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

Abstract

Described herein are compositions and kits containing recombinant adeno-associated viruses (rAAVs) with increased transduction enrichment in the brain and, in some cases, decreased transduction in the liver. The rAAV compositions described herein encapsidate transgenes, such as therapeutic nucleic acids. Gene therapy using rAAVs is described. Methods for treating brain-related diseases and conditions are also described. The present invention provides AAV capsid proteins, for example, comprising a sequence provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378.

Description

[Background technology]

[0001] background Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications due to their ability to transduce both dividing and non-dividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them attractive for therapeutic applications such as gene therapy. However, there is a need to significantly improve the performance of existing AAV serotypes to be selectively and efficiently expressed in different cell types upon systemic delivery to a subject. This need is particularly acute when AAV must be expressed in the brain.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in XML format and is incorporated by reference herein 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 [Means for solving the problem]

[0003] Summary of the Invention Disclosed herein are rAAVs with engineered peptide insertions and substitutions into the capsid structure through iterative rounds of selection in non-human primates (NHPs) that result in variants with increased transduction as measured in the brain and / or decreased expression in the liver compared to wild-type rAAVs based on the variants.

[0004] The present invention provides rAAVs that exhibit extensive brain transduction but reduced liver transduction. After IV injection, unmodified rAAVs, such as those derived from AAV9 (SEQ ID NO: 1), may not exhibit sufficient tissue enrichment to treat many human diseases through delivery of AAV cargo. The directed evolution of AAV9 as described herein has provided modified rAAVs that exhibit increased viral tissue enrichment in the brain. Therefore, engineered rAAVs as described herein are particularly useful for delivering DNA cargo to brain tissue. Furthermore, when current rAAVs are administered to patients in amounts high enough to provide effective disease treatment, off-target enrichment in certain tissues, such as the liver, can cause immune response problems. Therefore, in certain embodiments, the modified rAAVs disclosed herein have been selected not only for increased brain transduction but also for reduced liver transduction.

[0005] The present invention provides, in certain embodiments, an AAV capsid protein comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I.

[0006] Certain embodiments of the present invention include modified AAV capsid proteins, wherein the capsid protein comprises a peptide insertion / substitution comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I. The modified capsid proteins of the present invention may be characterized by increased brain transduction in a subject. Certain embodiments may provide modified AAV capsid proteins, wherein the capsid protein comprises a peptide insertion / substitution comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I, and the modified capsid proteins are characterized by decreased liver transduction in a subject. When increased or decreased transduction in a particular tissue is discussed herein, it may be compared to an unmodified or wild-type AAV capsid protein.

[0007] The present disclosure further includes a pharmaceutical composition comprising an rAAV having a peptide insertion / substitution comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I, and a pharmaceutically acceptable excipient.

[0008] Aspects disclosed herein provide a method for treating a disease or condition in a subject, comprising administering a therapeutically effective amount of a pharmaceutical preparation comprising the AAV capsid protein or AAV capsid of the present disclosure. In some embodiments, the disease or condition is a brain disease or condition of the subject's brain. Relatedly, the present invention includes the use of rAAV in the manufacture of a medicament for treating or preventing a disease or medical condition.

[0009] Other aspects of the present invention will become apparent from the detailed description and claims that follow.

[0010] 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. [Brief explanation of the drawings]

[0011] [Figure 1-1] Figure 1 shows the inserted and substituted amino acid sequences of AAV capsid proteins that were found to have increased brain enrichment and / or decreased liver enrichment in non-human primate brain compared to the parental capsid. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 1-9] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of Disclosure In certain aspects, the present disclosure provides modified rAAVs that have reduced expression levels in the liver and increased expression levels in the brain when compared to the parent AAV (e.g., AAV9).

[0013] In certain aspects, the present disclosure provides rAAVs having peptide insertions and / or substitutions comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I.

[0014] Embodiments disclosed herein include a compound having an amino acid sequence of Formula I X 1 -X 2 -GHIX 3 -I(I) (SEQ ID NO: 2) (In the formula, X 1 is an amino acid selected from R, A, and F, and X 2 is an amino acid selected from D, N, and A, and X 3 is an amino acid selected from The present invention provides an AAV capsid comprising an AAV capsid protein comprising:

[0015] In some embodiments, the AAV capsid protein is X 1 is R.

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

[0017] In some embodiments, the AAV capsid protein is X 2 is N.

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

[0019] In some embodiments, the peptide insertion and / or replacement sequence is 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).

[0020] In some embodiments, the insertion and / or flanking substitution sequences are represented by the peptide sequences listed in Table 1. [Table 1]

[0021] In certain embodiments, the parent AAV is AAV9. In some embodiments, the parent AAV comprises SEQ ID NO: 1. In various embodiments, the AAV capsid protein comprises a 7-mer insertion inserted into the parent AAV between amino acids 588 and 589 of the parent AAV, wherein positions 587-597 (using AA position numbering including the insertion) 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.

[0022] The AAV capsid protein may comprise amino acid substitutions compared to the parent AAV, including one or more of A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H, where the position number refers to the amino acid position in the parent AAV prior to any insertion.

[0023] In some embodiments, the sequence of 450-460 or 461 (depending on the presence of the optional insertion) is represented by a peptide sequence listed in Table 2 or selected from the group consisting of SEQ ID NOs: 379-622. [Table 2]

[0024] Embodiments of the invention may include AAV capsid proteins 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.

[0025] Generally, insertions comprise 5, 6, 7, or 8 amino acid sequences (5-mer, 6-mer, 7-mer, or 8-mer, respectively) inserted into or substituted into the 588 loop of the parent AAV capsid protein. Embodiments provided herein provide amino acid insertions comprising 7 or 8 amino acid polymers (7-mers or 8-mers) inserted at AA588-589, which may additionally comprise substitutions of one or two amino acids at amino acid positions adjacent to the 7-mer sequence (e.g., AA587-588 and / or AA589-590, using the parent amino acid position numbering) to create 11 amino acid polymers (11-mers) in the 588 loop of the parent AAV capsid protein. Column 4 of Figure 1 lists the 7-mer or 8-mer AA insertion sequences at 588-589, along with the two flanking amino acids on each side of the insertion (corresponding to positions AA587, 588, 589, and 590 in the parent capsid). The flanking amino acids may contain substitutions compared to the parent AAV capsid protein. The first column in Figure 1 lists the AA sequence at positions 450-460 compared to the parent capsid (or 461, in the case of an optional insertion compared to the parent capsid).

[0026] In some embodiments, the inserted amino acid sequence is at least 71.4% identical to the amino acid sequence provided in Tables 1-3, Figure 1, and / or Formula I. In some embodiments, the inserted amino acid sequence is at least 86.7% identical to the amino acid sequence provided in Tables 1-3, Figure 1, and / or Formula I.

[0027] Also disclosed herein are methods and kits for making therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising the rAAV particles, for the treatment of diseases or conditions affecting the brain.

[0028] The present disclosure provides an engineered AAV capsid that has increased viral transduction in the brain.The AAV capsid can encapsidate a viral vector that carries heterologous nucleic acid, for example, encoding a therapeutic gene expression product.The transduction of heterologous nucleic acid in the brain can be achieved when the AAV capsid of the present disclosure is encapsidated into a subject's systemic delivery.The AAV capsid disclosed herein is advantageous for many uses in gene therapy for treating human diseases, including but not limited to disorders of the central nervous system.

