TWI813851B - Frataxin expression constructs having engineered promoters and methods of use thereof - Google Patents

Abstract

The disclosure relates to compositions and methods for altering, e.g., enhancing, the expression of frataxin (FXN), whetherin vitro and/orin vivo including, but not limited to, the exploitation of engineered promoters. Such compositions include delivery via administration of an adeno-associated viral (AAV) particle. The compositions and methods of the present disclosure are useful in the treatment of subjects diagnosed with, or suspected of having Friedreich's ataxia or another neuromuscular or neurological condition resulting from a deficiency in the quantity and/or function of frataxin or associated with decreased expression or protein levels of frataxin.

Description

FRATAXIN expression construct with engineered promoter and methods of using the same

The present invention relates to compositions and methods based on Fataxin related to enhancing the expression of Fataxin (FXN), at least in part, in vitro or in vivo through the use of novel engineered promoters. Such syntaxin-based compositions can be delivered in an adeno-associated virus (AAV) vector. In other embodiments, a Fataxin-based composition, such as an AAV-Fataxin composition, is used to treat an individual in need thereof, such as one diagnosed with Friedrich's ataxia or affected by Fataxin. in human subjects with other neurological conditions caused by deficiencies in the amount and/or function of the neurotransmitter, or as a research tool to study such diseases or conditions in cellular or animal models of such diseases or conditions.

Friedreich's ataxia (FA), first described by German physician Nikolas Friedreich in the 1860s, is an autosomal recessive disorder that causes progressive damage to the nervous system. See Parkinson et al., Journal of Neurochemistry , 2013, 126 (Suppl 1), 103-117, the contents of which are incorporated by reference in their entirety. It usually begins in adolescence and persists until age 25. See Campuzano et al., Science, 271.5254 (March 8, 1996): 1423, the contents of which are incorporated by reference in their entirety. FA is usually caused by degeneration of neural tissue in the spinal cord due to reduced expression of the mitochondrial protein fascin (FXN) in sensory neurons that direct muscle movement in the arms and legs (via connections to the cerebellum). See Koeppen, Arnulf; J Neurol Sci. , 2011 Apr 15; 303(1-2): 1-12, the contents of which are incorporated by reference in their entirety. The spinal cord becomes thin and peripheral nerve cells lose some of their myelin, the insulating covering on some nerve cells that helps conduct nerve impulses. Initial symptoms of FA include poor coordination (such as gait disturbance), poor balance, leg weakness, reduced walking, loss of coordination, dysphonia, nystagmus, impaired sensation, kyphoscoliosis, and foot deformities. See Parkinson et al., Journal of Neurochemistry , 2013, 126 (Suppl 1), 103-117. FA is also associated with scoliosis, heart disease, and diabetes. The disease generally progresses until a wheelchair is required for mobility. The incidence of FA in the Caucasian population ranges from about 1/20,000 to about 1/50,000, and the carrier frequency in the European population is inferred to be about 1/120. See Nageshwaran and Festenstein, Frontiers in Neurology , vol. 6, Art. 262 (2015); Campuzano et al., Science, 271.5254 (March 8, 1996): 1423, each of which is cited in full. incorporated herein.

Expansion of the intronic GAA trinucleotide repeat sequence in the FXN gene is a genetic cause of reduced expression of fascin in FA. See Parkinson et al., Journal of Neurochemistry , 2013, 126 (Suppl 1), 103-117. Over time, this deficiency leads to the aforementioned symptoms, as well as frequent fatigue due to effects on cell metabolism.

Sclerosis and degeneration are most frequent in the dorsal root ganglia, spinocerebellar tracts, lateral corticospinal tracts, and posterior columns. See Sandi et al., Frontiers in Genetics , Volume 5, Art. 165 (June 2014), the contents of which are incorporated by reference in their entirety.

