Selected adeno-associated virus compositions with preferred brain, spinal cord and / or cardiac expression levels

Abstract

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

Description

Background Art

[0001] Recombinant adeno-associated virus (rAAV) is widely used as a carrier for gene delivery in therapeutic applications due to its ability to transduce both dividing and non-dividing cells, its ability to exist in infected cells for a long time in the form of free DNA, and its low immunogenicity. These characteristics make it attractive for applications in therapeutic applications (such as gene therapy). However, it is necessary to significantly improve the performance of existing AAV serotypes to selectively and effectively express in different cell types when delivered to subjects throughout the body. When AAV must be expressed in the brain, spinal cord and / or heart, this demand is particularly urgent. Summary of the invention

[0002] Disclosed herein are rAAVs that are engineered to transduce enrichment in capsid structures by iterative multiple rounds of selection in non-human primates (NHPs), thereby producing variants with increased transduction enrichment when measured in the brain, spinal cord, and / or heart. Specifically, when compared to rAAVs with native AAV sequences, these variants produce increased tissue-specific transduction enrichment. In addition, during the development of the rAAVs of the present invention, variants with increased transduction enrichment in different or non-target tissues were found. These variants provide information for further opening of rAAVs that specifically target the brain, spinal cord, and / or heart, because they reveal variants and motifs to be avoided to prevent non-tissue-specific transduction enrichment.

[0003] The present invention provides rAAVs with broad transduction to the brain, spinal cord, and / or heart. In certain embodiments, the present invention provides rAAVs with broad transduction to the brain, spinal cord, and / or heart.

[0004] In one aspect, the present invention provides a peptide insertion sequence comprising Table 1, Figure 1 and / or the amino acid sequence shown in any one of Formulae I-III or consisting thereof.

[0005] Another aspect of the present invention is a modified capsid protein, wherein the capsid protein comprises Table 1, Figure 1 AAV capsid protein having an amino acid sequence or a peptide consisting of the amino acid sequence shown in any one of Formulas I-III inserted therein is characterized by enrichment of brain, spinal cord and / or heart transduction in a subject. Another aspect of the present invention is a modified capsid protein, wherein the peptide has a peptide comprising Table 1, Figure 1 The AAV capsid protein having an amino acid sequence shown in or consisting of any one of Formulas I-III inserted therein is characterized by being enriched for brain, spinal cord and / or cardiac transduction in a subject.

[0006] In addition, the present disclosure also includes pharmaceutical compositions, which contain Table 1, Figure 1and / or a peptide having an amino acid sequence shown in any one of Formulae I to III or consisting thereof, and a pharmaceutically acceptable excipient.

[0007] Aspects disclosed herein provide methods of treating a disease or condition in a subject, the methods comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising an AAV capsid protein or AAV capsid disclosed herein. In some embodiments, the disease or condition is a disease or condition of the brain, spinal cord and / or heart of the subject. Relatedly, the invention includes the use of rAAV in the preparation of a medicament for treating or preventing the disease or medical condition.

[0008] Other aspects of the invention will be apparent from the ensuing detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description setting forth illustrative embodiments and the accompanying drawings in which the principles of the present invention are utilized, and in which:

[0010] Figure 1 The present invention provides AAV capsid protein insertions and substitutions found in non-human primate brain, spinal cord and / or heart after two rounds of evaluation of the engineered AAV library. Initially, 39,000 individual insertion sequences were evaluated, of which Figure 1 The 975 inserts listed in show the greatest tissue-specific enrichment. The sequences of the 7-mer amino acid sequence inserts as described herein, which were inserted after residue #587 of SEQ ID NO: 1 (native AAV2 sequence), including the 4 amino acids surrounding the insertion site, are provided as SEQ ID NO: 2-975. The corresponding 7-mer sequences are also Figure 1 976-1949. The figure shows the average enrichment of AAVs with the indicated inserts in different types of tissues, including tissues of the brain, spinal cord cisterns (SCP), heart, and / or liver. DETAILED DESCRIPTION

[0011] In one aspect, the present disclosure provides rAAVs with high expression levels in the brain, spinal cord, and / or heart. In one aspect, the present disclosure provides rAAVs with high expression levels in the brain. In one aspect, the present disclosure provides rAAVs with high expression levels in the spinal cord. In one aspect, the present disclosure provides rAAVs with high expression levels in the heart. In one aspect, the present disclosure provides rAAVs with low expression levels in the liver. In one aspect, the present disclosure provides rAAVs developed to specifically avoid certain peptide sequences and / or motifs, thereby avoiding non-target tissue expression.

[0012] In one aspect, the present disclosure provides a method comprising: Figure 1 And / or the amino acid sequence shown in any one of formula I-III or the peptide consisting of it is inserted and / or substituted modified capsid protein. In some aspects, the disclosure includes a modified capsid protein with an amino acid sequence or a peptide consisting of at least 70% identity with a sequence selected from SEQ ID NO: 976-1949. In some aspects, the disclosure includes a modified capsid protein with an amino acid sequence or a peptide consisting of it having at least 80% identity with a sequence selected from SEQ ID NO: 2-975. In some aspects, the disclosure includes a modified capsid protein with an amino acid sequence or a peptide consisting of it having at least 90% identity with a sequence selected from SEQ ID NO: 2-975 or a peptide inserted and / or substituted.

[0013] In preferred aspects, the present disclosure provides modified capsid proteins with peptide insertions and / or substitutions that confer tissue-specific enrichment. In preferred aspects, the modified capsid proteins provide tissue-specific enrichment in the heart, brain, and / or spinal cord, while providing lower enrichment in other tissues such as the liver. In some embodiments, such modified capsid proteins include those having Figure 1 In some aspects, such as Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 3, 4, 5 and 255. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 978, 977, 979 and 1229. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 638, 640 and 641. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 638, 640 and 641. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 1612, 1614 and 1615. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO: 524 and 596. In some embodiments, Figure 1 The amino acid sequence described in is selected from SEQ ID NO:1498 and 1570.

[0014] In some embodiments, the insert sequence is represented by the peptide sequences listed in Table 1.

[0015]

[0016] In certain aspects, the present invention provides a modified capsid protein, wherein the modified capsid protein comprises a seven amino acid insertion having a sequence having at least 70% identity in amino acid sequence to a sequence selected from one of SEQ ID NOs:976-1949, the amino acid sequence having a sequence having at least 98% identity to amino acid 217 to amino acid 736 of the AAV capsid protein provided in SEQ ID NO:1.

[0017] In certain aspects, the modified AAV capsid protein is characterized by at least one of increased specificity and / or increased transduction efficiency relative to the native AAV capsid protein of SEQ ID NO: 1 when measured in the brain, heart, or spinal cord of a subject when delivered systemically to the subject. In certain aspects, the modified AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the brain of a subject.

[0018] In preferred aspects, the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the brain. In certain aspects, the seven amino acid insertion of the modified capsid protein of the invention comprises a sequence having at least 70% identity to a sequence selected from one of SEQ ID NOs: 976-1411. In certain aspects, the AAV capsid protein of the invention characterized by increased specificity and / or increased transduction efficiency when measured in the brain comprises a 7-mer amino acid insertion comprising a sequence of formula [I]:

[0019] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [I]) (SEQ ID NO: 1951), wherein:

[0020] X 1 is an amino acid selected from L, I and V; X 2 is an amino acid selected from L, V and M; X 3 is an amino acid selected from G, S and P; X 4 is an amino acid selected from the group consisting of P, G, S, T and H; X 5 is an amino acid selected from the group consisting of S, L, T, A, H and Q; X 6 is an amino acid selected from L, M, P, S, T and G; and / or X 7 is an amino acid selected from the group consisting of S, T, A, L, Q, P and V.

[0021] In some embodiments, the AAV capsid protein comprises an amino acid sequence of formula [I], wherein X 2 In some embodiments, the AAV capsid protein comprises an amino acid sequence of formula [I], wherein X 3 is G. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula [I], wherein X 6 is L. In certain embodiments, the seven amino acid insertion is one of SEQ ID NOs: 976-1411. In certain embodiments, the capsid protein comprises a sequence of one of SEQ ID NOs: 2-437.

[0022] In preferred aspects, the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the heart of a subject. In certain aspects, the seven amino acid insertion of the modified capsid protein of the invention comprises a sequence having at least 70% identity to a sequence selected from one of SEQ ID NOs: 1412-1612. In certain aspects, the AAV capsid protein of the invention characterized by increased specificity and / or increased transduction efficiency when measured in the heart comprises a 7-mer amino acid insertion comprising a sequence of formula [II]:

[0023] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [II]) (SEQ ID NO: 1952), wherein:

[0024] X 1 is an amino acid selected from L, I and V; X 2 is an amino acid selected from L, V and M; X 3 is an amino acid selected from G, S and P; X 4 is an amino acid selected from the group consisting of P, G, S, T and H; X 5 is an amino acid selected from the group consisting of S, L, T, A, H and Q; X 6 is an amino acid selected from L, M, P, S, T and G; and / or X 7 is an amino acid selected from the group consisting of S, T, A, L, Q, P and V.

[0025] In some embodiments, the AAV capsid protein comprises an amino acid sequence of formula [I], wherein X 2 In some embodiments, the AAV capsid protein comprises an amino acid sequence of formula [I], wherein X 3is G. In some embodiments, the AAV capsid protein comprises the amino acid sequence of formula [I], wherein X 6 is L. In certain embodiments, the seven amino acid insertion is one of SEQ ID NOs: 1412-1612. In certain embodiments, the capsid protein comprises the sequence of one of SEQ ID NOs: 438-638.

[0026] In preferred aspects, the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the spinal cord of a subject. In certain aspects, the seven amino acid insertion of the modified capsid protein of the invention comprises a sequence having at least 70% identity to a sequence selected from one of SEQ ID NOs: 1613-1949. In certain aspects, the AAV capsid protein of the invention characterized by increased specificity and / or increased transduction efficiency when measured in the spinal cord comprises a 7-mer amino acid insertion comprising a sequence of formula [III]:

[0027] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [III]) (SEQ ID NO: 1950), wherein:

[0028] X 1 is an amino acid selected from L, S and T; X 2 is an amino acid selected from L, P, S and T; X 3 is an amino acid selected from S, T, G, P and L; X 4 is an amino acid selected from S, T, L and P; X 5 is an amino acid selected from S, L, T and G; X 6 is an amino acid selected from L, P, S and T; and / or X 7 is an amino acid selected from S, P, and E. In certain embodiments, the seven amino acid insertion is one of SEQ ID NOs: 1613-1949. In certain embodiments, the capsid protein comprises a sequence of one of SEQ ID NOs: 639-975.

[0029] In a preferred aspect, the modified AAV capsid protein of the invention comprising insertions / substitutions as described herein is a modified AAV2 capsid protein.

[0030] Also disclosed herein are methods and kits for producing therapeutic recombinant AAV (rAAV) particles, as well as methods for treating diseases or conditions affecting, for example, the brain, CNS, spinal cord and / or heart, and pharmaceutical compositions or formulations comprising these rAAV particles. The rAAV particles of the present invention and their pharmaceutical compositions or formulations provide increased specificity and / or increased transduction efficiency compared to rAAV comprising native AAV capsid protein sequences. Thus, it is found that the rAAV of the present invention has significant utility in treating tissue-specific conditions, such as conditions of the brain, spinal cord and / or heart.