[0029] Also provided herein are 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 comprising AAV viral capsids encoding VP1, VP2, and VP3, at least one of which is modified to produce the AAV capsid proteins of the present disclosure. The provided recombinant AAV vectors can be derived from an AAV serotype (e.g., AAV9) or a variant AAV serotype comprising an insertion of the present invention.

[0030] AAV capsid

[0031] Provided herein are modified adeno-associated (AAV) virus capsid compositions useful for integrating transgenes into target cells or the environment (in a subject when administered systemically to the subject).

[0032] rAAV contains an AAV capsid that can be engineered to encapsidate heterologous nucleic acids (e.g., therapeutic nucleic acids, gene editing machinery). The AAV capsid is composed of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid. VP1 covers the entire VP2 protein plus an approximately 137 amino acid N-terminal region (VP1u), and VP2 covers the entire VP3 protein plus an approximately 65 amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid 138 (e.g., AA139-736).

[0033] Without wishing to be bound by theory, it is understood that the parent AAV capsid sequence comprises VP1 region.In certain embodiments, the parent AAV capsid sequence comprises VP1, VP2 and / or VP3 region, or any combination thereof.The parent VP1 sequence can be considered synonymous with the parent AAV capsid sequence.

[0034] The AAV VP3 structure contains a highly conserved region common to all serotypes: a core eight-stranded β-barrel motif (βB-βI) and a small α-helix (αA). The loop region inserted between the β-strands consists of the unique HI loop between β-strands H and I, the DE loop between β-strands D and E, and nine variable regions (VRs) that form the top of the loops. These VRs, such as the AA588 loop, are found on the capsid surface and may be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity.

[0035] In some embodiments, the rAAV variants of the invention comprise an AAV capsid protein having a peptide insertion at residues corresponding to amino acids 588-589 of the AAV9 native sequence of SEQ ID NO:1.

[0036] The AAV capsid contains AAV capsid proteins (e.g., VP1, VP2, and VP3), each of which has an insertion, such as within the 588 loop of the parent AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains the site for heparan sulfate binding in AAV2 and is suitable for peptide display. The only known receptor for AAV9 is the N-linked terminal galactose and AAV receptor (AAVR), although numerous indications indicate that there are others. Modifications of the AAV9 588 loop to confer increased transgene transduction in a target in vivo environment are presented herein.

[0037] The present invention provides, in certain aspects, peptide insertions in AAV588 loops comprising or consisting of an amino acid sequence set forth in any one of Tables 1-3, FIG. 1, and / or Formula I.

[0038] Disclosed herein is an AAV capsid comprising an AAV capsid protein with an insertion in the 588 loop, which confers higher transduction in brain cell types (for example, brain endothelial cells, neurons, astrocytes).In particular, the AAV capsid protein disclosed herein enables rAAV-mediated transduction of heterologous nucleic acid (for example, transgene) in the brain of a subject.The AAV capsid of the present disclosure can be formulated as a pharmaceutical composition.In addition, the AAV capsid can be isolated and purified for use in various applications.

[0039] 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, the rAAV or variant AAV proteins confer increased localization of the rAAV in target tissues compared to the parent AAV capsid or capsid protein contained therein.

[0040] AAV capsid protein Disclosed herein are recombinant AAV (rAAV) capsids comprising AAV capsid proteins engineered with modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the disclosed rAAV capsid proteins are produced using the methods disclosed herein. In some embodiments, the AAV capsid proteins are used in methods for delivering therapeutic nucleic acids (e.g., transgenes) to a subject. In some cases, the rAAV capsid proteins have desired AAV expression, making them particularly suitable for certain therapeutic applications, e.g., for treating diseases or disorders in a subject, such as those disclosed herein.

[0041] The rAAV capsid protein is engineered for optimized expression in the CNS, e.g., brain, of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid protein is engineered to contain an insertion as provided in Tables 1-3, Figure 1, and / or Formula I. The rAAV capsid protein containing an insertion as provided in Tables 1-3, Figure 1, and / or Formula I is engineered to achieve efficient transduction of the encapsidated transgene. In particular, the rAAV capsid protein has increased expression in the subject's brain.

[0042] The engineered AAV capsid proteins described herein, in some cases, have an insertion of an amino acid heterologous to the parent AAV capsid protein at an amino acid position within the 588 loop. In some embodiments, the amino acid is not endogenous to the parent AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid at the same or equivalent amino acid position as the replacement insertion in a different AAV capsid protein.

[0043] The heptamer described herein is advantageously generated using polymerase chain reaction (PCR) with degenerate primers, where each of the seven amino acids is encoded by the deoxyribose nucleic acid (DNA) sequence NNK. "N" is any of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method of generating random heptamer amino acid sequences allows for 1.28 billion possible combinations at the protein level. Certain heptamer sequences are subsequently modified by single amino acid substitutions or insertions, in some cases resulting in octamer sequences for insertion, as seen in Figure 1.

[0044] The rAAV capsid proteins of the present disclosure may contain amino acid insertions in the amino acid sequence of the AAV capsid protein. The AAV capsid from which the engineered AAV capsid proteins of the present disclosure are generated is referred to as the "parent" AAV capsid. The complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, and the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862, 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., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), the AAV-11 genome is provided in Virology, 330(2):375-383 (2004), a portion of the AAV-12 genome is provided in GenBank accession number DQ813647, and a portion of the AAV-13 genome is provided in GenBank accession number EU285562.

[0045] 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 may be a variant AAV capsid protein. For example, the variant may contain a heterologous amino acid in the amino acid sequence of the AAV capsid protein. The heterologous amino acid may not naturally occur in the AAV capsid protein. The heterologous amino acid may naturally occur in a different AAV capsid protein. In some cases, the parent AAV capsid is described in U.S. Patent Application Publication No. 2020 / 0165576, U.S. Provisional Patent Application Publication No. 62 / 832,826, and PCT / US20 / 20778, the contents of each of which are incorporated herein.

[0046] In some cases, the parent AAV is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of the AAV9 VP1 capsid protein (>tr|Q6JC40|Q6JC40_9VIRU capsid protein VP1 OS=adeno-associated virus 9 OX=235455 GN=cap PE=1 SV=1) comprises SEQ ID NO: 1. [ka] In some cases, the parent AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO:1.

[0047] AAV capsid proteins from natural AAV serotypes, such as AAV9, which have tropism that includes the liver, activate the innate immune response and, in some cases, can cause a severe inflammatory response in subjects, leading to multiple organ failure. By improving transduction of natural AAV serotypes to target in vivo tissues (e.g., the brain) and potentially reducing transduction of off-target tissues (e.g., the liver), the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent activation of the innate immune response.

[0048] In some cases, the parent AAV capsid protein includes the entire VP1 region (e.g., amino acids 1-736) provided in SEQ ID NO: 1. In some cases, the parent AAV capsid protein includes amino acids 217-736 in SEQ ID NO: 1, which is a common region found in the VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein includes amino acids 64-736 in SEQ ID NO: 1, which is a common region found in VP1 and VP2. The parent AAV capsid protein sequences are 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200-736, 210-736, 220-736, 230-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, 450-736, 460-736, 470-736, 480-736, 490-736, 500-736, 510-736, 520-736, 530-736, 540-736, 550-736, 560-736 In some embodiments, the rAAV variant may comprise an AAV capsid protein comprising 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-fold symmetry axis of the corresponding parent AAV capsid protein.