Progressive destruction of the dorsal root ganglion causes dorsal root thinning, dorsal column degeneration, transsynaptic atrophy of nerve cells in Clarke's column and dorsal spinocerebellar fibers, atrophy of the gracilis and cuneate nuclei, and Neuropathy of sensory nerves. See Koeppen, Arnulf; J Neurol Sci ., 2011 Apr 15; 303(1-2): 1-12, the contents of which are incorporated by reference in their entirety. Lesions of the dentate nucleus consist of progressive and selective atrophy of the larger glutamate-stimulating neurons and degeneration of the synaptic terminals of the cortical nucleus containing gamma-aminobutyric acid (GABA). The smaller GABA-stimulating neurons and their projection fibers in the dentate-olivary tract were spared. Atrophy of Betz cells and corticospinal tracts constitutes the second lesion. There is currently no effective treatment for FA, and patients are most often simply monitored for symptom management.

Accordingly, there is a long-standing need in the art to develop pharmaceutical compositions and methods for treating FXN-related disorders and to ameliorate protein deficiencies in patients with FA. Adeno-associated viruses (AAV) have emerged as one of the most extensively studied and exploited viral particles for the delivery of therapeutically effective polypeptides to mammalian cells. See, e.g., Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999), each of which Their contents are incorporated into this article by full reference. Therefore, this modality is well suited for the treatment of FA and the delivery of fataxin and fataxin-related proteins and peptides.

In some aspects, the present invention provides an AAV viral genome comprising at least one inverted terminal repeat (ITR) and a payload region, wherein the payload region encodes a syntaxin. In some embodiments, the AAV viral genome includes a 5' ITR, an engineered promoter, a payload region, and a 3' ITR. The encoded fataxin may be a human ( Homo sapiens ) fataxin, a macaque ( long-tailed macaque ) fataxin or a common macaque ( rhesus macaque ) fataxin, a synthetic (non-naturally occurring) fataxin, or other Derivatives, such as variants that retain one or more functions of wild-type cotaxin. In some embodiments, the fataxin can be at least partially humanized.

The engineered promoter of the AAV viral genome can be derived from the cytomegalovirus (CMV) promoter, the chicken beta-actin (CBA) promoter, or the FXN (FXN) promoter. In some embodiments, the engineered promoter is a promoter variant or derivative of the parent promoter sequence.

In some embodiments, the engineered promoter is derived from the CMV promoter.

In some embodiments, the engineered promoter is derived from a CBA promoter.

In some embodiments, the engineered promoter is derived from the FXN promoter.

An engineered promoter of an AAV viral genome as described herein may comprise the sequence set forth in any of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to any of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to any of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to any of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter can consist of any of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter is derived from a CMV promoter and can comprise the sequence set forth in any of SEQ ID NOs: 1743-1751, 1767, and 1772-1774. In some embodiments, the engineered promoter comprises SEQ ID NO: 1777. In some embodiments, the engineered promoter is derived from a CBA promoter and can comprise the sequence set forth in any of SEQ ID NOs: 1734-1742, 1760-1766, 1768, and 1775-1776. In some embodiments, the engineered promoter is derived from the FXN promoter and can comprise the sequence set forth in any of SEQ ID NOs: 1752-1759 and 1769-1770.

In some embodiments, the engineered promoter comprises the sequence set forth in SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter consists of SEQ ID NO: 1738.

In some embodiments, the engineered promoter includes the sequence set forth in SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter consists of SEQ ID NO: 1740.

In some embodiments, the engineered promoter comprises the sequence set forth in SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter consists of SEQ ID NO: 1742.

In some embodiments, the engineered promoter includes the sequence set forth in SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter consists of SEQ ID NO: 1750.

In some embodiments, the engineered promoter comprises the sequence set forth in SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence that has at least 90% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence that has at least 99% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter consists of SEQ ID NO: 1756.

Engineered promoters as described herein can have a length of 50 to 1400 nucleotides (nt). In some embodiments, the engineered promoter is derived from the CMV promoter and is 50 to 700 nt in length. In some embodiments, the engineered promoter is derived from the CMV promoter and is 109 nt in length. In some embodiments, the engineered promoter is derived from a CBA promoter and is 100 to 700 nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 100 to 400 nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 100 nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 200 to 350 nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 260 nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 332 nt in length. In some embodiments, the engineered promoter is derived from the FXN promoter and is 200 to 1400 nt in length. In some embodiments, the engineered promoter is 950 to 1150 nt in length. In some embodiments, the engineered promoter is derived from the FXN promoter and is 1060 nt in length.