[0031] Disclosed herein are AAV capsids engineered to increase viral transduction in the brain, spinal cord, and / or heart. AAV capsids can encapsidate viral vectors with heterologous nucleic acids encoding, for example, therapeutic gene expression products. Transduction of heterologous nucleic acids in the CNS can be achieved by delivering the AAV capsid of the present disclosure that encapsidates heterologous nucleic acids to a subject systemically. AAV capsids disclosed herein are advantageous for many applications of gene therapy for treating human diseases (including but not limited to disorders of the brain, spinal cord, and / or heart).

[0032] Also provided herein is a recombinant AAV vector comprising a nucleic acid sequence encoding an AAV capsid protein of the present disclosure. For example, a viral vector of the present disclosure comprises a nucleic acid sequence comprising an AAV viral Cap (capsid) encoding VP1, VP2 and VP3, at least one of which is modified to produce an AAV capsid protein of the present disclosure. The recombinant AAV vector provided can be derived from an AAV serotype (e.g., AAV2) or include an inserted variant AAV serotype of the present invention. In a preferred aspect, the AAV capsid protein is a variant or modification of an AAV2 capsid protein.

[0033] AAV capsid

[0034] Provided herein are modified adeno-associated (AAV) viral capsid compositions that can be used to integrate a transgene into target cells or the environment of a subject when systemically administered to the subject.

[0035] rAAV comprises an AAV capsid that can be engineered to encapsidate heterologous nucleic acids (e.g., therapeutic nucleic acids, gene editing machines). The AAV capsid is composed of three AAV capsid protein monomers VP1, VP2 and VP3. Sixty copies of these three VP proteins interact with each other at a ratio of 1:1:10 to form a viral capsid. In addition to the approximately 137 amino acid N-terminal region (VPlu), VP1 also covers the entire VP2 protein, and in addition to the approximately 65 amino acid N-terminal region (VP1 / 2 common region), VP2 also covers the entire VP3. These three capsid proteins share the conservative amino acid sequence of VP3, and in some cases, the conservative amino acid sequence is a region starting from amino acid position 138 (e.g., AA139-736).

[0036] Although not wishing to be bound by theory, it is understood that the parent AAV capsid sequence comprises a VP1 region. In certain embodiments, the parent AAV capsid sequence comprises a VP1, VP2 and / or VP3 region or any combination thereof. The parent VP1 sequence can be considered synonymous with the parent AAV capsid sequence.

[0037] The AAV VP3 structure contains highly conserved regions common to all serotypes, a core eight-stranded β-barrel motif (βB-βI) and a small α-helix (αA). The loop region inserted between the β strands consists of a unique HI loop between β strands H and I, a DE loop between β strands D and E, and nine variable regions (VRs) forming the top of the loop. These VRs are present on the capsid surface and can be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity. Residue #587 of the native AAV2 capsid protein (SEQ ID NO: 1) is located at the uppermost portion of one of these variable loop regions.

[0038] Thus, in some aspects, the rAAV variants of the invention comprise an AAV capsid protein having a peptide insertion at residues corresponding to amino acids 587-588 of the AAV2 native sequence of SEQ ID NO:1.

[0039] The AAV capsid comprises AAV capsid proteins (e.g., VP1, VP2, and VP3), each of which has an insertion in the loop at residue #587 of the parent AAV2 capsid protein structure (AAV2 VP1 numbering). The loop at residue #587 contains the heparan sulfate proteoglycan (HSPG) binding site of AAV2 and is suitable for peptide display. HSPG is the main receptor for AAV2, but there are many indications that there are other receptors. Modifications to the loop at residue #587 ("587 loop") of AAV2 are shown herein to confer increased transgene transduction in a tissue-specific manner in the target in vivo environment.

[0040] In one aspect, the invention provides a peptide insertion at the AAV 587 loop comprising Table 1, Figure 1 and / or the amino acid sequence shown in any one of Formulae I-III or consisting thereof.

[0041] Disclosed herein are AAV capsids comprising an AAV capsid protein having an insertion at loop 587 that confers higher transduction enrichment of CNS cell types (e.g., brain endothelial cells, neurons, astrocytes). Specifically, certain AAV capsids of the invention specifically target certain types of CNS cell types, such as brain cells and / or spinal cord cells. In some aspects, certain AAV capsids of the invention specifically target cells of the heart.

[0042] Specifically, the AAV capsid proteins disclosed herein enable rAAV-mediated transduction of heterologous nucleic acids (e.g., transgenes) in certain types of cells of a subject. In preferred aspects, the cell type is selected from at least one of the following: cells of the brain, cells of the spinal cord, and / or cells of the heart. The AAV capsids disclosed herein can be formulated into pharmaceutical compositions. In addition, the AAV capsids can be isolated and purified while targeting specific cell types for various applications.

[0043] 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 contained therein increase the localization of the rAAV in target tissues (e.g., brain, spinal cord, and / or heart) compared to the parent AAV capsid or capsid protein.

[0044] AAV capsid protein

[0045] Disclosed herein are recombinant AAV (rAAV) capsids comprising AAV capsid proteins engineered to have modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are produced using the methods disclosed herein. In some embodiments, AAV capsid proteins are used in methods for delivering therapeutic nucleic acids (e.g., transgenics) to subjects. In some cases, rAAV capsid proteins have desired AAV expression, making them particularly suitable for certain therapeutic applications, e.g., treatment of a disease or condition of a subject as disclosed herein.

[0046] Certain rAAV capsid proteins are engineered to achieve optimized expression in the CNS (e.g., brain and / or spinal cord) of a subject when rAAV is administered systemically to the subject. Certain rAAV capsid proteins are engineered to achieve optimized expression in the heart of a subject when rAAV is administered systemically to the subject. In preferred aspects, rAAV capsid proteins are engineered to have reduced expression in hepatocytes compared to other cell types, particularly the brain, spinal cord, and / or heart.

[0047] The described rAAV capsid proteins are engineered to include Table 1, Figure 1 And / or the insertion provided in Formula I-III. Including Table 1, Figure 1 The inserted rAAV capsid protein provided in and / or Formula I-III is engineered to achieve efficient transduction of the encapsidated transgene. Specifically, the expression enrichment of the rAAV capsid protein in the brain, spinal cord and / or heart of the subject is increased.

[0048] In some cases, the engineered AAV capsid proteins described herein have an insertion of an amino acid that is heterologous to the parent AAV capsid protein at an amino acid position in the 587 loop. In some embodiments, at the inserted amino acid position, the amino acid is not endogenous to the parent AAV capsid protein. The amino acid can be a naturally occurring amino acid in the same or equivalent amino acid position as the substituted insertion in a different AAV capsid protein.

[0049] Typically, insertion is included in the five, six or seven amino acid sequences (respectively 5-mers, 6-mers or 7-mers) inserted or replaced at the 587 loop in the parent AAV capsid protein. The various aspects provided herein provide amino acid insertions, which are included in the seven amino acid polymers (7-mers) inserted at AA587-588, and can additionally include the replacement of one or two amino acids at the amino acid positions (e.g., AA586-587 and / or AA588-589) of the 7-mer sequence flanking the 11 amino acid polymers (11-mers) at the 587 loop of the parent AAV capsid protein. The 7-mers described herein are advantageously produced using polymerase chain reaction (PCR) using degenerate primers, wherein each of the seven amino acids is encoded by a deoxyribonucleic acid (DNA) sequence NNK. "N" is any one of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method for generating random 7-mer amino acid sequences enables 1.28 billion possible combinations to be achieved at the protein level.

[0050] The rAAV capsid protein of the present disclosure comprises an amino acid insertion in the amino acid sequence of the AAV capsid protein. The AAV capsid that produces the engineered AAV capsid protein of the present disclosure is referred to as the "parental" 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 Srivastava et al., Journal of Virology, 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; 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 Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004); portions of the AAV-12 genome are provided in Genbank Accession No. DQ813647; portions of the AAV-13 genome are provided in Genbank Accession No. EU285562.

[0051] In some cases, the parent AAV is derived from an AAV having a serotype selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. The AAV capsid protein "derived" from another serotype can be a variant AAV capsid protein. The variant may include, for example, heterologous amino acids in the amino acid sequence of the AAV capsid protein. Heterologous amino acids may be non-naturally present in the AAV capsid protein. Heterologous amino acids may be naturally present in different AAV capsid proteins. In some cases, the parent AAV capsid is described in U.S. Patent Publication 2020 / 0165576 and U.S. Patent Application Serial No. 62 / 832,826 and PCT / US20 / 20778, the contents of each of which are incorporated herein.

[0052] In some cases, the parent AAV is AAV2. In some cases, the amino acid sequence of the AAV2 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of the AAV2 VP1 capsid protein is provided in SEQ ID NO: 1. (*NR*QAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL). In some cases, the parent AAV is AAV2. In some cases, the amino acid sequence of the AAV2 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of the AAV2 VP1 capsid protein is provided in SEQ ID NO: 1. V The 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. The 7-mer insertion is located between the *NR*.

[0053] Tropical AAV capsid proteins from natural AAV serotypes such as AAV2 activate innate immune responses, which in some cases cause severe inflammatory responses in subjects, which may lead to multi-organ failure, including the liver. By increasing the transduction enrichment of natural AAV serotypes to target in vivo tissues (e.g., brain, spinal cord and / or heart), the rAAV particles of the present disclosure reduce the immunogenic nature of AAV-mediated transgene delivery and prevent the activation of innate immune responses.

[0054] In some cases, the parent AAV capsid protein comprises the entire VP1 region (e.g., amino acids 1-736) provided in SEQ ID NO: 1. In some cases, the parent AAV capsid protein comprises amino acids 217-736 of SEQ ID NO: 1, which is a common region found in VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein comprises amino acids 64-736 of SEQ ID NO: 1, which is a common region found in VP1 and VP2. The parent AAV capsid protein sequence can comprise amino acids 1-736 of SEQ ID NO: 1. NO: 1 is selected from the following amino acids: 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 In some aspects, the rAAV variant comprises an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1. In some cases, the amino acid is inserted at the three (3)-fold symmetry axis of the corresponding parent AAV capsid protein.

[0055] Disclosed herein is the insertion of an amino acid sequence in an AAV capsid protein. With reference to the sequence number designation "587-588" of AAV2 (e.g., AAV VP1), the present invention also includes insertions at similar positions in other AAV serotypes. As used herein, "AA587-588" indicates that the insertion of an amino acid (or amino acid sequence) is immediately after the amino acid (AA) at position 587 in the amino acid sequence of the parent AAV VP capsid protein (VP1 numbering), and immediately before the AA at position 588.

[0056] It is envisioned that the insertions disclosed herein (Table 1, Figure 1 and / or Formulas I-III) can be inserted at AA587-588 in the amino acid sequence of the parent AAV2 capsid protein, its variants, or the equivalent amino acid position of a parent AAV of a different serotype (e.g., AAV1, AAV3, AAV9, etc.).

[0057] In some cases, the insertions described herein may comprise a 7-mer insertion at AA 587-588. It is contemplated that any 7-mer insertion disclosed herein may comprise an 11-mer, except for substitutions at amino acid positions 586-589 with any amino acid.

[0058] Disclosed herein are AAV capsid proteins having an insertion described above in a parent AAV capsid protein that imparts increased transduction enrichment to the CNS of a subject even when delivered systemically. One of the many advantages of certain AAV capsid proteins described herein is their ability to target tissues and cells within the CNS. The tissue may be the brain. Non-limiting examples of CNS cells include neurons and glial cells. Glial cells may be selected from oligodendrocytes, ependymal cells, astrocytes, and microglia. In preferred aspects, the AAV capsid proteins described herein target tissues and cells of the brain and / or spinal cord. In certain aspects, the AAV capsid proteins described herein target tissues and cells of the heart.