[0049] Insertions of amino acid sequences into AAV capsid proteins are disclosed herein. Where the sequence numbering designation "588-589" is used with respect to AAV9, e.g., AAV VP1, the present invention also encompasses insertions in similar positions in other AAV serotypes. As used herein, "AA588-589" indicates that the insertion of an amino acid (or amino acid sequence) occurs immediately after the amino acid (AA) at position 588 and immediately before the AA at position 589 in the amino acid sequence of the parent AAV VP capsid protein (VP1 numbering). Amino acids 587-591 contain an "AQAQA"-containing motif as set forth 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 amino acid sequence of the AAV9 capsid with a 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 positions AA588-589 or AA587-590 (substituting amino acids AA587-590) in the amino acid sequence of the parent AAV9 capsid protein, variants thereof, or the equivalent amino acid position in a parent AAV of a different serotype (e.g., AAV1, AAV2, AAV3, etc.). In certain embodiments, the aforementioned "AQAQ" sequence adjacent to the insertion may contain one or more substitutions. In any AAV capsid protein sequence disclosed herein, the amino acid at position 449 may be R or K. The sequence may contain one or more substitutions and / or insertions between positions 450 and 460 (inclusive). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0050] The insertions described herein may, in some cases, include a 7-mer or 8-mer insertion at AA 588-589. In addition to substitutions with any amino acid at amino acid positions 587-590 [AQAQ], it is contemplated that any 7-mer or 8-mer insertion disclosed herein may constitute an 11-mer or 12-mer sequence selected from Figure 1 or Table 1.

[0051] Disclosed herein is an AAV capsid protein that has the above-mentioned insertion in the parent AAV capsid protein, which provides increased transduction in the brain of a subject even when delivered systemically.One of the many advantages of the AAV capsid protein described herein is their ability to target tissues and cells in the brain.Tissue can be 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 detargeting effect on liver tissue compared with parent AAV capsid proteins.

[0052] In some cases, the AAV capsid protein comprises an insertion / substitution of at least or about 7, 8, 9, 10, 11, or 12 amino acids of the amino acid sequence of Tables 1-3, FIG. 1, and Formula I at amino acid positions 588-589 or 587-590 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAV capsid protein has increased viral transduction enrichment in the brain. In some cases, the AAV capsid protein has decreased viral transduction enrichment in the liver.

[0053] The rAAV capsid proteins of the present disclosure can also have amino acid sequence substitutions at amino acids 452-458 of the parent AAV9 capsid protein or variants thereof, as described in WO 2020 / 068990. Exemplary substitutions at amino acids 452-458 of the parent AAV9 capsid protein can be found in the second column of Figure 1 or in Table 2.

[0054] The rAAV capsid protein described herein can be isolated and purified.AAV can be isolated and purified by standard methods in the art, such as by column chromatography, iodixanol gradient, or cesium chloride gradient.Methods for purifying AAV from helper virus are known in the art, and can include, for example, the methods disclosed in Clark et al., Hum.Gene Ther.,10(6):1031-1039(1999), Schenpp and Clark, Methods Mol.Med.69:427-443(2002), U.S. Patent No. 6,566,118 and WO 98 / 09657.

[0055] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, may in some cases be formulated into pharmaceutical preparations which further comprise a pharmaceutically acceptable carrier.

[0056] The rAAV capsid protein can be conjugated to nanoparticles, second molecules, or viral capsid proteins.In some cases, the nanoparticles or viral capsid proteins encapsidate the therapeutic nucleic acid described herein.In some cases, the second molecule is a therapeutic agent, such as a small molecule, an antibody, an antigen-binding fragment, a peptide, or a protein, such as those described herein.

[0057] "Percent identity" is the percent of symbols that actually match. Percent similarity is the percent of symbols that are similar. Symbols across a gap are ignored. Similarity is scored when the score matrix value for a pair of symbols exceeds or equals a similarity threshold of 0.50. The scoring matrix used in version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0058] The sequence identity / similarity values ​​provided herein may refer to values ​​obtained using the BLAST+ 2.5.0 suite of programs using default settings (blast.ncbi.nlm.nih.gov) (Camacho, C. et al. (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0059] As those skilled in the art can understand, BLAST search assumes that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequences, which may be homopolymer tracts, short-period repeats, or regions enriched with one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even if other regions of the proteins are not completely similar. Several low-complexity filter programs can be used to reduce such low-complexity alignments. For example, SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Ci-ayerie and States (1993) Comput. Chem. 17:191-201) low-complexity filters can be used alone or in combination.

[0060] The terms "substantial identity" and "substantially identical" refer to a polypeptide or nucleic acid having between 55-100% sequence identity to a reference sequence, at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity, or any percentage value within the range of 55-100% sequence identity to a reference sequence. The percent sequence identity may occur over a specified comparison window. Optimal alignment may be confirmed or performed using the Needleman and Wunsch homology alignment algorithm described above.

[0061] For example, an inserted sequence may include, but is not limited to, a sequence that is not exactly the same as a sequence disclosed herein, but has, in addition to the substitutions explicitly set forth for the various sequences listed herein, additional substitutions of amino acid residues that do not substantially impair the activity or properties of the sequences described herein, such as those predicted by homology software, for example, the BLOSUM62 matrix.

[0062] AAV particles The rAAV particles having the insert sequences described herein have increased transduction enrichment in the brain.In some cases, the increased transduction enrichment includes 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more increase.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.

[0063] The rAAV particles with the insert sequence described herein have increased expression enrichment in the brain.Detecting whether rAAV has more or less target specificity in an in vivo environment includes measuring the level of gene expression product (for example, RNA or protein) expressed from the heterologous nucleic acid encapsidated by rAAV in tissue samples obtained from subject.Preferred methods for measuring the expression of gene expression product include next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0064] heterologous nucleic acid Disclosed herein are therapeutic nucleic acids useful for treating or preventing a disease or condition, or a symptom of a disease or condition. In some embodiments, the therapeutic nucleic acid encodes a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.), and gene editing components for use in treating, preventing, and / or ameliorating a disease or disorder, or a symptom of a disease or disorder. In some cases, the therapeutic nucleic acid is placed into a subject's organism, cell, tissue, or organ by an rAAV, such as those disclosed herein.

[0065] Disclosed herein are rAAVs, each comprising a viral vector (e.g., a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector comprises two inverted terminal repeat (ITR) sequences, each approximately 145 bases long, flanking the transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid and, in some cases, a promoter in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter can initiate transcription of the therapeutic nucleic acid in the nucleus of the 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 ITRs are from AAV2. In some cases, the ITRs are from AAV9.

[0066] Disclosed herein are transgenes that can comprise any number of nucleotides. In some cases, a transgene can comprise fewer than about 100 nucleotides. In some cases, a transgene can comprise at least about 100 nucleotides. In some cases, a transgene can comprise at least about 200 nucleotides. In some cases, a transgene can comprise at least about 300 nucleotides. In some cases, a transgene can comprise at least about 400 nucleotides. In some cases, a transgene can comprise at least about 500 nucleotides. In some cases, a transgene can comprise at least about 1000 nucleotides. In some cases, a transgene can comprise at least about 5000 nucleotides. In some cases, a transgene can comprise more than 5000 nucleotides. In some cases, a transgene can comprise between about 500 and about 5000 nucleotides. In some cases, a transgene comprises about 5000 nucleotides. In any of the cases disclosed herein, a transgene can comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene may be single-stranded. In some cases, the transgene may be double-stranded.

