SEQ ID NO: 11), AQRNGHILIAQ (SEQ ID NO: 12), AQRNGNILIAK (SEQ ID NO: 13), and AQRNGQILIAK (SEQ ID NO: 14).

8. The AAV capsid protein according to claim 1, comprising AAV9 as the parental AAV.

9. The AAV capsid protein according to claim 8, wherein the parental AAV comprises SEQ ID NO:

1.

10. The AAV capsid protein according to claim 9, comprising a heptameric insertion or an octameric insertion inserted into the parental AAV between amino acid 588 and amino acid 589 of the parental AAV, wherein the sequence at positions 587-597 or 587-598 of the AAV capsid protein is selected from the sequences provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378.

11. The AAV capsid protein according to claim 1, wherein 60 copies of the AAV capsid protein are assembled into the AAV capsid.

12. The AAV capsid protein according to claim 1, wherein the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid.

13. The AAV capsid protein according to claim 1, further characterized in that When measured in the brain tissue of the subject upon systemic delivery to the subject, the transduction enrichment is increased relative to AAV9.

14. The AAV capsid protein according to claim 1, further characterized in that When measured in the liver tissue of the subject upon systemic delivery to the subject, the transduction enrichment is decreased relative to AAV9.

15. The AAV capsid protein according to claim 7, wherein the AAV capsid protein further comprises an amino acid substitution relative to the parental AAV, the amino acid substitution comprising one or more of the following: A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H.

16. An AAV capsid protein comprising the sequences provided in Table 2 or selected from the group consisting of SEQ ID NOs: 379-622.

17. The AAV capsid protein according to claim 16, which comprises AAV9 as the parental AAV.

18. The AAV capsid protein according to claim 17, wherein the parental AAV comprises SEQ ID NO:

1.

19. The AAV capsid protein according to claim 18, which comprises one or more substitutions at positions 452-460 and an optional insertion at any one of positions 452-453 to 458-459 relative to the parental AAV, wherein the sequence of positions 450-460 or 450-461 of the AAV capsid protein is selected from those provided in Table 2 or from the group consisting of SEQ ID NOs: 379-622.

20. The AAV capsid protein according to claim 16, wherein 60 copies of the AAV capsid protein are assembled into the AAV capsid.

21. The AAV capsid protein according to claim 16, wherein the AAV capsid protein is present in VP1, VP2 and VP3 of the AAV capsid.

22. The AAV capsid protein according to claim 16, further characterized in that When measured in the brain tissue of the subject upon systemic delivery to the subject, the transduction enrichment is increased relative to AAV9.

23. The AAV capsid protein according to claim 16, further characterized in that When measured in the liver tissue of the subject upon systemic delivery to the subject, the transduction enrichment is decreased relative to AAV9.

24. An AAV capsid protein, which comprises: a first sequence, which is provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378; and a second sequence, which is provided in Table 2 or selected from the group consisting of SEQ ID NOs: 379-622.

25. The AAV capsid protein according to claim 24, wherein the first sequence comprises an insertion sequence X of formula I 1 -X 2 -G-H-I-X 3 -I(I)(SEQ ID NO:2) wherein X 1 is an amino acid selected from R, A, and F; X 2 is an amino acid selected from D, N, and A; and X 3 is an amino acid selected from L and F.

26. The AAV capsid protein according to claim 25, wherein X 1 is R.

27. The AAV capsid protein according to claim 2, wherein X 1 is A.

28. The AAV capsid protein according to claim 25, wherein X 2 is N.

29. The AAV capsid protein according to claim 25, wherein X 3 is L.

30. The AAV capsid protein according to claim 24, wherein the first sequence is selected from the group consisting of: AQRDGHILIAK (SEQ ID NO:3), AQANGHILIAK (SEQ ID NO:4), AQANGHILIAR (SEQ ID NO:5), AQFNGHILIAK (SEQ ID NO:6), AQRAGHILIAP (SEQ ID NO:7), AQRNGHIFIAH (SEQ ID NO:8), AQRNGHIFIAK (SEQ ID NO:9), AQRNGHIFIAR (SEQ ID NO:10), AQRNGHILIAK (SEQ ID NO:11), AQRNGHILIAQ (SEQ ID NO:12), AQRNGNILIAK (SEQ ID NO:13) and AQRNGQILIAK (SEQ ID NO:14).

31. The AAV capsid protein according to claim 24, which comprises AAV9 as the parental AAV.

32. The AAV capsid protein according to claim 31, wherein the parental AAV comprises SEQ ID NO:

1.

33. The AAV capsid protein according to claim 32, which comprises a heptameric insertion or an octameric insertion inserted between amino acid 588 and amino acid 589 of the parental AAV, wherein positions 587-597 or 587-598 of the AAV capsid protein are selected from the sequences provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378.

34. The AAV capsid protein according to claim 33, which comprises one or more substitutions at positions 452-460 relative to the parental AAV and an optional insertion at any one of positions 452-453 to 458-459, wherein positions 450-460 or 450-461 of the AAV capsid protein are selected from the sequences provided in Table 2 or selected from the group consisting of SEQ ID NOs: 379-622.

35. The AAV capsid protein according to claim 24, wherein 60 copies of the AAV capsid protein are assembled into the AAV capsid.

36. The AAV capsid protein according to claim 24, wherein the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid.

37. The AAV capsid protein according to claim 24, further characterized in that When measured in the brain tissue of the subject upon systemic delivery to the subject, the transduction enrichment is increased relative to AAV9.

38. The AAV capsid protein according to claim 24, further characterized in that When measured in the liver tissue of the subject upon systemic delivery to the subject, the transduction enrichment is decreased relative to AAV9.

39. The AAV capsid protein according to claim 30, wherein the AAV capsid protein further comprises an amino acid substitution relative to the parental AAV, and the amino acid substitution comprises one or more of the following: A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H.

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