[0059] In some cases, the AAV capsid protein comprises Table 1, Figure 1 and / or an insertion of at least or about five, six, or seven amino acids of the amino acid sequence of Formulae I-III. In some cases, the AAV capsid protein has increased viral transduction in the brain.

[0060] The rAAV capsid protein of the present disclosure may also have a substitution of the amino acid sequence at amino acid positions 452-458 in the parent AAV2 capsid protein or a variant thereof, as described in WO2020068990.

[0061] The rAAV capsid protein described herein can be separated and purified. AAV can be separated 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 viruses are known in the art and may include methods disclosed in, for example, Clark et al., Human Gene Therapy (Hum.Gene Then), 10 (6): 1031-1039 (1999); Schenpp and Clark, Methods Mol.Med., 69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0062] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, can be formulated into a pharmaceutical formulation, which in some cases further comprises a pharmaceutically acceptable carrier.

[0063] The rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In some cases, the nanoparticle or viral capsid protein will encapsidate the therapeutic nucleic acid described herein. In some cases, the second molecule is a therapeutic agent, such as a small molecule, an antibody, an antigen binding fragment, a peptide, or a protein, such as those described herein.

[0064] "Percent identity" is the percentage of symbols that actually match. Percent similarity is the percentage of similar symbols. Symbols that span gaps are ignored. When the scoring matrix value of a pair of symbols is greater than or equal to 0.50 (similarity threshold), similarity is scored. The scoring matrix used in the 10th version of the Wisconsin Genetics Software Package is BLOSUM62 (see: Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[0065] The sequence identity / similarity values ​​provided herein may refer to the values ​​obtained using the BLAST+2.5.0 program suite using default settings (blast.ncbi.nlm.nih.gov) (Camacho, C. et al., (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0066] Those of ordinary skill in the art will appreciate that BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins contain regions of non-random sequences, which may be homopolymer regions, short-period repeats, or regions rich in one or more amino acids. Such low-complexity regions can be aligned between unrelated proteins, even if other regions of the protein are completely different. Many 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.

[0067] The terms "substantial identity" and "substantially identical" indicate that a polypeptide or nucleic acid comprises a sequence having 55%-100% sequence identity to a reference sequence, at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity relative to the reference sequence, or any percentage of values ​​within the range of 55%-100% sequence identity. The percentage sequence identity may occur over a specified comparison window. The optimal alignment may be determined or performed using the homology alignment algorithm of Needleman and Wunsch described above.

[0068] For example, an inserted sequence may include, but is not limited to, a sequence that is not completely identical to a sequence disclosed herein, but has, in addition to the substitutions explicitly described 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, as predicted by homology software (e.g., the BLOSUM62 matrix).

[0069] AAV particles

[0070] rAAV particles with insertion sequences described herein have increased transduction enrichment in the brain, spinal cord and / or heart. In some cases, relative to rAAV particles with native AAV2 capsid protein (SEQ ID NO: 1), the increased transduction enrichment comprises an increase of 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more. In some cases, the increased transduction enrichment is at least 1 times. In some cases, the increased transduction enrichment is at least 2 times. In some cases, the increased transduction enrichment is at least 4 times.

[0071] rAAV particles with insertion sequences described herein have increased expression enrichment in the brain, spinal cord and / or heart. Detecting whether rAAV has more or less expression includes measuring the level of gene expression products (e.g., RNA or protein) expressed by the heterologous nucleic acid encapsidated by rAAV in a tissue sample obtained from a subject. Suitable methods for measuring the expression of gene expression products include next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0072] Increased expression in cells of the brain, spinal cord and / or heart is caused by Figure 1 The enrichment values ​​provided are indicated in . Figure 1 The expression levels in liver are also provided, and for many sequences, these expression levels are much lower than the values ​​in brain, spinal cord and / or heart. This reveals the tissue or cell type specificity conferred by the modified capsid proteins and rAAV disclosed herein.

[0073] Heterologous Nucleic Acid

[0074] Disclosed herein are therapeutic nucleic acids that can be used to treat or prevent a disease or condition or a symptom of the disease or condition. In some embodiments, these therapeutic nucleic acids encode therapeutic gene expression products. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.) and gene editing components for treating, preventing and treating and / or improving a disease or condition or a symptom of a disease or condition. In some cases, these therapeutic nucleic acids are placed in an organism, a cell, a tissue or an organ of a subject by rAAVs as disclosed herein.

[0075] 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 of about 145 bases each, and the two ITR sequences flank a transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid, and in some cases, comprises a promoter arranged in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter is capable of initiating transcription of the therapeutic nucleic acid in the nucleus of a target cell. The ITR sequence 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, ITR is from AAV2. In some cases, ITR is from AAV9.

[0076] Transgenics that can include any number of nucleotides are disclosed herein. In some cases, the transgenic can include less than about 100 nucleotides. In some cases, the transgenic can include at least about 100 nucleotides. In some cases, the transgenic can include at least about 200 nucleotides. In some cases, the transgenic can include at least about 300 nucleotides. In some cases, the transgenic can include at least about 400 nucleotides. In some cases, the transgenic can include at least about 500 nucleotides. In some cases, the transgenic can include at least about 1000 nucleotides. In some cases, the transgenic can include at least about 5000 nucleotides. In some cases, the transgenic can include more than 5,000 nucleotides. In some cases, the transgenic can include about 500 to about 5000 nucleotides. In some cases, the transgenic includes about 5000 nucleotides. In any case disclosed herein, the transgenic can include DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgenic can be single-stranded. In some cases, the transgenic can be double-stranded.

[0077] Disclosed herein are transgenics that can be used to regulate the expression or activity of a target gene or its gene expression product. In some cases, the transgenic is encapsidated by the rAAV capsid protein of the rAAV particles described herein. In some cases, the rAAV particles are delivered to a subject to treat a disease or condition disclosed herein in the subject. In some cases, the delivery is systemic.

[0078] Transgenics disclosed herein can be used to express endogenous genes at a level similar to that of healthy or normal individuals. This is particularly useful in treating diseases or conditions related to low expression or insufficient expression of gene expression products. In some embodiments, transgenics disclosed herein can be used to overexpress endogenous genes so that the expression level of endogenous genes is higher than that of healthy or normal individuals. In addition, transgenics can be used to express exogenous genes (e.g., such as antibodies, peptides, nucleic acids, or gene editing components). In some embodiments, therapeutic gene expression products can change, enhance, increase or induce the activity of one or more endogenous biological processes in cells. In some embodiments, transgenics disclosed herein can be used to reduce endogenous genes, for example, the expression of dominant negative genes. In some embodiments, therapeutic gene expression products can change, inhibit, reduce, prevent, eliminate or weaken the activity of one or more endogenous biological processes in cells. In some aspects, the increase in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. On the one hand, the protein product of the gene targeted can increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some respects, the minimizing of gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. On the one hand, the protein product of the gene targeted can reduce by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0079] When an endogenous sequence (endogenous or partial transgene) is expressed 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 functions of these full-length or partial sequences include increasing the serum half-life of a polypeptide expressed by a transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0080] Transgenics can be inserted into endogenous genes so that all, some or none of the endogenous genes are expressed. For example, transgenics described herein can be inserted into endogenous loci so that some (to the N-terminal and / or C-terminal of the transgenic) endogenous sequences or none of the endogenous sequences are expressed as, for example, fusions with transgenics. In other cases, transgenics (e.g., with or without additional coding sequences of endogenous genes) are integrated into any endogenous loci, such as safe harbor loci. For example, a commissure protein (FXN) transgenic can be inserted into an endogenous FXN gene. Transgenics can be inserted into any gene (e.g., a gene as described herein).

[0081] At least one advantage of the present disclosure is that in fact any therapeutic nucleic acid can be used to express any therapeutic gene expression product. In some cases, the therapeutic gene expression product is a therapeutic protein or peptide (e.g., an antibody, an antigen binding fragment, a peptide or a protein). In one embodiment, the length of the protein encoded by the therapeutic nucleic acid is between 50-5000 amino acids. In some embodiments, the encoded protein length is between 50-2000 amino acids. In some embodiments, the encoded protein length is between 50-1000 amino acids. In some embodiments, the encoded protein length is between 50-1500 amino acids. In some embodiments, the encoded protein length is between 50-800 amino acids. In some embodiments, the encoded protein length is between 50-600 amino acids. In some embodiments, the encoded protein length is between 50-400 amino acids. In some embodiments, the encoded protein length is between 50-200 amino acids. In some embodiments, the encoded protein length is between 50-100 amino acids. In some embodiments, the length of the encoded peptide is between 4-50 amino acids. In some embodiments, the encoded protein is a tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide. In some embodiments, the encoded protein comprises a peptide of 2-30 amino acids (e.g., 5-30, 10-30, 2-25, 5-25, 10-25 or 10-20 amino acids). In some embodiments, the encoded protein comprises a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids or a peptide of no more than 50 amino acids (e.g., no more than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids).

[0082] Non-limiting examples of therapeutic proteins or peptides include adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoiesis stimulating agents, glutamate 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, armillary proteins, armillary protein receptors, serotonin transporters, serotonin uptake inhibitors, serotonin receptors, serine protease inhibitors, serine protease inhibitor receptors, and tumor suppressors. In certain embodiments, the therapeutic protein or peptide is selected from the following: brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropin, interferon-γ (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-β (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, growth hormone, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17 and IL-18.

[0083] 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 factors such as effector nucleases (TALENs). In a non-limiting example, a subject with Huntington's disease is identified. Then, a first amount of rAAV is systemically administered to the subject, which encapsidates a viral vector encoding a ZFN that is engineered to inhibit transcription of the Huntington protein (HTT) gene. rAAV will include a modified AAV capsid protein, and the modified AAV capsid protein includes Table 1, Figure 1and / or the amino acid sequence provided in any one of Formulas I-III, thereby allowing the ZFN to be correctly targeted to the brain while reducing expression in off-target organs such as the liver. If necessary, a second or third dose of rAAV is administered to the subject until a therapeutically effective amount of ZFN is expressed in the brain of the subject.

[0084] Therapeutic nucleic acids can include non-protein coding genes, for example, sequences encoding antisense RNA, RNAi, shRNA and microRNA (miRNA), miRNA sponges or decoys, for conditional gene deletion recombinase delivery, conditional (recombinase-related) expression including those required for gene editing components described herein. Non-protein coding genes can also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA and / or long non-coding RNA (IncRNA). In some cases, non-protein coding genes can regulate the expression or activity of target genes or gene expression products. For example, RNA described herein can be used to inhibit gene expression in CNS. In some cases, the inhibition of gene expression refers to at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% inhibition. In some cases, the protein product of the targeted gene can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene can be a wild-type gene or a gene with at least one mutation. The targeted protein can be a wild-type protein or a protein with at least one mutation.

[0085] Therapeutic nucleic acid can regulate the expression or activity of a gene or a gene expression product expressed by the gene that is relevant to a disease or condition of the brain, spinal cord and / or heart. For example, in some cases, the therapeutic nucleic acid is a modified version of a gene or a gene described herein. In some cases, the gene or gene expression product is suppressed. In some cases, the gene or gene expression product is enhanced.

[0086] In another example, the therapeutic nucleic acid comprises an effector gene expression product, such as a gene editing component that is specific to the gene targeted therein. Non-limiting examples of genes include target genes or gene expression products selected from the following: ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, inositol alpha (SGCA), glutamate decarboxylase 65 (GAD65), glutamate decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), motor neuron survival gene 1, STXBP1, telomere (SMN1), factor X (FIX), retinoid isomerase (Retinoid isohydrolase (RPE65), sarcoplasmic / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), anti-tubulin agent, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the following: PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX110, PEX12, PEX13, PEX14, PEX16, PEX19 and PEX26. In some cases, the gene or gene expression product is inhibited. In some cases, the gene or gene expression product is enhanced.