[0067] The present paper discloses a transgene that is useful for regulating the expression or activity of target gene or its gene expression product.In some cases, the transgene is encapsidated by the rAAV capsid protein of the rAAV particle described herein.In some cases, the rAAV particle is delivered to a subject to treat the disease or symptoms disclosed herein in the subject.In some cases, the delivery is systemic.

[0068] The transgenes disclosed herein are useful for expressing endogenous genes at levels similar to those of healthy or normal individuals. This is particularly useful for treating diseases or conditions associated with underexpression or lack of expression of a gene expression product. In some embodiments, the transgenes disclosed herein are useful for overexpressing endogenous genes such that the expression level of the endogenous gene exceeds that of a healthy or normal individual. Additionally, transgenes can be used to express exogenous genes (e.g., active agents such as antibodies, peptides, nucleic acids, or gene editing components). In some embodiments, the therapeutic gene expression product can alter, enhance, increase, or induce the activity of one or more endogenous biological processes in a cell. In some embodiments, the transgenes disclosed herein are useful for reducing the expression of endogenous genes, e.g., dominant-negative genes. In some embodiments, the therapeutic gene expression product can alter, inhibit, reduce, prevent, eliminate, or impair the activity of one or more endogenous biological processes in a cell. In some embodiments, an increase in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the protein product of the targeted gene may be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In some embodiments, a decrease in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. In certain embodiments, the protein product of the targeted gene may be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%.

[0069] When an endogenous sequence (part of an endogenous or transgene) is expressed together with a 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 function of these full-length or partial sequences include extending the serum half-life of a polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0070] A transgene can be inserted into an endogenous gene such that all, some, or none of the endogenous gene is expressed. For example, a transgene as described herein can be inserted into an endogenous locus such that only a portion of the endogenous sequence (e.g., the N-terminus and / or C-terminus of the transgene) is expressed, e.g., as a fusion with the transgene. In other cases, a transgene (e.g., with or without additional coding sequence of the endogenous gene) can be integrated into any endogenous locus, e.g., 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., a gene as described herein.

[0071] 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 5,000 amino acids in length. In some embodiments, the encoded protein is between 50 and 2,000 amino acids in length. In some embodiments, the encoded protein is between 50 and 1,000 amino acids in length. In some embodiments, the encoded protein is between 50 and 1,500 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 to 30 amino acids, such as 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 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 50 amino acids or less, such as 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids or less.

[0072] Non-limiting examples of therapeutic proteins or peptides include adrenergic agents, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoietic agents, glutamic acid decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartate antagonists, plexins, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphoring, semaphorin receptors, serotonin transporter proteins, serotonin uptake inhibitors, serotonin receptors, serpins, serpin receptors, and tumor suppressors. In certain embodiments, the therapeutic protein or peptide is selected from the group consisting of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), 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 (TGF), and phosphoinositide (PG). The inhibitor is selected from transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, somatotropin, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.

[0073] The therapeutic gene expression product may include a gene editing component. Non-limiting examples of gene editing components include those required for CRISPR / Cas, artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), and transcription factor-like effector nucleases (TALENs). In a non-limiting example, a subject with Huntington's disease is identified. The subject is then systemically administered a first dose of rAAV that encapsidates a viral vector encoding a ZFN engineered to suppress transcription of the huntingtin (HTT) gene. The rAAV contains a modified AAV capsid protein comprising an amino acid sequence as provided in any one of Tables 1-3, Figure 1, and Formula I, allowing for proper targeting of the ZFN to the nervous system while reducing expression in off-target organs such as the liver. If necessary, the subject is administered a second or third dose of rAAV until a therapeutically effective amount of the ZFN is expressed in the subject's nervous system.

[0074] Therapeutic nucleic acids may include sequences encoding non-protein-coding genes, such as antisense RNA, RNAi, shRNA, and microRNA (miRNA), miRNA sponges, or decoys, including those required for conditional gene deletion and recombinase delivery for conditional (recombinase-dependent) expression, as described herein. Non-protein-coding genes may also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (lncRNA). In some cases, non-protein-coding genes can regulate the expression or activity of target genes or gene expression products. For example, the RNAs described herein can be used to inhibit gene expression in the 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 either a wild-type gene or a gene with at least one mutation. The targeted protein can be either a wild-type protein or a protein with at least one mutation.

[0075] Therapeutic nucleic acid can regulate the expression or activity of gene or gene expression product expressed from gene that is involved in brain disease or disorder.For example, in some cases, therapeutic nucleic acid is a modified version of gene or gene described herein.In some cases, gene or gene expression product is inhibited.In some cases, gene or gene expression product is enhanced.

[0076] In another example, the therapeutic nucleic acid comprises an effector gene expression product, such as a gene editing component, specific for targeting a gene therein. Non-limiting examples of genes include ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and survival of motor neurons. Neuron 1, STXBP1, telomere (SMNl), factor X (FIX), retinoid isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG-binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SODl), iduronate 2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some cases, the gene or gene expression product is inhibited. In some cases, the gene or gene expression product is enhanced.

[0077] AAV vectors The embodiments disclosed herein include plasmid vectors that contain the nucleic acid sequences encoding the AAV capsids and AAV capsid proteins described herein.The AAV vectors described herein are useful for rAAV assembly and viral packaging of heterologous nucleic acids.In addition, AAV vectors can encode transgenes that contain heterologous nucleic acids.

[0078] In some cases, AAV vectors may contain a transgene encoding a heterologous gene expression product (e.g., a therapeutic gene expression product, a recombinant capsid protein, etc.). The transgene is in cis with two inverted terminal repeats (ITRs) flanking the transgene. The transgene may contain a therapeutic nucleic acid encoding the therapeutic gene expression product. Due to the limited packaging capacity of rAAV (approximately 5 kB), in some cases, longer transgenes may be split between two AAV vectors (the first one with a 3' splice donor and the second one with a 5' splice acceptor). Upon coinfection of cells, concatemers form, which are spliced ​​together to express the full-length transgene.

[0079] A transgene is generally inserted so that its expression is driven by the endogenous promoter at the integration site, i.e., the promoter that drives the expression of the endogenous gene into which the transgene is inserted.In some cases, the transgene includes a promoter and / or enhancer, such as a constitutive promoter or an inducible or tissue / cell-specific promoter.As non-limiting examples, the promoter can be a CMV promoter, a CMV-β-actin-intron-β-globin hybrid promoter (CAG), a CBA promoter, a FRDA or FXN promoter, a UBC promoter, a GUSB promoter, an NSE promoter, a synapsin promoter, a MeCP2 promoter, a GFAP promoter, an H1 promoter, a U6 promoter, an NFL promoter, an NFH promoter, an SCN8A promoter, or a PGK promoter. As non-limiting examples, the promoter may be a tissue-specific expression element, including, but not limited to, human elongation factor 1 α-subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC). The transgene may contain tissue-specific expression elements for neurons, such as, but not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk, and EAAT2 promoters. The transgene may contain tissue-specific expression elements for astrocytes, such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may contain tissue-specific expression elements for oligodendrocytes, such as, but not limited to, the myelin basic protein (MBP) promoter.