[0087] AAV vectors

[0088] Aspects disclosed herein include plasmid vectors comprising nucleic acid sequences encoding the AAV capsid and AAV capsid proteins described herein. The AAV vectors described herein can be used for the assembly of rAAV and viral packaging of heterologous nucleic acids. In addition, the AAV vectors can encode transgenes comprising heterologous nucleic acids.

[0089] AAV vectors can include transgenes that encode heterologous gene expression products (e.g., therapeutic gene expression products, recombinant capsid proteins, etc.) in some cases. Transgenes are cis with two reverse terminal repeats (ITRs) flanking the transgenes. Transgenes can include therapeutic nucleic acids encoding therapeutic gene expression products. Due to the limited packaging capacity of rAAV (about 5kB), in some cases, longer transgenes can be split between two AAV vectors, the first having a 3' splice donor and the second having a 5' splice acceptor. After cell co-infection, concatemers are formed, which are spliced ​​together to express full-length transgenes.

[0090] The transgenic gene is usually inserted so that its expression is driven by an endogenous promoter at the integration site (i.e., a promoter that drives the expression of the endogenous gene into which the transgenic gene is inserted). In some cases, the transgenic gene comprises a promoter and / or an enhancer, such as a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter can be a CMV promoter, a CMV-β-actin-intron-β-globin hybrid promoter (CAG), a CBA promoter, a FRDA or FXN promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synaptophysin promoter, a MeCP2 promoter, a GFAP promoter, a H1 promoter, a U6 promoter, a NFL promoter, a NFH promoter, a SCN8A promoter, or a PGK promoter. As a non-limiting example, the promoter can be a tissue-specific expression element, including but not limited to human elongation factor 1α subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derived CAG, β-glucuronidase (GUSB) and ubiquitin C (UBC). Transgenics can include tissue-specific expression elements for neurons, such as but not limited to neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptophysin (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. Transgenics can include tissue-specific expression elements for astrocytes, such as but not limited to 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.

[0091] In some embodiments, the promoter is less than 1 kb. The length of the promoter can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more. The length of the promoter can be between 200-300, between 200-400, between 200-500, between 200-600, between 200-700, between 200-800, between 300-400, between 300-500, between 300-600, between 300-700, between 300-800, between 400-500, between 400-600, between 400-700, between 400-800, between 500-600, between 500-700, between 500-800, between 600-700, between 600-800, or between 700-800. The promoter can provide expression of the therapeutic gene expression product in a target tissue, such as, but not limited to, the CNS, for a period of time.The expression of the therapeutic gene expression product can continue for the following time periods: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months 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, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 or more years. Expression of the payload may continue 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.

[0092] The AAV vector may comprise the genome of a helper virus. The assembly of recombinant AAV (rAAV) and the packaging of transgenes containing heterologous nucleic acids into rAAV require helper virus proteins. Helper virus genes are adenovirus genes E4, E2a and VA that assist AAV replication when expressed in cells. In some embodiments, the AAV vector comprises E2. In some embodiments, the AAV vector comprises E4. In some embodiments, the AAV vector comprises VA. In some cases, the AAV vector comprises one or any combination of helper virus proteins.

[0093] The target gene or gene expression product for transgenesis can be selected from the following: ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, inositol alpha (SGCA), glutamate decarboxylase 65 (GAD65), glutamate decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), survival of motor neuron 1, STXBP1, telomere (SMN1), factor X (FIX), Retinoid isomerohydrolase (RPE65), sarcoplasmic / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), anti-tubulin agent, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and aNLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the group consisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX110, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0094] The AAV vector may comprise a viral genome comprising a nucleic acid encoding a recombinant AAV (rAAV) capsid protein described herein. The viral genome may comprise a replication (Rep) gene encoding a Rep protein and a capsid (Cap) gene encoding an AAP protein in a first open reading frame (ORF1) or a Cap protein in a second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some cases, the Cap gene is modified to encode the modified AAV capsid protein described herein. The wild-type Cap gene encodes three proteins VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1-VP3 are modified. The AAV vector may comprise a nucleic acid encoding a wild-type Rep78, Rep68, Rep52, Rep40, and AAP protein.

[0095] In some cases, the AAV2 VP1 gene provided in SEQ ID NO: 1 is modified to include Figure 1 Any of SEQ ID NOs: 2-1949 in. The AAV vectors described herein can be used to produce variant AAV capsids by the methods described herein.

[0096] Methods for producing rAAV

[0097] Disclosed herein is a method for producing an AAV capsid comprising an AAV capsid protein and a viral vector encoding a therapeutic nucleic acid. The AAV capsid protein is produced by introducing a first vector, a second vector, and a third vector into a cell (e.g., an immortalized stem cell), wherein the first vector contains a transgenic cassette flanked by an inverted terminal repeat (ITR) sequence from a parental AAV virus (the transgenic cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell), the second vector encodes an AAV genome with an AAV capsid protein (encoding an AAV Rep gene and a modified Cap gene of the variant produced), and the third vector encodes helper viral proteins that are required for assembling an AAV capsid structure and packaging a transgene in a modified AAV capsid structure. The assembled AAV capsid can be isolated and purified from cells using suitable methods known in the art.

[0098] Also provided herein is a transgene contained in a recombinant AAV (rAAV) vector and encapsidated by the AAV capsid protein of the present disclosure. The transgene disclosed herein is delivered to a subject for various purposes, such as to treat a disease or condition of 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 encoding a therapeutic gene expression product that effectively regulates the activity or expression of itself or another target gene or gene expression product.

[0099] Aspects disclosed herein provide methods of making rAAV viruses or viral particles, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector containing a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette having a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell); (ii) a second vector encoding an AAV genome having an AAV capsid protein of the invention; and (iii) a vector encoding helper viral proteins required for assembling an AAV capsid structure and packaging a transgene in a modified AAV capsid structure; (b) expressing the AAV capsid proteins described herein in the cell; (c) assembling AAV particles comprising the AAV capsid proteins disclosed herein; and (d) packaging the AAV particles. In some cases, the cell is mammalian. In some cases, the cell is immortalized. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In some cases, human embryonic stem cells are human embryonic kidney 293 (HEK-293) cells. In some cases, the Cap gene is derived from the deoxyribonucleic acid (DNA) provided in SEQ ID NO:86. In some cases, 5'ITR and 3'ITR are derived from AAV2 serotype. In some cases, 5'ITR and 3'ITR are derived from AAV5 serotype. In some cases, 5'ITR and 3'ITR are derived from AAV9 serotype. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are trans. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are cis. In some cases, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence are trans.

[0100] In some cases, these methods comprise 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, rAAV particles are isolated, concentrated, and purified using suitable virus purification methods such as those described herein.

[0101] In some cases, the rAAV of the present disclosure is produced using the method described in the following: Challis, RC et al. "Natural Experiment Manual (Nat. Protoc.) 14, 379 (2019). In brief, triple transfection of HEK293T cells (ATCC) was performed using polyethyleneimine (PEI), and the virus was collected from the cell lysate and culture medium after 120 hours and purified by iodixanol. In a non-limiting example, rAAV is produced by triple transfection of precursor cells (e.g., HEK293T) cells using a standard transfection protocol (e.g., PEI). Viral particles are harvested from the culture medium after a certain period of time (e.g., 72 hours after transfection), and viral particles are harvested from cells and culture medium at a later time point (e.g., 120 hours after transfection). The virus present in the culture medium is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500mM sodium chloride, and the precipitated virus is added to the lysate prepared from the collected cells. Virus was purified by iodixanol (Optiprep, Sigma) stepwise gradient (15%, 25%, 40% and 60%). Virus was concentrated and prepared in PBS. Virus titer was determined by measuring the number of DNase1 resistance vector genome copies (VG) using qPCR and linearized genomic plasmid as a control.

[0102] The cell can be selected from humans, primates, mice, cats, dogs, pigs, sheep, cattle, horses, epine, goats and wolf host cells. In some cases, the cell is a progenitor cell or precursor cell, such as a stem cell. In some cases, the stem cell is a mesenchymal cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a fibroblast or other tissue-specific stem cell. The cell can be immortalized. In some cases, the immortalized cell is a HEK293 cell. In some cases, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in combination with any transgenic strain, which expresses a recombinase in the target cell type of interest, to develop an AAV capsid that more efficiently transduces the target cell population.

[0103] Treatment

[0104] Disclosed herein are methods for treating a disease or condition of a subject or a symptom of the disease or condition, comprising administering to the subject a therapeutically effective amount of one or more compositions disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions). In some embodiments, the composition is an rAAV capsid protein described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein described herein. In some embodiments, the rAAV particles encapsidate an AAV vector comprising a transgene (e.g., a therapeutic nucleic acid). In some embodiments, the composition is an rAAV capsid protein conjugated with a therapeutic agent disclosed herein as described herein. In some embodiments, the composition is a pharmaceutical composition comprising the rAAV particles and a pharmaceutically acceptable carrier. In some embodiments, the one or more compositions are administered alone to a subject (e.g., an independent therapy). In some embodiments, the composition is a first-line therapy for a disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy for a disease or condition.

[0105] Recombinant adeno-associated virus (rAAV)-mediated gene delivery utilizes the AAV mechanism of viral transduction for nuclear expression of free heterologous nucleic acids (e.g., transgenic, therapeutic nucleic acids). After delivery to the host's in vivo environment, rAAV will (1) bind or attach to cell surface receptors on target cells, (2) endocytosis, (3) travel to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid, (5) convert the heterologous nucleic acid from single-stranded DNA to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe the free heterologous nucleic acid in the nucleus of the host cell ("transduction"). rAAV engineered to have increased tissue-specific transduction enrichment (transcription of free heterologous nucleic acids in host cells) is ideal for gene therapy applications.

[0106] 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 a rAAV of the disclosure or a pharmaceutical formulation of the disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, administration is performed intracranially, intraventricularly, intraventricularly, intravenously, intraarterially, intranasally, intrathecally, intracisternal, or subcutaneously.

[0107] Provided herein is a method for treating a disease or condition associated with abnormal expression or activity of a target gene or its gene expression product, the method 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 disclosed herein. In some cases, the expression or activity of the target gene or gene expression product is reduced relative to the expression or activity in a normal (non-diseased) individual; and administering rAAV to the subject is sufficient to increase the expression or activity of the target gene or gene expression product. In some cases, the expression or activity of a gene or gene expression product is increased relative to the expression or activity in a normal individual; and administering rAAV to the subject is sufficient to reduce the expression or activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer's disease (caused in some cases by gain of function of presenilin 1 and / or presenilin 2 (encoded by genes PSEN1 and PSEN2, respectively)) is administered a rAAV disclosed herein that encapsidates a therapeutic nucleic acid, which is a silencing RNA (siRNA) or other RNAi that has a loss-of-function effect on PSEN1 mRNA.

[0108] Also provided are methods for preventing a disease or condition disclosed herein in a subject, comprising administering to the subject a therapeutically effective amount of a rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The rAAV vector can be encapsidated in a modified capsid protein or rAAV virus particle described herein. In some cases, the therapeutic gene expression product effectively regulates the activity or expression of a target gene or gene expression product.

[0109] Disclosed herein are methods of treating a disease or condition in a subject by administering a composition comprising a rAAV disclosed herein. An advantage of the rAAV disclosed herein is that the rAAV can be used to treat virtually any disease or condition that would benefit from transgenic 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, Battens disease, lysosomal storage disorders, glioblastoma multiforme, Rett syndrome, Leber's congenital amaurosis, late infantile neuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinal cord injury, traumatic brain injury, lysosomal storage disorders, and / or cardiovascular disorders.

[0110] In some cases, the disease or condition is localized to a specific in vivo environment of the subject, e.g., the brain, spinal cord, and / or heart. The compositions of the present disclosure are particularly useful in treating the diseases or conditions described herein because these compositions specifically or more effectively target the in vivo environment and deliver therapeutic nucleic acids that are engineered to modulate the activity or expression of a target gene expression product associated with the pathogenesis or pathology of the disease or condition.

[0111] Provided herein are methods of treating a disease or condition, or a symptom of the disease or condition, in a subject, comprising: (a) diagnosing a subject with a disease or condition that affects a target in vivo environment; and (b) treating the disease or condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions), wherein the composition is engineered to have increased expression.

[0112] Disclosed herein are methods of treating a subject suffering from a targeted disease or condition or a symptom of the disease or condition, the methods comprising: (a) administering a composition (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) to the subject; and (b) expressing a therapeutic nucleic acid into a targeted in vivo environment within the subject with increased transduction enrichment.

[0113] In some embodiments, the methods further comprise reducing or eliminating delivery of heterologous nucleic acids in off-target in vivo environments (such as the liver). In some embodiments, delivery is characterized by increased transduction enrichment (e.g., of the heterologous nucleic acid) in the brain, spinal cord, and / or heart.

[0114] In some embodiments, a method of treating a disease or condition affecting the brain, spinal cord, and / or heart comprises administering to the brain, spinal cord, and / or heart of a subject a rAAV particle comprising a rAAV capsid protein comprising at amino acid positions 587-588 of a parental AAV2 capsid protein of Table 1, Figure 1 and / or an insertion of about five, six, or seven amino acids in the amino acid sequence provided in Formulas I-III. In some embodiments, the method of treating a disease or condition affecting the brain, spinal cord, and / or heart comprises administering to the brain, spinal cord, and / or heart of a subject a rAAV particle comprising a rAAV capsid protein comprising an insertion of about five, six, or seven amino acids in the amino acid sequence and in Table 1, Figure 1 and / or one or more substitutions of the amino acids found at amino acid positions 586-589 provided in Formulas I-III. In some embodiments, the parent AAV capsid protein is an AAV2 capsid protein (eg, as provided in SEQ ID NO: 1).

[0115] Also provided are methods for regulating target gene expression products, which methods comprise administering a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) to a subject in need thereof. For example, the methods provided herein comprise administering to a subject an rAAV having an rAAV capsid protein, which encapsidates a viral vector comprising a heterologous nucleic acid that regulates the expression or activity of a target gene expression product.

[0116] The term "normal individual" refers to an individual who does not suffer from a disease or condition characterized by an alteration in the expression or activity of the gene or its gene expression product.

[0117] In some embodiments, the disease or condition of the CNS, brain and / or spinal cord is selected from the group consisting of: absent septum pellucidum, acid lipase disease, acid maltase deficiency, acquired epileptic aphasia, acute diffuse encephalomyelitis, attention deficit hyperactivity disorder (ADHD), Adie's Pupil, Adie's Syndrome, adrenoleukodystrophy, corpus callosum dysgenesis, agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS-neurological complications, Alexander disease, Disease), Alpers' Disease, Alternating Hemiparesis, Alzheimer's Disease, ALS, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Hypoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cyst, Arachnoiditis, Amold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxia and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit Hyperactivity Disorder, Autism Spectrum Disorder, Dysautonomia, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy Palsy), benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, Bemhardt-Roth Syndrome, Binswanger's Disease, blepharospasm, Bloch-Sulzberger Syndrome, brachial plexus birth injury, brachial plexus injury, Bradbury-Eggleston Syndrome, brain and spinal tumors, brain aneurysm, brain injury, lateral spinal cord compression-segmental cord syndrome (Brown-Sequard Syndrome), spinal bulbar muscular atrophy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), Canavan diseaseDisease), Carpal Tunnel Syndrome, Causalgia, Cavernous Tumor, Cavernous Hemangioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Spinal Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Head Disease, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavernous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculofacial-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth Syndrome, Classical Rhizoctonia Dysplasia Punctuata (RCDP), Cerebellar Tonsillar Herniation Malformation (Chiari Malformation), Cholesterol Ester Storage Disease, Chorea, Chorea Acanthosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome, Cockayne Syndrome Type II, Coffin-Lowry Syndrome Syndrome), syrinx, coma, complex regional pain syndrome, congenital facial palsy, congenital myasthenia, congenital myopathy, congenital cavernous malformation, cortical-basal degeneration, cranial arteritis, craniosynostosis, Cree encephalitis, Creutzfeldt-Jakob Disease, cumulative trauma disorder, Cushing's syndrome, cytomegalovirus infection, dancing eyes and dancing feet syndrome, atresia of the fourth ventricle (Dandy-Walker Syndrome), Dawson Disease, deafness, Demers syndrome, De-Ker palsy, dementia, multi-infarct dementia, semantic dementia, subcortical dementia, dementia with Lewy bodies, dentine-cerebellar ataxia, dentate nuclear atrophy, dermatomyositis, developmental movement disorder, Devic's syndromeSyndrome), diabetic neuropathy, diffuse sclerosis, Dravet syndrome, Duchenne muscular dystrophy, autonomic dysfunction, dysgraphia, dyslexia, dysphagia, dyskinesia, cerebellar myoclonic dyssynergia, progressive cerebellar dyssynergia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalitis lethargica, encephalocele, encephalopathy, encephalopathy (familial infantile), cerebral trigeminal angiomatosis, epilepsy, epileptic hemiplegia, Erb's palsy, Ou-Dubonnet palsy and DeGelina-Krompke palsy, essential tremor, extrapontine myelinolysis Familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralysis, familial spastic paralysis, Farber disease, febrile seizures, fibromuscular dysplasia, Fisher syndrome, floppy baby syndrome, foot drop, fragile X syndrome, Friedreich's ataxia, frontotemporal dementia (FTD), Gaucher disease, generalized gangliosidosis, Guillain-Strauss syndrome, Guillain-Strauss syndrome, giant axonal neuropathy, giant cell arteritis, giant cell inclusion disease, glioblastoma, glomuscular dysplasia Leukodystrophy, glossopharyngeal neuralgia, glycogen storage disease, Guillain-Barré syndrome, Harrington-Strauss disease, head injury, headache, migraine continua, hemifacial spasm, alternating hemiplegia, hereditary neuropathy, hereditary spastic paraplegia, hereditary ataxia polyneuritis, herpes zoster, herpes zoster oticus, Hirayama syndrome, Hodgkin-Ellison syndrome, schizophrenia, HTLV-1 associated myelopathy, Hughes syndrome, Huntington's disease, hydrocephalus, hydrocephalus, hydrocephalus-normal pressure, hydromyelia, hypercortisolism, hypersomnia, hypertonia, hypotonia , hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, pigment incontinence, infantile hypotonia, infantile axonal dystrophy, infantile phytanic acid storage disease, infantile Refsum's disease (IRD), infantile spasms, inflammatory myopathy, anencephaly, intestinal lipodystrophy, intracranial cysts, intracranial hypertension, Isaacs syndrome, Joubert syndrome, Kearns-Sell syndrome, Kennedy disease, Kingsborne syndrome, Kleine-Levin syndrome, Klippel-Feil syndrome, Klippel-Trenaunay syndrome (KTS), Klippel-Feil syndrome, congenital venous malformation hypertrophy syndrome (KTS), Kliiver-Bucy syndrome, Korsakoff's Amnesic Syndrome, Krabbe disease, Kugelberg-Welander diseaseDisease), Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Compression, Dorsolateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Dementia with Lewy Bodies, Lipid Storage Diseases, Lipid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus - Neurological Sequelae, Lyme Disease - Neurological Complications, Machado-Joseph Disease, Megacephaly, Maple Syrup Sydenhamopathy, Megacephaly, Melkersson-Rosenthal Syndrome Syndrome), Meningitis, Meningitis and Encephalitis, Menkes Disease, Menkes Syndrome, Paresthesia, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller-Fisher Syndrome, Mini-stroke, Mitochondrial Myopathy, Moebius Syndrome, Monosomy, Motor Neuron Disease, Moyamoya Disease, Mucolipidemia, Mucopolysaccharidosis, Mucopolysaccharidosis II, Multi-infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Congenital Myasthenia, Myasthenia Gravis, Diffuse Myelinolytic Sclerosis, Infantile Myoclonic Encephalopathy, Myoclonus, Myopathy, Myopathy-Congenital, Myoclonus Diseases - thyrotoxicity, myotonia, myotonia congenita, myotonic dystrophy, narcolepsy, neuroacanthocytosis, neurodegeneration with brain iron accumulation, neurofibromatosis, neuroleptic malignant syndrome, neurologic complications of AIDS, neurologic complications of Lyme disease, neurologic consequences of cytomegalovirus infection, neurologic manifestations of Pompe disease, neurologic sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorders, hereditary neuropathies, neurosarcoidosis, neurosyphilis, neurotoxicity, cavernous nevus, Niemann-Pick disease, O'Sullivan-McLeod syndrome, occipital neuralgia, Ohtahara syndromeSyndrome), olivopontocerebellar atrophy, opsoclonus-myoclonus, orthostatic hypotension, overuse syndrome, chronic pain, pantothenate kinase-related neurodegeneration, paraneoplastic syndrome, paresthesia, Parkinson's disease, paroxysmal choreoathetosis, paroxysmal migraine, Parry-Romberg disease, Pierce-Moller disease, Pena-Schukel II syndrome, perineural cysts, periodic paralysis, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorder, phenylketonuria, phytanic acid storage disease, Pick's disease, pinched nerve, piriformis syndrome, pituitary tumor, polymyositis, Pompe disease, brain perforation, postpoliomyelitis syndrome, postherpetic neuralgia, postinfectious encephalomyelitis, postural hypotension, postural tachycardia syndrome, postural tachycardia syndrome , Prader-Willi syndrome, primary odontoid atrophy, primary lateral sclerosis, primary progressive aphasia, prion disease, progressive hemifacial atrophy, progressive motor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing poliomyelitis dystrophy, progressive supranuclear palsy, prosopagnosia, pseudo-Torch syndrome, pseudo-toxoplasmosis syndrome, pseudotumor cerebri, psychogenic movements, Ramsay-Hunt syndrome I, Ramsay-Hunt syndrome II, Memsheimer's encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease-infantile, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rheumatic encephalitis, Holliday syndrome, sacral nerve root cyst, Saint Vitus choreaDance), salivary gland disease, Sandhoff disease, Skild's disease, schizencephaly, Setelberg disease, seizure disorder, semantic dementia, septooptic dysplasia, severe myoclonic epilepsy of infancy (SMEI), shaken baby syndrome, herpes zoster, Shy-Dreifenbach syndrome, Sjögren's syndrome, sleep apnea, sleeping sickness, Sue's syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinocerebellar ataxia, spinal cord Cerebellar atrophy, spinocerebellar degeneration, Stille-Richardson-Olszewski syndrome, stiff-person syndrome, striatonigral degeneration, stroke, Stych-Weber syndrome, STXBP1 encephalopathy, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, transient, unilateral neuralgic (SUNCT) headache, dysphagia, Sydenham's chorea, syncope, syphilitic myelosclerosis, syringomyelia, syringomyelia, systemic lupus erythematosus, kyphoscoliosis, Tangier disease , tardive dyskinesia, Tarlov cyst, Tay-Sachs disease, temporal arteritis, tethered cord syndrome, Thomson myotonia, thoracic outlet syndrome, thyrotoxic myopathy, Tourette syndrome, Todd's palsy, Tourette syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paralysis, Troyer syndrome, tuberous sclerosis, vascular erectile tumors, central nervous system vasculitis syndrome, von Economou disease, von Hippel-Lindau disease (VHL), von Hippel-Lindau syndrome, von Recklinghausen disease, dorsolateral medullary syndrome, Werdnig-Hoffmann disease, Wernicke-Korsakoff syndrome, Wester syndrome, Whiplash, Whipple disease, Williams syndrome, Wilson disease, Wolman disease, X-linked spinal and bulbar muscular atrophy, and Zellweger syndrome.