[0080] In some embodiments, the promoter is less than 1 kb. 0, 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 in length. The promoter can have a length of between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800. The promoter can provide expression of the therapeutic gene expression product over a period of time in a targeted tissue, such as, but not limited to, the brain.Therapeutic gene expression products were measured at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days , 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 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 The term may be between 1 year, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 ​​years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years. Expression of the payload can be 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.

[0081] AAV vectors can contain the genome of a helper virus. Helper virus proteins are necessary for the assembly of recombinant AAV (rAAV) and the packaging of transgenes containing heterologous nucleic acids into rAAV. Helper virus genes are adenovirus genes E4, E2a, and VA, which support AAV replication when expressed in cells. In some embodiments, AAV vectors contain E2. In some embodiments, AAV vectors contain E4. In some embodiments, AAV vectors contain VA. In some cases, AAV vectors contain one or any combination of helper virus proteins.

[0082] Target genes or gene expression products for use in transgenes include ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), and survival of motor neurons. Neuron)1, STXBP1, telomere (SMNl), factor X (FIX), retinoid isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG-binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SODl), iduronate 2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0083] The AAV vector may comprise a viral genome including a nucleic acid encoding a recombinant AAV (rAAV) capsid protein described herein. The viral genome may comprise a Rep gene encoding a replication (Rep) protein and a Cap gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some cases, the Cap gene is modified to encode a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three of VP1 through VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.

[0084] In some cases, the AAV9 VP1 gene provided in SEQ ID NO:641 shown in Table 4 can be modified to encode any of the insertions and / or substitutions found in Figure 1. The AAV vectors described herein can be used to generate variant AAV capsids by the methods described herein. [Table 4-1] [Table 4-2]

[0085] How to make rAAV Disclosed herein is a method for producing AAV capsids, including AAV capsid proteins and viral vectors encoding therapeutic nucleic acids. The AAV capsid proteins are produced by introducing into cells (e.g., immortalized stem cells) a first vector containing a transgene cassette (the transgene cassette has a promoter sequence that drives the transcription of a heterologous nucleic acid in the nucleus of a target cell) flanked by inverted terminal repeat (ITR) sequences from the parent AAV virus, a second vector encoding an AAV genome (encoding the AAV Rep gene and a modified Cap gene for the variant being produced) containing the AAV capsid proteins, and a third vector encoding helper virus proteins required for the assembly of the AAV capsid structure and the packaging of the transgene into the modified AAV capsid structure. The assembled AAV capsid can be isolated and purified from the cells using suitable methods known in the art.

[0086] Also provided herein is a transgene that is contained in a recombinant AAV (rAAV) vector and is encapsidated by the AAV capsid protein of the present disclosure.The transgene disclosed herein is delivered to a subject for various purposes, such as treating a disease or symptom in the subject.The transgene can be a gene editing component that regulates the activity or expression of a target gene or gene expression product.Alternatively, the transgene is a gene that encodes a therapeutic gene expression product that is effective in regulating the activity or expression of itself or another target gene or gene expression product.

[0087] Embodiments disclosed herein provide a method for producing a rAAV virus or viral particle, the method comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector comprising a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parent AAV virus (the transgene cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell); (ii) a second vector encoding an AAV genome having an AAV capsid protein of the present invention; and (iii) a vector encoding helper virus proteins required for assembly of an AAV capsid structure and packaging of a transgene into the modified AAV capsid structure; (b) expressing in the cell the AAV capsid proteins described herein; (c) assembling AAV particles comprising the AAV capsid proteins disclosed herein; and (d) packaging the AAV particles. Optionally, the cell is mammalian. Optionally, the cell is immortalized. Optionally, the immortalized cell is an embryonic stem cell. 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 (HEK-293). In some cases, the Cap gene is derived from the deoxyribose nucleic 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 present in trans. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are present in cis. In some cases, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are present in trans.

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

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

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

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

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

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

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

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

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

[0097] In some cases, the disease or condition is localized to a particular in vivo environment in a subject, for example, the brain. The compositions of the present disclosure are particularly useful for treating the diseases or conditions described herein because they specifically or more efficiently target the in vivo environment and deliver therapeutic nucleic acids engineered to regulate the activity or expression of target gene expression products involved in the etiology or pathology of the disease or condition.

[0098] Provided herein are methods of treating a disease or condition, or a symptom of a disease or condition, in a subject, comprising: (a) diagnosing a subject 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), which has been engineered to have increased specificity for the target in the in vivo environment.

[0099] Disclosed herein are targeted methods for treating a disease or condition or a symptom of a disease or condition in a subject, 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 in the subject that has increased transduction enrichment.

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

[0101] In some embodiments, a method of treating a disease or condition affecting the brain comprises administering rAAV particles to the brain in a subject, wherein the rAAV particles comprise an rAAV capsid protein comprising an insertion of about 5, 6, 7, or 8 amino acids of the amino acid sequence provided in Tables 1-3, Figure 1, and Formula I at amino acid positions 588-589 of the parent AAV capsid protein. In some embodiments, a method of treating a disease or condition affecting the brain comprises administering rAAV particles to the brain in a subject, wherein the rAAV particles comprise an rAAV capsid protein comprising an insertion of about 5, 6, 7, or 8 amino acids of the amino acid sequence as well as one or more substitutions at amino acids [AQAQ] found at amino acid positions 587-590 as provided in Tables 1-3, Figure 1, and Formula I. In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein (e.g., provided in SEQ ID NO: 1).