[0118] In some embodiments, the drug formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some cases, the therapeutic gene expression product effectively regulates the activity or expression of a target gene or gene expression product selected from the following: ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMT1, TCF4, RAI1, PEX1, ARSA, EIF2B5, EIF2B1, EIF2B2, NPC1, ADAR, MFSD8, STXBP1, PRICKLE2, PRRT2, IDUA, STX1B, inositol alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), glial cell line derived neurotrophic factor (GDNF), motor neuron survival gene 1, STXBP1, telomere (SMN1 ), factor X (FIX), retinoid isomerase (RPE65), sarcoplasmic reticulum / endoplasmic reticulum Ca2+-ATPase (SERCA2a), glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, frataxin (FXN), huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), anti-tubulin agent, copper-zinc superoxide dismutase (SOD1), iduronate 2 sulfatase (hIDS), glucosylceramidase beta (GBA), fragile X mental retardation 1 (FMR1), NPC intracellular cholesterol transporter 1 (NPC1), SCN1A, C9orf72, NPS3, and NLRP3 inflammasome. In some embodiments, the peroxisome biogenesis factor (PEX) is selected from the group consisting of PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX110, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0119] In some aspects, other examples of genes involved in CNS, brain and / or spinal cord 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.

[0120] In some embodiments, the cardiac disease or condition includes any disease of the cardiopulmonary system, including but not limited to heart failure, local ischemia, arrhythmia, myocardial infarction, congestive heart failure, transplant rejection, abnormal cardiac contractility, non-ischemic cardiomyopathy, mitral valve regurgitation, refractory myocardial ischemia, non-ischemic heart failure, aortic valve stenosis or regurgitation, abnormal Ca2+ metabolism, and disorders of cardiac-specific proteins or proteins that can effectively regulate cardiac activity or physiology.

[0121] In some embodiments, genes involved in cardiac disease include those expressing proteins involved in the regulation of calcium cycling in cardiomyocytes, such as the sarcoplasmic endoplasmic reticulum Ca2+ ATPase pump.

[0122] In some embodiments, the cells of the heart or cardiac tissue comprise tissues or cells of any part of the cardiopulmonary system. In certain aspects, the cells of the heart or cardiac tissue comprise cardiomyocytes / tissues, cells / tissues of the cardiac vasculature, and cells / tissues present in the heart valves. The cells of the heart may include cardiomyocytes, epithelial cells, endothelial cells, fibroblasts, conducting tissue cells, cardiac pacemaker cells, and neurons.

[0123] In some cases, the therapeutic gene expression product comprises a gene editing component. In some cases, the gene editing component is selected from: artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), transcription factor-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / Cas enzymes and CRISPR / Cas guide RNAs.

[0124] In some cases, the expression of a gene or the expression or activity of a gene expression product is inhibited by administering the composition to a subject. In some cases, the expression of a gene or the expression or activity of a gene expression product is enhanced by administering the composition to a subject.

[0125] Formulations, Dosages and Routes of Administration

[0126] Disclosed herein are methods comprising delivering rAAV particles that encapsidate heterologous nucleic acids to the brain, spinal cord, and / or heart of a subject, the rAAV particles comprising (i) increased transduction of the heterologous nucleic acid in the brain, spinal cord, and / or heart, wherein the rAAV particles have a rAAV capsid protein that comprises the amino acid positions 587-588 of the parental AAV2 capsid protein of Table 1, Figure 1 and / or an insertion of five, six or seven amino acids in the amino acid sequence provided in Formulas I-III, or comprising a rAAV capsid protein comprising an insertion of about five, six or seven amino acids in the amino acid sequence and in the amino acid sequence provided in Tables 1, Figure 1and / or one or more substitutions of the amino acids found at amino acid positions 586-589 provided in Formulas I-III.

[0127] Typically, the methods disclosed herein include administering a therapeutic rAAV composition by systemic administration. In some cases, these methods include administering a therapeutic rAAV composition by intravenous ("iv") administration. Therapeutic rAAV compositions can be administered by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transcutaneous 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 approach, dosage, time point and duration of the therapeutic agent can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of any one or both of the acute and chronic symptoms of the disease or condition. Other delivery routes include, but are not limited to, intracranial administration, lateral ventricular administration and intravascular administration.

[0128] The effective dose and dosage of the pharmaceutical composition for preventing or treating the diseases or conditions disclosed herein are defined by the observed beneficial response associated with the disease or condition or the symptoms of the disease or condition. The beneficial response includes preventing, alleviating, stopping or curing the disease or condition or the symptoms of the disease or condition. In some embodiments, the beneficial response can be measured by detecting the measurable improvement of the presence, level or activity of the subject's biomarkers, transcriptome risk profile or intestinal microbiome. As used herein, "improvement" refers to the presence, level or activity to the presence, level or activity observed in normal individuals (e.g., individuals without diseases or conditions). In the case where the therapeutic rAAV composition is ineffective or does not fully alleviate the disease or condition or the symptoms of the disease or condition, the dosage and / or route of administration can be changed, or another agent can be administered to the subject together with the therapeutic rAAV composition. In some embodiments, when the patient begins the regimen of the therapeutic rAAV composition, the patient also gradually stops (e.g., the dose is gradually reduced) the second treatment regimen.

[0129] In some cases, a dose of a pharmaceutical composition can contain infectious particles at a concentration of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 or 10 17 In some cases, the concentration of infectious particles was 2 × 107 , 2×10 8 , 2×10 9 , 2×10 10 , 2×10 11 , 2×10 12 , 2×10 13 , 2×10 14 , 2×10 15 , 2×10 16 or 2×10 17 In some cases, the concentration of infectious particles was 3 × 10 7 , 3×10 8 , 3×10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 13 , 3×10 14 , 3×10 15 , 3×10 16 or 3×10 17 In some cases, the concentration of infectious particles was 4 × 10 7 , 4×10 8 , 4×10 9 , 4×10 10 , 4×10 11 , 4×10 12 , 4×10 13 , 4×10 14 , 4×10 15 , 4×10 16 or 4×10 17 In some cases, the concentration of infectious particles was 5 × 10 7 , 5×10 8 , 5×10 9 , 5×10 10 , 5×10 11 , 5×10 12 , 5×10 13 , 5×10 14 , 5×10 15 , 5×10 16 or 5×10 17 In some cases, the concentration of infectious particles was 6 × 10 7 , 6×10 8 , 6×10 9 , 6×10 10 , 6×10 11 , 6×10 12 , 6×10 13 , 6×10 14 , 6×1015 , 6×10 16 or 6×10 17 In some cases, the concentration of infectious particles was 7 × 10 7 ,7×10 8 ,7×10 9 ,7×10 10 ,7×10 11 ,7×10 12 ,7×10 13 ,7×10 14 ,7×10 15 ,7×10 16 or 7×10 17 In some cases, the concentration of infectious particles was 8 × 10 7 , 8×10 8 , 8×10 9 , 8×10 10 , 8×10 11 , 8×10 12 , 8×10 13 , 8×10 14 , 8×10 15 , 8×10 16 or 8×10 17 In some cases, the concentration of infectious particles was 9 × 10 7 ,9×10 8 ,9×10 9 ,9×10 10 ,9×10 11 ,9×10 12 ,9×10 13 ,9×10 14 ,9×10 15 ,9×10 16 or 9×10 17 .

[0130] In some embodiments, disclosed herein are formulations of pharmaceutically acceptable excipients and carrier solutions suitable for delivering rAAV compositions described herein, as well as suitable dosages and treatment regimens for using specific compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition can be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and as such, a variety of dosages and treatment regimens may be desired.

[0131] In some embodiments, the pharmaceutical form of the rAAV-based viral composition suitable for injection includes 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, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. Microbial action can be prevented by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferred to include an isotonic agent, for example, sugar or sodium chloride.

[0132] In some cases, for the use of injectable aqueous solution, if necessary, the solution can be suitably buffered, and the liquid diluent is first isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Depending on the symptom of the experimenter being treated, some changes will inevitably occur in dosage. The personnel responsible for administration will determine the appropriate dosage for individual subjects under any circumstances. In addition, for human use, formulations should meet the sterility, pyrogenicity and general safety and purity standards required by the FDA Office of Biological Products Standards.

[0133] Disclosed herein are sterile injectable solutions containing rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the desired amount into an appropriate solvent together with several other ingredients enumerated above as needed, and then filtering and sterilizing. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from their previously sterile filtered solutions. Injectable solutions can be advantageous for systemic administration (e.g., by intravenous or intrathecal administration).

[0134] The appropriate dose and dosage for administration to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, the disease condition and its severity, the identity of the subject in need of treatment (e.g., weight, sex, age), and may be determined based on the specific circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0135] The amount of rAAV composition and the time of administration of such composition will be within the capabilities of those skilled in the art having the benefit of the present teachings. However, it is likely that administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, e.g., a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to a patient receiving such treatment. This is made possible at least in part by the fact that certain target cells (e.g., cells of the brain, spinal cord, and / or heart) do not divide, thereby eliminating the need for multiple or chronic administration.

[0136] In certain embodiments, the data obtained from cell culture assays and animal studies can be used to formulate therapeutically effective daily dosage ranges and / or therapeutically effective unit doses for mammals (including humans). In certain embodiments, dosage ranges and / or unit doses vary within the range, depending on the dosage form employed and the route of administration utilized.

[0137] Combination therapy

[0138] Therapeutic rAAV can be used alone or in combination with another therapeutic agent (collectively referred to as a "therapeutic agent"). In some cases, a therapeutic rAAV as used herein is administered alone. The therapeutic agents can be administered together or sequentially in a combination therapy. The combination therapy can be administered on the same day, or can be administered one or more days, weeks, months, or years apart.

[0139] Additional therapeutic agents may include small molecules. Additional therapeutic agents may include antibodies or antigen binding fragments. Additional therapeutic agents may include lipid nanoparticle-based therapies, antisense oligonucleotide therapies, and other viral therapies.

[0140] Additional therapeutic agents may include cell-based therapies. 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. Any one of NK cells or CAR-NK cells or a combination of NK cells and CAR-NK cells may be used in combination with the methods disclosed herein. In some embodiments, NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSC), cord blood, or cell lines. NK cells and CAR-NK cells may include cytokine receptors and suicide genes. Cell-based therapies may include stem cell therapy. Stem cell therapy may be embryonic stem cells or somatic stem cells. Stem cells may be separated from donors (allogeneic) or separated from subjects (autologous). Stem cells may be adipose-derived stem cells (eASC), hematopoietic stem cells (HSC), mesenchymal stem (stromal) cells (MSC), or induced pluripotent stem cells (iPSC) derived from cells of subjects.

[0141] Reagent test kit

[0142] Kits comprising the compositions disclosed herein are disclosed herein. Kits for treating or preventing diseases or conditions of the brain, spinal cord and / or heart are also disclosed herein. In some cases, the disease or condition is cancer, a pathogen infection, a lung disease or condition, a nervous system disease, a muscle disease or an immune disorder, such as a disease or condition described herein.

[0143] In one embodiment, the kit may include a therapeutic or preventive composition containing an effective amount of rAAV particles and a composition of a recombinant AAV (rAAV) capsid protein disclosed herein, the rAAV particles encapsidating a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., a therapeutic nucleic acid). In another embodiment, the kit may include a therapeutic or preventive composition containing an effective amount of cells modified by rAAV described herein in a unit dosage form expressing a therapeutic nucleic acid ("modified cells"). In some embodiments, the kit includes a sterile container that may contain a therapeutic composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs.

[0144] In some cases, the test kit further comprises cells. In some cases, the cells are mammalian. In some cases, the cells are immortalized. In some cases, the immortalized cells are embryonic stem cells. In some cases, the embryonic stem cells are human embryonic stem cells. In some cases, the human embryonic stem cells are human embryonic kidney 293 (HEK-293) cells. In some cases, the test kit further comprises an AAV vector, which comprises a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the AAV vector is an episome.

[0145] In some cases, the rAAV is provided with instructions for administering the rAAV to a subject having a disease or condition (e.g., a disease of the brain, spinal cord, and / or heart) or at risk of developing the disease or condition. The instructions can generally include information about the use of the composition for treating or preventing a disease or condition.

[0146] 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; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions can be printed directly on the container (if present), or as a label applied to the container or as a separate sheet, brochure, card, or folder provided in or with the container. In some cases, the instructions provide procedures for administering rAAV alone to a subject. In some cases, these instructions provide that the rAAV is formulated for systemic delivery.

[0147] definition

[0148] The terms used herein are for the purpose of describing a particular situation only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, where the terms "including / includes", "having / has / with" or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0149] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, for example, limitations of the measurement system. For example, depending on the practice of a given value, "about" can mean within 1 or more than 1 standard deviation. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean an acceptable error range for that specific value unless otherwise stated.

[0150] As used herein, "consisting essentially of" when used to define compositions and methods shall mean excluding other elements having any significance to the combination for the stated purpose. Thus, a composition consisting essentially of elements as defined herein would not exclude other materials or steps that do not materially affect one or more of the basic and novel characteristics of the claimed disclosure, such as a composition for treating skin conditions such as acne, eczema, psoriasis, and rosacea.

[0151] The terms "homologous", "homology" or "homology percentage" as used herein generally refer to an amino acid sequence or nucleic acid sequence having the same or similar sequence as a reference sequence. As of the date of filing of this application, the latest version of BLAST can be used to determine the homology percentage of a sequence.

[0152] The term "increase" is used herein to generally refer to an increase in a statistically significant amount. In certain embodiments, the term "increase" means an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, 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% increase, or any increase between 10%-100%. Other examples of "increase" include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times compared to a reference level.

[0153] The term "reduce (decreased / decrease)" is generally used to represent the reduction of statistically significant amount in this article.In certain embodiments, "reduce" means reduced by at least 10% compared with a reference level, for example, reduced by 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% reduction (for example, non-existent level or undetectable level compared with a reference sample) or any reduction between 10-100%.In the context of markers or symptoms, these terms mean the statistically significant reduction of such levels.Reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to the level accepted in the normal range as an individual not suffering from a given disease.

[0154] The term "subject" is any organism. In some cases, the organism is a mammal. Non-limiting examples of mammals include any member of the mammal class: humans, non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs, etc.; domestic animals, such as rabbits, dogs and cats, etc.; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. On the one hand, the mammal is a human. As used herein, the term "animal" includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, such as a rodent, such as a rat or a mouse. In one embodiment, a "non-human primate" is a mammal, such as a monkey. In some cases, a subject is a patient, which, as used herein, may refer to a subject diagnosed with a specific disease or condition.

[0155] As used herein, the term "gene" refers to a nucleic acid fragment (also referred to as a "coding sequence" or "coding region") that encodes a single protein or RNA, optionally together with associated regulatory regions (such as promoters, operators, terminators, etc.), which may be located upstream or downstream of the coding sequence.

[0156] As used herein, the term "adeno-associated virus" or "AAV" refers to an adeno-associated virus or a derivative thereof. Non-limiting examples of AAV include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), avian AAV, cattle AAV, canine AAV, horse AAV, primate AAV, non-primate AAV and sheep AAV. In some cases, AAV is described as "primate AAV", which refers to AAV that infects primates. Similarly, AAV can infect cattle (e.g., "cattle AAV", etc.). In some cases, AAV is wild-type or naturally occurring. In some cases, the AAV is recombinant.

[0157] As used herein, the term "AAV capsid" refers to a capsid protein or peptide of an adeno-associated virus. In some cases, the AAV capsid protein is configured to encapsidate genetic information (e.g., transgenic, therapeutic nucleic acid, viral genome). In some cases, the AAV capsid of the present disclosure is a modified AAV capsid relative to the corresponding parent AAV capsid protein.

[0158] As used herein, the term "tropism" refers to a quality or property of an AAV capsid, which quality or property may include an increased or decreased enrichment for expressing encapsidated genetic information into an in vivo environment relative to a second in vivo environment. In some cases, the in vivo environment is a cell type. In some cases, the in vivo environment is an organ or organ system.

[0159] The term "AAV vector" as used herein refers to a nucleic acid polymer encoding genetic information associated with a virus. The AAV vector can be a recombinant AAV vector (rAAV), which refers to an AAV vector produced using a recombinant genetics method. In some cases, the rAAV vector comprises at least one heterologous polynucleotide (e.g., a polynucleotide other than a wild-type or naturally occurring AAV genome, such as a transgene).

[0160] As used herein, the term "AAV particle" refers to an AAV virus, a virion, an AAV capsid protein, or a component thereof. In some cases, the AAV particle is modified relative to a parent AAV particle.

[0161] The term "gene product" of "gene expression product" refers to the 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).

[0162] As used herein, the term "heterologous" refers to a genetic element (eg, coding region) or gene expression product (eg, RNA, protein) derived from an entity that is genotypically different from the rest of the entity to which it is being compared.

[0163] As used herein, the term "endogenous" refers to a genetic element (eg, coding region) or gene expression product (eg, RNA, protein) that is naturally present in or associated with an organism or a specific cell within an organism.

[0164] As used herein, the terms "treat", "treating" and "treatment" refer to the alleviation or elimination of a disease, disease or condition; or one or more symptoms associated with a disease, disease or condition; or the alleviation or elimination of the cause of the disease, disease or condition itself. The desired therapeutic effect may include, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0165] The term "therapeutically effective amount" refers to that amount of a compound or therapy which, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of a disorder, disease, or condition of a disease; or an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or clinician.

[0166] 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 human and animal tissues or organs at a reasonable benefit / risk ratio without producing excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications. See Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th ed.; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004).

[0167] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein and other chemical components such as a diluent or carrier. A pharmaceutical composition can facilitate administration of a compound to an organism. There are a variety of techniques for administering a compound in the art including, but not limited to, systemic administration.

[0168] Non-limiting examples of "samples" include any material from which nucleic acids and / or proteins can be obtained. As non-limiting examples, this includes whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph fluid, fecal extract, cheek swab, cells or other body fluids or tissues, including but not limited to tissues obtained by surgical biopsy or surgical resection. Alternatively, the sample can be obtained by a primary patient-derived cell line, or can be an archived patient sample in the form of a stored sample or a fresh frozen sample.

[0169] The term "in vivo" is used to describe events that occur within the body of a subject.

[0170] The term "in vitro" is used to describe events that occur in a container used to hold laboratory reagents, such that the material is separated from the biological source from which the material was obtained. In vitro assays may encompass cell-based assays, in which living or dead cells are employed. In vitro assays may encompass cell-free assays, in which intact cells are not employed.

[0171] The term "CNS" or "central nervous system" means a tissue selected from the group consisting of the brain, thalamus, cortex, dura mater, lateral ventricles, medulla, 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.

[0172] The term "systemic delivery" is defined as a route of administration in which a drug or other substance enters the circulatory system so that the entire body is affected. Administration can be performed by enteral administration (drugs are absorbed by the gastrointestinal tract) or parenteral administration (usually injection, infusion or implantation). "Circulatory system" includes both the blood circulatory system and the cerebrospinal fluid circulatory system. Examples of systemic administration for CNS include intra-arterial, intravenous or intrathecal injections. Other examples include administration to the cerebrospinal fluid at any position in the spinal cord (i.e., but not limited to the lumbar vertebrae) or brain (i.e., but not limited to the cerebellomedullary cistern). The terms "systemic administration" and "systemic delivery" are used interchangeably.

[0173] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0174] Examples

[0175] Example 1

[0176] Methods for Identifying Modified Capsid Proteins in Cynomolgus Macaques

[0177] The therapeutic applicability of engineered adeno-associated viruses (AAVs) is primarily concerned with the extent to which their transduction profiles (including tissue-specific transduction profiles) are translated into human applications. Although previous engineering efforts have focused on in vitro or in vivo rodent screening platforms because of the ease of use and flexibility of screening platforms, screening efforts conducted directly in non-human primates (NHPs) are more likely to identify viruses that are translated. The present invention selected cynomolgus monkeys (an old world NHP) for engineering efforts. The engineering efforts are focused on the region at amino acid position #587 on the surface of the AAV2 capsid, which is one of the most exposed loops on the capsid surface. The loop is a variable region between natural AAV serotypes and plays a role in receptor binding. This article and others have studied the insertion of peptides between positions 587 and 588 and between 588 and 589 in the past, and novel receptor binding and significantly altered capsid tropism have been produced. A viral capsid library was established by randomly inserting 7 amino acids at this site in AAV2 in order to obtain new tropism for NHP brain, heart and / or spinal cord. The first library was introduced into macaques and the expression levels of the variants were screened in different tissue types (i.e., brain, spinal cord, heart, and liver). Of the billions of variants introduced in the first library, 39,000 variant sequences were identified for the second round of screening. As in the first round, these 39,000 were studied for tissue-specific expression, and the best 975 were selected for inclusion in this paper. Figure 1 in the variants described in .

[0178] Plasmids . The first round of viral DNA libraries was generated by amplifying a fragment of the AAV2 capsid genome between amino acids 450-599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 587 and 588 with all possible variations. The resulting library insert was then introduced into the rAAV-ACap-in-rev-RNA plasmid by Gibson assembly as previously described. The resulting capsid DNA library rAAV-Cap-Cag-GFP11 contains a diversity of billions of variants at the amino acid level. As described above, the second round of viral DNA libraries contained 39,000 variants at the amino acid level, with 3 barcode replicas for each variant selected from the variants screened in round 1.

[0179] The AAV2 REP-AAP-ACAP plasmid transfected into HEK293T cells to provide the Rep gene for library virus production prevented the production of wild-type AAV2 capsids following plausible recombination events between this plasmid and the library plasmid containing the library insert at each stage during viral library production.

[0180] Virus production . Recombinant AAV was produced according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple transfected with four vectors using polyethyleneimine (PEI). The first vector was a rAAV-Cap-in-cis-Lox library flanked by inverted terminal repeat (ITR) sequences from the parental AAV virus. The second vector was an AAV2 / 9REP-AAP-ACAP plasmid. The third vector contained nucleic acids encoding auxiliary viral proteins required for viral assembly and packaging of heterologous nucleic acids into modified capsid structures. The fourth was a pUC-18 plasmid, which was included to achieve the correct PEI / DNA ratio for optimal transfection enrichment. Only 10 ng of rAAV-Cap-in-cis-Lox library DNA (per 150 mm plate) was transfected to reduce the possibility of multiple library DNAs entering the same cell. Viral particles were harvested from cells and culture medium 60 hours after transfection. The virus that is present in the culture medium is concentrated by 8% polyethylene glycol and 500mM sodium chloride precipitation, and the virus through precipitation is added in the lysate prepared by collected cells. Through iodixanol (Optiprep, Sigma) stepwise gradient (15%, 25%, 40% and 60%) virus purification. The virus is concentrated and deployed in PBS. Determine the virus titer by using qPCR and the quantity of the linearized genomic plasmid measurement DNase1 resistance vector genome copy (VG) as a control.