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

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

[0104] In some embodiments, the brain disease or condition is selected from the group consisting of: absence of the septum pellucidum, acid lipase disease, acid maltase deficiency, acquired epileptiform aphasia, acute disseminated encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Addie pupil, Addie syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Goutières syndrome disorder, AIDS-neurological complications, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), anencephaly, aneurysm, Angelman syndrome, hemangiomatosis, anoxia, antiphospholipid syndrome, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's 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, Behçet's disease, Bell's palsy, benign idiopathic blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bernhard-Ross syndrome, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, at birth Brachial plexus injury, brachial plexus injury, Bradbury-Eggleston syndrome, brain and spinal cord tumors, cerebral aneurysm, brain injury, Brown-Séquard syndrome, spinal-bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarctions and leukoencephalopathy (CADASIL), Canavan disease, carpal tunnel syndrome, burning pain, cavernoma, cavernous hemangioma, cavernous malformation, central cervical spinal cord syndrome, central spinal cord syndrome, central pain syndrome, central pontine myelinolysis, head injury, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, Cerebral hypoxia, cerebral convulsion, cerebro-cutaneous-facial-skeletal syndrome (COFS), Charcot-Marie-Tooth disease, Charcot-Marie-Tooth syndrome, classic rhizomelic chondrodysplasia punctata (RCDP), Chiari malformation, cholesterol ester storage disease, chorea, acanthocytic chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome, Cockayne syndrome type II, Coffin-Lowry syndrome, agenesis of the corpus callosum, coma, complex regional pain syndrome, congenital ophthalmoplegia, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformation,Corticobasal degeneration, cranial arteritis, craniosynostosis, Klee encephalitis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalic inclusion disease, cytomegalovirus infection, Dancing eyes, dancing feet syndrome, Dandy-Walker syndrome, Dawson's disease, hearing loss, Domorsia syndrome, Dejarin-Klumpke palsy, dementia, dementia-multiinfarct, dementia-semantic, dementia-subcortical, dementia with Lewy bodies, dentate cerebellar ataxia, dentate atrophy, dermatomyositis, developmental coordination disorder, Devic's syndrome, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, Duchenne muscular dystrophy Fee, autonomic neuropathy, dysgraphia, dyslexia, dysphagia, dyspraxia, myoclonic cerebellar dyssynergia, progressive cerebellar dyssynergia, myotonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalopathy (familial infantile), trigeminal angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Erb-Duchenne-Krampke palsy, essential tremor, extrapontine myelin sheath Disintegrative syndrome, Fabry disease, Fahr syndrome, syncope, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, hypotonic infantile syndrome, foot drop, fragile X syndrome, Friedreich ataxia, frontotemporal dementia (FTD), Gaucher disease, systemic gangliosidosis, Gerstmann syndrome group, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, giant cell inclusion body disease, glioblastoma, globoid cell leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Hallervorden-Spatz disease, head injury, headache, persistent hemicrania, hemifacial spasm, alternating hemiplegia, hereditary neuropathies, hereditary spastic paraplegia, hereditary polyneuropathies, herpes zoster Zoster), herpes zoster oticus, Hirayama syndrome, Holmes-Adie syndrome, holoprosencephaly, HTLV-1-associated myelopathy, Hughes syndrome, Huntington's disease, hydranencephaly, hydrocephalus, hydrocephalus-normotensive, hydromyelopathy, hyperadrenocorticism, hypersomnia, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease (IRD), infantile spasms, inflammatory myopathy, forencephaly prolapse, intestinal lipodystrophy, intracranial cyst, increased intracranial pressure, Isaacs syndrome, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Kleine-Levin syndrome, Klippel-Fehr syndrome, Klippel-Trenaunay syndrome (KTS), Klüver-Bucy syndrome, Korsakoff amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, kuru, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Lateral Femoral Cutaneous Nerve Pain Entrapment, Lateral Cord Syndrome, Learning Disability, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Cerebral Leukodystrophy, Levin-Critchley Syndrome, Dementia with Lewy Bodies, Lipid Storage Disease, Lipoproteinosis, Ligliosis, Locked-In Syndrome, Lou Gehrig's Disease, Lupus - Neurological Sequelae, Lyme Disease - Neurological Complications, Machado-Joseph Disease, Megalencephaly, Maple Syrup Urine Disease, Megalencephaly, Merkelson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalopathy, Menkes Disease, Menkes Syndrome, Dysesthesias and Femoral Neuralgia (Meralgia)Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller-Fisher Syndrome, Minor Stroke, Mitochondrial Myopathy, Moebius Syndrome, Unilateral Muscular Atrophy, Motor Neuron Disease, Moyamoya Disease, Mucolipidosis, Mucopolysaccharidosis, Mucopolysaccharidosis II, Multiple Obstructive Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia - Congenital, Myasthenia Gravis, Myeloablative Diffuse Sclerosis, Infantile Myoclonic Encephalopathy, Myoclonus, Myopathy, Myopathy - Congenital, Myopathy - Thyrotoxicity, Myotonia, Myotonia Congenita, Myotonic Dystrophy Strophy, narcolepsy, chorea acanthocytosis, neurodegeneration with cerebral iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of Lyme disease, neurological effects of cytomegalovirus infection, neurological symptoms of Pompe disease, neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorders, neuropathy - congenital, neurosarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Otahara syndrome, olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndromes, Pain - chronic, pantothenate kinase-related neurodegeneration, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal chorea, paroxysmal migraine, Parry-Romberg disease, Pelizaeus-Merzbach disease, Pena-Choqueir II syndrome, perineural cyst, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phenylketonuria, phytanic acid storage disease, Pick's disease, pincer nerve, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, porencephaly, post-polio syndrome, postherpetic neuralgia, infectious encephalomyelitis, orthostatic hypotension, postural orthostatic tachycardia syndrome, orthostatic tachycardia syndrome, Prader-Willi syndrome, primary dentate atrophy, primary Lateral sclerosis, primary progressive aphasia, prion disease, progressive hemiface atrophy, progressive gait ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing gray matter dystrophy, progressive supranuclear palsy, prosopagnosia, pseudotorch syndrome, pseudotoxoplasmosis syndrome, pseudotumor cerebri, psychogenic movement disorder, Ramsay-Hunt syndrome I, Ramsay-Hunt syndrome II, Rasmussen encephalopathy, reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease-infancy, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye syndrome, rheumatic encephalopathy, Riley-Day syndrome, sacral nerve root cyst, chorea (Saint Vitus)Dance), salivary gland disease, Sandhoff disease, Schilder's disease, schizencephaly, Seitelberger's disease, seizure disorder, semantic dementia, septo-optic dysplasia, severe myoclonic epilepsy in infants (SMEI), shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleep disorders, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor tumor, 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 spinal sclerosis, syringomyelia, spinal cord emptying Sinus disease, systemic lupus erythematosus, tabes dorsalis, Tangier disease, tardive dyskinesia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen myotonia, thoracic outlet syndrome, thyrotoxic myopathy, painful tics, Todd's palsy, Tourette's syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myositis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer's syndrome, tuberous sclerosis, vascular erectile dysfunction tumors, Selected from vasculitis syndromes of the central nervous system, von Economo disease, von Hippel-Lindau disease (VHL), von Hippel-Lindau syndrome, von Recklinghausen disease, Wallenberg syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, West syndrome, whiplash, Whipple disease, Williams syndrome, Wilson disease, Wolman disease, X-linked spinal and bulbar muscular atrophy and Zellweger syndrome.

[0105] In some embodiments, the pharmaceutical formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some cases, the therapeutic gene expression product is selected from the group consisting of ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), survival of motor neurons (SMR), and the like. Neuron)1, STXBP1, telomere (SMNl), factor X (FIX), retinoid isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), prog The compounds are effective in regulating the activity or expression of target genes or gene expression products selected from ranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SODl), iduronate 2-sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPCl), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0106] In some embodiments, other examples of genes involved in 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.

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

[0108] In some cases, expression of the gene or expression or activity of the gene expression product is inhibited by administering the composition to a subject. In some cases, expression of the gene or expression or activity of the gene expression product is enhanced by administering the composition to a subject.

[0109] Formulation, dosage, and route of administration Disclosed herein are methods comprising delivering rAAV particles that encapsidate a heterologous nucleic acid to the brain in a subject, wherein the rAAV particles (i) increase transduction of the heterologous nucleic acid in the brain, wherein the rAAV particles have an rAAV capsid protein comprising an insertion of 5, 6, 7, or 8 amino acids of the amino acid sequence provided in Tables 1-3, FIG. 1, and Formula I at amino acid positions 588-589 of the parent AAV capsid protein, and one or more substitutions for amino acids [AQAQ] found at amino acid positions 587-590 as provided in Tables 1-3, FIG. 1, and Formula I. In various embodiments, the rAAV capsid protein can comprise one or more substitutions at amino acid positions 452-458, alone or in combination with the foregoing modifications.

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

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

[0112] In some cases, the dose of the pharmaceutical composition is at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or 10 17 In some cases, the concentration of infectious particles is 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 × 1016 , or 2 × 10 17 In some cases, the concentration of infectious particles is 3 x 10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 , or 3 × 10 17 In some cases, the concentration of infectious particles is 4 x 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 x 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 x 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 x 10 7 , 7×10 8 , 7×109 , 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 x 10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 , or 8 × 10 17 In some cases, the concentration of infectious particles is 9 x 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 is.

[0113] In some embodiments, the present disclosure provides formulations of pharmaceutically acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable administration and treatment regimens for using certain compositions described herein in various treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition may be adjusted to obtain a suitable dosage for any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations are taken into account by those skilled in the art when preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0114] In some embodiments, pharmaceutical forms of rAAV-based virus compositions suitable for injectable use 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, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, such as sugars or sodium chloride.

[0115] In some cases, for administration of injectable aqueous solutions, the solution may be appropriately buffered as necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose.These particular aqueous solutions are 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 in any case determine the appropriate dose for each individual subject.In addition, for human administration, preparations should meet the sterility, pyrogenicity, and general safety and purity standards required by the FDA Office of Biologics standards.

[0116] Sterile injectable solutions containing the rAAV compositions disclosed herein are prepared by incorporating the rAAV compositions disclosed herein in the required amount into a suitable solvent, optionally with some of the other ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For the preparation of sterile powders for sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which obtains a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Injectable solutions can be advantageous for systemic administration, for example, by intravenous or intrathecal administration.

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

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

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

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

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

[0122] The additional therapeutic agent may include a cell-based therapy. Exemplary cell-based therapies include, but are not limited to, immune effector cell therapy, chimeric antigen receptor T cell (CAR-T) therapy, natural killer cell therapy, and chimeric antigen receptor natural killer (NK) cell therapy. Either NK cells, CAR-NK cells, or a combination of both NK cells and CAR-NK cells may be used in combination with the methods disclosed herein. In some embodiments, the NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSCs), umbilical cord blood, or cell lines. The NK cells and CAR-NK cells may contain cytokine receptors and suicide genes. The cell-based therapy may include stem cell therapy. The stem cell therapy may be embryonic or somatic stem cells. The stem cells may be isolated from a donor (allogeneic) or from 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 interest.

[0123] kit The present specification discloses a kit comprising the composition disclosed herein.The present specification also discloses a kit for treating or preventing brain disease or symptoms.In some cases, the disease or symptoms is cancer, pathogen infection, lung disease or symptoms, neurological disease, muscular disease, or immune disorder, such as those described herein.

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

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

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

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

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

[0129] 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 measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to convention for a given value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean an acceptable error range for the particular value.

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

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

[0132] The term "increased" or "increase" is generally used herein to mean an increase by a statically significant amount. In some embodiments, the term "increased" or "increase" refers to 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%, or any increase between 10 and 100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0133] The term "reduced" or "reduction" is generally used herein to mean a statistically significant reduction. In some embodiments, "reduced" or "reduction" refers to a reduction of at least 10% compared to a reference level, e.g., a reduction 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 a 100% reduction compared to a reference level (e.g., a non-existent or undetectable level compared to a reference level), or any reduction between 10% and 100%. In the context of a marker or symptom, these terms refer to a statistically significant reduction in such a level. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more than 40%, preferably to a level that is accepted as being within the normal range for individuals without a given disease.

[0134] The term "subject" refers to any living organism. In some instances, 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, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and the like. In certain aspects, a mammal is a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, e.g., a rodent such as a rat or a mouse. In one embodiment, a "non-human primate" is a mammal, e.g., a monkey. In some instances, a subject is a patient, which as used herein may refer to a subject diagnosed with a particular disease or disorder.

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

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

[0137] 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 (for example, transgene, therapeutic nucleic acid, viral genome). In some cases, the AAV capsid of the present disclosure is a modified AAV capsid compared to the corresponding parent AAV capsid protein.

[0138] The term "tropism" as used herein refers to the quality or characteristics of an AAV capsid, which may include specificity for the expression of encapsidated genetic information in an in vivo environment and / or increased or decreased enrichment of said expression, compared to a second in vivo environment. The in vivo environment may optionally be a cell type. The in vivo environment may optionally be an organ or organ system.

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

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

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

[0142] As used herein, the term "heterologous" refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

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

[0144] As used herein, the terms "treat," "treating," and "treatment" refer to alleviating or suppressing a disorder, disease, or condition, or one or more symptoms associated with a disorder, disease, or condition; or to alleviating or eradicating the cause of the disorder, disease, or condition itself. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis.

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

[0146] The terms "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 may be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds. The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

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

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

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

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

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

[0152] The term "systemic delivery" is defined as the route of administration of a drug or other substance into the circulatory system so that the entire body is affected. Administration can occur via enteral administration (drug absorption through the gastrointestinal tract) or parenteral administration (generally via injection, infusion, or implantation). "Circulatory system" includes both the blood and cerebrospinal fluid circulatory systems. 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 spine (i.e., not limited to the lumbar region) or brain (i.e., not limited to the cisterna magna). The terms "systemic administration" and "systemic delivery" are used interchangeably.

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

[0154] Example Example 1 Methods for identifying modified capsid proteins in cynomolgus monkeys A major concern regarding the therapeutic applicability of engineered adeno-associated viruses (AAVs) is how well their transduction profiles translate to human applications. Previous technical efforts have focused on in vitro or in vivo rodent screening platforms due to their ease and flexibility, but direct screening efforts in nonhuman primates (NHPs) offer a much higher probability of identifying transforming viruses. For this technical effort, we selected the cynomolgus monkey, an archaic NHP. We focused our efforts on a region of the AAV9 capsid surface located at amino acid 588, a variable region among native AAV serotypes and one of the most exposed loops on the capsid surface that plays a role in receptor binding. Insertion of a peptide between positions 588 and 589 has been previously investigated, resulting in novel receptor binding (AAV-PHP.B / AAV-PHP.eB binding to Ly6a on rodent brain endothelium to promote blood-brain barrier crossing and high brain transduction) and dramatically altered capsid tropism. Hoping to discover novel tropism for the brain of NHPs, we created a library of viral capsids by randomly inserting seven amino acids into this site within AAV9. Two rounds of screening through this process identified a rAAV with a specific seven-amino acid peptide insertion that conferred high brain tropism. This rAAV was then used as a parent capsid for generating mutational substitutions and insertions in rounds 3 and 4 of screening.

[0155] Plasmids. The first-round viral DNA library was generated by amplifying a section of the AAV9 capsid genome between amino acids 450 and 599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 588 and 589, harboring all possible variations. The resulting library inserts were then introduced into the rAAV-ΔCap-in-rev-RNA plasmid via Gibson assembly as previously described. The resulting capsid DNA library, rAAV-Cap-Cag-GFP11, contained a diversity of approximately 1.28 billion variants at the amino acid level. The second-round viral DNA library was generated similarly to the first round, except that instead of the NNK degenerate primer inserted at 588, a synthetic oligo pool (Twist Biosicence) was used to generate only selected variants in the UBC-Cap-DNA and CAG-Cap-DNA constructs bearing the CAP. The third and fourth round viral DNA libraries were generated similarly to the second round, except that synthetic oligo pools of selected variants were ordered from IDT and used to generate capsids with amino acid insertions and substitutions in the 588 and / or 452 loops. 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, two barcoded replicates of each variant were used.