[0181] animal . The cynomolgus monkey procedure was approved by the IACUC committee of Envol Biomedical. Cynomolgus monkeys were born and raised in the Envol Biomedical colony and were housed in family groups under standard conditions. They were fed ad libitum and were enriched as part of Envol Biomedical's primate enrichment program for NHPs. For AAV infusion, animals were screened for endogenous neutralizing antibodies (Nab). None of the screened animals showed any detectable blocking reaction when the serum was diluted 1:10. The group was then housed alone for several days and adapted to the new room before injection. The animals were restrained and the test articles were administered by intravenous infusion for 10 minutes. Two young monkeys were used in each of the 1st and 2nd rounds of screening. Activity and behavior were monitored daily during life.

[0182] DNA / RNA recovery and sequencing The first and second round virus libraries were divided into 1-3×10 13 vg / kg animal was injected into cynomolgus monkeys, and the rAAV genome was recovered two weeks after injection. The animals were euthanized, and the brain, spinal cord, heart and liver, as well as other peripheral tissues, were recovered, quickly frozen, and placed at -80°C for long-term storage. For round 1, the brain was divided into seven brain regions, 100 mg each, and for round 2, the brain was divided into thirteen brain regions. 20-100 mg of each brain part, spinal cord and liver were homogenized in buffer using MagMAX DNA ULTRA (A25597) and Bead Ruptor 96 (OMNI), and viral DNA was isolated according to the manufacturer's recommended protocol. The recovered viral DNA was treated with RNase and purified with a Zymo DNA cleaning and concentrator kit (D4033). The viral genome was enriched by 25 cycles of PCR amplification, with primers flanking the 587-588 insertion site in the capsid genome, using 50% of the total extracted viral DNA as a template. After Zymo DNA purification, samples were diluted 1:10 to 1:1000 according to tissue type, and each dilution was further amplified around the variable region of the library with 10 cycles of PCR. Subsequently, the samples were further amplified for more than 10 cycles using custom primers with Illumina indexes. The amplified products were run on 2% low melting point agarose gel (Thermo Fisher Scientific, 16520050) to better separate and recover the 210 bp band.

[0183] For the second round of libraries only, 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, such as viral DNA extracted from tissues to add adapters and indexes for Illumina next-generation sequencing, and purified after gel electrophoresis. This viral library DNA, along with viral DNA extracted from tissues, was deeply sequenced using Illumina's NextSeq 2000 system.

[0184] NGS data alignment and processing . Raw fastq files from NGS runs were processed using a custom script (Capsida CapSeq tool). For the first-round libraries, the pipeline used to process these datasets involved filtering to remove low-quality reads, utilizing a quality score for each sequence and removing bias caused by PCR-induced mutations or high GC content. The filtered datasets were then aligned using a perfect string matching algorithm and adjustments were made to improve alignment quality. Read counts for each sequence were extracted and presented by tissue, at which point all sequences found in the brain, spinal cord, and / or heart were compiled to form the second-round libraries.

[0185] For the second round of libraries, the read counts by tissue were also tabulated similarly. Then, the read count 1 was added to each sequence to remove the 0 value, all brain regions of each sequence were added, and the read sequences of each codon copy of the given 7-mer amino acid sequence were added to obtain a single value for each peptide insertion. Finally, the read count data was normalized relative to counts per million (Cpm). The enrichment value of each capsid variant was calculated by normalized cpm from [tissue of interest] / cpm from the virus library.

[0186] Although 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. Without departing from the present invention, many variations, changes and substitutions will now occur to those skilled in the art. It should be understood that each alternative of the embodiments of the present invention described herein can be used to practice the present invention. The appended claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

[0187] 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 having at least 70% identity to a sequence selected from SEQ ID NOs: 976-1949.

2. The AAV capsid protein of claim 1, comprising a sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 2-975.

3. The AAV capsid protein of claim 2, comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 2-975.

4. The AAV capsid protein of claim 3, comprising a sequence selected from the group consisting of SEQ ID NOs: 2-975.

5. The AAV capsid protein of claim 1, wherein the capsid protein comprises a seven amino acid insertion having a sequence having at least 70% identity in amino acid sequence to a sequence selected from one of SEQ ID NOs: 976-1949, the amino acid sequence having a sequence at least 98% identical to amino acid 217 to amino acid 736 of the AAV capsid protein provided in SEQ ID NO:

1.

6. The AAV capsid protein of claim 5, wherein the seven amino acid insertion is inserted between amino acid positions 587 and 588 of SEQ ID NO:

1.

7. The AAV capsid protein of claim 6, wherein the AAV capsid protein is characterized by at least one of increased specificity and / or increased transduction efficiency relative to the native AAV capsid protein of SEQ ID NO: 1 when measured in the brain, heart or spinal cord of the subject when delivered systemically to the subject.

8. The AAV capsid protein of claim 7, wherein the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the brain.

9. The AAV capsid protein of claim 8, wherein the seven amino acid insertion comprises a sequence having at least 70% identity to a sequence selected from one of SEQ ID NOs: 976-1411.

10. The AAV capsid protein of claim 9, wherein the seven amino acid insertion comprises a sequence of formula [I]: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula 1) (SEQ ID NO: 1950), wherein: X 1 is an amino acid selected from L, I and V; X 2 is an amino acid selected from L, V and M; X 3 is an amino acid selected from G, S and P; X 4 is an amino acid selected from the group consisting of P, G, S, T and H; X 5 is an amino acid selected from the group consisting of S, L, T, A, H and Q; X 6 is an amino acid selected from L, M, P, S, T and G; and / or X 7 is an amino acid selected from the group consisting of S, T, A, L, Q, P and V.

11. The AAV capsid protein according to claim 10, wherein X 2 It's L.

12. The AAV capsid protein according to claim 10, wherein X 3 It's G.

13. The AAV capsid protein according to claim 10, wherein X 6 It's L.

14. The AAV capsid protein of claim 9, wherein the seven amino acid insertion is one of SEQ ID NOs: 976-1411.

15. The AAV capsid protein of claim 14, wherein the capsid protein comprises a sequence of one of SEQ ID NOs: 2-437.

16. The AAV capsid protein of claim 7, wherein the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the heart.

17. The AAV capsid protein of claim 16, wherein the seven amino acid insertion comprises a sequence having at least 70% identity to a sequence selected from one of SEQ ID NOs: 1412-1612.

18. The AAV capsid protein of claim 17, wherein the seven amino acid insertion comprises a sequence of formula [I]: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [I]) (SEQ ID NO: 1951), wherein: X 1 is an amino acid selected from the group consisting of S, N, T and L; X 2 is an amino acid selected from the group consisting of T, N, V ​​and S; X 3 is an amino acid selected from the group consisting of R, K, T and S; X 4 is an amino acid selected from the group consisting of K, S, T, A and R; X 5 is an amino acid selected from the group consisting of L, I, S, R and G; X 6 is an amino acid selected from the group consisting of P, S, Q and L; and / or X 7 is an amino acid selected from the group consisting of P, A and S.

19. The AAV capsid protein of claim 17, wherein the seven amino acid insertion is one of SEQ ID NOs: 1412-1612.

20. The AAV capsid protein of claim 19, wherein the capsid protein comprises a sequence of one of SEQ ID NOs: 438-638.

21. The AAV capsid protein of claim 7, wherein the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the heart.

22. The AAV capsid protein of claim 21, wherein the seven amino acid insertion comprises a sequence that is at least 70% identical to a sequence selected from one of SEQ ID NOs: 976-1411.

23. The AAV capsid protein of claim 22, wherein the seven amino acid insertion comprises a sequence of formula [II]: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [II]) (SEQ ID NO: 1952), wherein: X 1 is an amino acid selected from L, I and V; X 2 is an amino acid selected from L, V and M; X 3 is an amino acid selected from G, S and P; X 4 is an amino acid selected from the group consisting of P, G, S, T and H; X 5 is an amino acid selected from the group consisting of S, L, T, A, H and Q; X 6 is an amino acid selected from L, M, P, S, T and G; and / or X 7 is an amino acid selected from the group consisting of S, T, A, L, Q, P and V.

24. The AAV capsid protein according to claim 23, wherein X 2 It's L.

25. The AAV capsid protein according to claim 23, wherein X 3 It's G.

26. The AAV capsid protein according to claim 23, wherein X 6 It's L.

27. The AAV capsid protein of claim 22, wherein the seven amino acid insertion is one of SEQ ID NOs: 976-1411.

28. The AAV capsid protein of claim 27, wherein the capsid protein comprises a sequence of one of SEQ ID NOs: 2-437.

29. The AAV capsid protein of claim 7, wherein the AAV capsid protein is characterized by increased specificity and / or increased transduction efficiency relative to the capsid protein of SEQ ID NO: 1 when measured in the spinal cord.

30. The AAV capsid protein of claim 29, wherein the seven amino acid insertion comprises a sequence that is at least 70% identical to a sequence selected from one of SEQ ID NOs: 1613-1949.

31. The AAV capsid protein of claim 29, wherein the seven amino acid insertion comprises a sequence of formula [III]: X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (Formula [III]) (SEQ ID NO: 1950), wherein: X 1 is an amino acid selected from L, S and T; X 2 is an amino acid selected from the group consisting of L, P, S and T; X 3 is an amino acid selected from the group consisting of S, T, G, P and L; X 4 is an amino acid selected from the group consisting of S, T, L and P; X 5 is an amino acid selected from the group consisting of S, L, T and G; X 6 is an amino acid selected from L, P, S and T; and / or X 7 is an amino acid selected from the group consisting of S, P and E.

32. The AAV capsid protein of claim 31 , wherein the seven amino acid insertion is one of SEQ ID NOs: 1613-1949.

33. The AAV capsid protein of claim 14, wherein the capsid protein comprises a sequence of one of SEQ ID NOs: 639-975.

34. The AAV capsid protein of any one of claims 1 to 33, wherein the AAV is AAV2.

35. The AAV capsid protein of any one of claims 1 to 34, wherein 60 copies of the AAV capsid protein are assembled into an AAV capsid.

36. The AAV capsid protein of any one of claims 1 to 35, wherein the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid.

37. An AAV capsid comprising the AAV capsid protein of any one of claims 1 to 36.

38. The AAV capsid of claim 37, wherein the AAV capsid is chimeric.

39. The AAV capsid of any one of claims 37 to 38, which is isolated and purified.

40. The AAV capsid of any one of claims 37 to 39, formulated as a pharmaceutical formulation for systemic administration to treat a disease or condition.

41. A nucleic acid sequence encoding a peptide sequence as provided in SEQ ID NO: 2-1949.

42. A recombinant vector comprising a nucleic acid encoding the AAV capsid protein according to any one of claims 1 to 40.

43. A kit comprising: a) a first vector, which comprises the recombinant vector according to claim 42; b) a second vector encoding a helper virus protein; and c) a third vector comprising a therapeutic nucleic acid encoding an expression product of the therapeutic gene.

44. A method of treating a disease or condition in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising the AAV capsid protein of any one of claims 1 to 40.

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