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

[0157] Virus production. Recombinant AAV was generated according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple-transfected with four vectors using polyethyleneimine (PEI). The first vector was the rAAV-Cap-in-cis-Lox library, flanked by inverted terminal repeat (ITR) sequences from the parent AAV virus. The second vector was the AAV2 / 9 REP-AAP-ΔCAP plasmid. The third vector contained nucleic acids encoding helper virus proteins required for virus assembly and packaging of the heterologous nucleic acid into the modified capsid structure. The fourth was a pUC-18 plasmid, included to achieve the correct PEI / DNA ratio for optimal transfection enrichment. To reduce the possibility of multiple library DNAs entering the same cells, only 10 ng of rAAV-Cap-in-cis-Lox library DNA (per 150 mm plate) was transfected. 60 h post-transfection, viral particles were collected from the cells and 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 on an iodixanol (Optiprep, Sigma) step gradient (15%, 25%, 40%, and 60%). The virus is concentrated and formulated in PBS. The virus titer is determined by measuring the number of DNase I-resistant vector genome copies (VG) using qPCR and a linearized genome plasmid as a control.

[0158] Animals. Cynomolgus monkey procedures were approved by Envol Biomedical's IACUC committee. Cynomolgus monkeys were born and raised in the Envol Biomedical colony and housed in family groups under standard conditions. They fed ad libitum and received enrichment as part of Envol Biomedical's NHP primate enrichment program. For AAV infusion, animals were screened for endogenous neutralizing antibodies (Nabs). None of the screened animals showed a detectable blocking reaction at a 1:10 dilution of serum. They were then housed for several days to acclimate to the new room before injection. The animals were restrained, and the test substance was administered via intravenous infusion over 10 minutes. Two infant monkeys were used in each of rounds 1 and 2 of screening, and three infant monkeys were used in rounds 3 and 4 of screening. Activity and behavior were monitored daily throughout their lives.

[0159] DNA / RNA recovery and sequencing. Viral libraries are 1–3 × 10 13rAAV was injected into cynomolgus monkeys at a dose of 0.01 mg / kg animal, and rAAV genomes were recovered two weeks after injection. Animals were euthanized, and the brain, spinal cord, and liver were harvested, snap-frozen, and placed in long-term storage at -80°C, along with other peripheral tissues such as the heart, spleen, adrenal glands, kidneys, and quads. Brains were separated into 11–13 brain regions. 100 mg of each brain region was homogenized in buffer using MagMAX DNA ULTRA (A25597) and Bead Ruptor 96 (OMNI, INC). Viral DNA was isolated according to the manufacturer's recommended protocol. The recovered viral DNA was treated with RNase A and purified using the Zymo DNA Clean and Concentrator Kit (D4033). Viral genomes were enriched by 25 cycles of PCR amplification using primers flanking the AA452–AA588 region of the capsid genome, using 50% of the extracted total viral DNA as a template. After Zymo DNA purification, samples were diluted 1:10 to 1:1000 depending on tissue type, and each dilution was further amplified around the library variable region using 10 cycles of PCR. Samples were then amplified for an additional 10 cycles using custom primers with Illumina Indices. The amplified products were run on a 2% low-melting-point agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 600 bp band.

[0160] For the second-, third-, and fourth-round libraries, 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 by two PCR amplification steps, as was the viral DNA extracted from tissue, and purified after gel electrophoresis to add adapters and indexes for Illumina next-generation sequencing. This viral library DNA, along with the viral DNA extracted from tissue, was then sent for deep sequencing using an Illumina NextSeq 2000 system.

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

[0162] For the second, third, and fourth round libraries, the read counts per tissue were similarly tabulated. A read count of 1 was then added to each sequence to remove values ​​of 0. All brain regions for each sequence were summed together, and the read sequences for each codon duplication of a given capsid amino acid sequence were summed together to give a single value for each variant. Finally, the read count data were normalized to counts per million (Cpm). The enrichment value for each capsid variant was calculated using the normalized cpm from [tissue of interest] / cpm from the viral library.

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

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

Claims

1. An AAV capsid protein comprising a sequence provided in Table 1 or selected from the group consisting of SEQ ID NOs: 3-378.

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

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

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

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

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

7. 2. The AAV capsid protein of 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 of claim 1 , which comprises AAV9 as a parent AAV.

9. The AAV capsid protein of claim 8, wherein the parent AAV comprises SEQ ID NO:

1.

10. 10. The AAV capsid protein of claim 9, comprising a 7-mer or 8-mer insertion inserted into said parental AAV between amino acids 588 and 589 of said parental AAV, wherein positions 587-597 or 587-598 of said AAV capsid protein are selected from the sequences provided in Table 1, or selected from the group consisting of SEQ ID NOs: 3-378.

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

12. The AAV capsid proteins of claim 1, which are present in VP1, VP2, and VP3 of the AAV capsid.

13. 2. The AAV capsid protein of claim 1, further characterized by increased transduction enrichment compared to AAV9 when measured in brain tissue in a subject when systemically delivered to the subject.

14. 2. The AAV capsid protein of claim 1, further characterized by reduced transduction enrichment compared to AAV9 when measured in liver tissue in a subject when systemically delivered to the subject.

15. 8. The AAV capsid protein of claim 7, further comprising amino acid substitutions including one or more of A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H compared to the parent AAV.

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

17. The AAV capsid protein of claim 16, which comprises AAV9 as the parent AAV.

18. The AAV capsid protein of claim 17, wherein the parent AAV comprises SEQ ID NO:

1.

19. 19. The AAV capsid protein of claim 18, comprising one or more substitutions at positions 452-460 and optional insertions at any of positions 452-453 through 458-459 relative to the parent AAV, 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.

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

21. The AAV capsid proteins of claim 16, which are present in VP1, VP2, and VP3 of the AAV capsid.

22. 17. The AAV capsid protein of claim 16, further characterized by increased transduction enrichment compared to AAV9 when measured in brain tissue in a subject when delivered systemically to the subject.

23. 17. The AAV capsid protein of claim 16, further characterized by reduced transduction enrichment compared to AAV9 when measured in liver tissue in a subject when systemically delivered to the subject.

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

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

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

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

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

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

30. 25. The AAV capsid protein of 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 of claim 24, comprising AAV9 as the parent AAV.

32. The AAV capsid protein of claim 31 , wherein the parent AAV comprises SEQ ID NO:

1.

33. 33. The AAV capsid protein of claim 32, comprising a 7-mer or 8-mer insertion inserted into the parental AAV between amino acids 588 and 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. 34. The AAV capsid protein of claim 33, comprising one or more substitutions at positions 452-460 and optional insertions at any of positions 452-453 through 458-459 relative to the parent AAV, 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. 25. The AAV capsid protein of claim 24, wherein 60 copies of the AAV capsid protein are assembled into the AAV capsid.

36. 25. The AAV capsid protein of claim 24, which is present in VP1, VP2, and VP3 of the AAV capsid.

37. 25. The AAV capsid protein of claim 24, further characterized by increased transduction enrichment compared to AAV9 when measured in brain tissue in a subject when delivered systemically to the subject.

38. 25. The AAV capsid protein of claim 24, further characterized by reduced transduction enrichment compared to AAV9 when measured in liver tissue in a subject when systemically delivered to the subject.

39. 31. The AAV capsid protein of claim 30, further comprising amino acid substitutions including one or more of A587H, A587D, A587K, Q590K, Q590P, Q590R, or Q590H compared to the parent AAV.

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