In some embodiments, the engineered promoter includes an enhancer region.

The engineered promoter and payload region encoding the cotaxin can be incorporated into the AAV viral genome.

In some embodiments, in addition to the engineered promoter and payload region, the AAV viral genome also includes a 5' ITR, an enhancer, an intron, and at least one miR-binding site (e.g., one, two or three miR-binding sites). site), polyA sequence, stuffer sequence and 3' ITR. In some embodiments, the AAV viral genome contains multiple miR binding sites ("miR binding site series") that may occur contiguously or be separated by one or more nucleotides. In some embodiments, the 5' ITR and/or the 3' ITR is an AAV2 ITR.

In some embodiments, the viral genome contains at least one ITR sequence. In some embodiments, the ITR may be an AAV2 ITR. In some embodiments, the 5' ITR can be an AAV2 ITR. In some embodiments, the 3' ITR can be an AAV2 ITR. In some embodiments, the 5' and/or the 3' ITR can be 141 nt in length. In some embodiments, the 5' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1811. In some embodiments, the 3' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1812.

In some embodiments, ITR-to-ITR sequences comprise intron/exon regions. In some embodiments, the intron/exon region may be an enhancer sequence. As a non-limiting example, the enhancer sequence may include two or more subcomponents, such as (but not limited to) iel exons (e.g., exon 1), iel introns (e.g., intron 1 ), human beta-hemoglobulin intron (eg, intron 2) and/or human beta-hemoglobulin exon (eg, exon 3), or fragments thereof. In some embodiments, the intron/exon region comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1815-1821 . In some embodiments, the enhancer comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1815-1821. In some embodiments, the enhancer comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1777. In some embodiments, the intron/exon region comprises one or more human beta-hemoglobin sequences, e.g., at least 90%, at least 95%, at least 99% identical to SEQ ID NO: 1820 and/or 1821 or a 100% identical sequence. In some embodiments, the intron can comprise the sequence set forth in any of SEQ ID NOs: 1815-1821. In some embodiments, the intron has a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1816. In some embodiments, the intron may consist of SEQ ID NO: 1816.

In some embodiments, the series of miR binding sites includes at least one miR122 binding site sequence. In some embodiments, the at least one miR122 binding site comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1827. In some embodiments, the at least one miR122 binding site consists of SEQ ID NO: 1827. In some embodiments, the AAV vector genome comprises three copies of a miR122 binding site, such as three copies of SEQ ID NO: 1827 or a variant thereof with at least 90% sequence identity. In some embodiments, the series of miR binding sites can comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1826. In some embodiments, the series of miR binding sites can consist of SEQ ID NO: 1826.

In some embodiments, the polyA sequence is a human growth hormone (hGH) polyA sequence. In some embodiments, the viral genome comprises an hGH polyA sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1828. In some embodiments, the polyA sequence consists of SEQ ID NO: 1828.

In some embodiments, the AAV viral genome further includes stuffer sequences, such as albumin stuffer sequences. In some embodiments, the filler sequence may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1829-1842. In some embodiments, the stuffer sequence may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1838. In some embodiments, the stuffer sequence may consist of SEQ ID NO: 1838. In some embodiments, the stuffer sequence may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1839. In some embodiments, the stuffer sequence may consist of SEQ ID NO: 1839. In some embodiments, the stuffer sequence may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1840. In some embodiments, the stuffer sequence may consist of SEQ ID NO: 1840. In some embodiments, the stuffer sequence may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1841. In some embodiments, the filler sequence may consist of SEQ ID NO: 1841.

In some embodiments, the AAV viral genome may comprise the sequence set forth in any of SEQ ID NOs: 1778-1810. In some embodiments, the AAV viral genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identical to any of SEQ ID NOs: 1778-1810. In some embodiments, the AAV viral genome comprises 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, Sequences with 90-95%, 90-99% or 90-100% sequence identity. An AAV viral genome in which the encoded cotaxin is a Cynomolgus sp. cotaxin may comprise the sequence set forth in any one of SEQ ID NOs: 1778-1795. In some embodiments, the AAV viral genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identical to any of SEQ ID NOs: 1778-1795. In some embodiments, the AAV viral genome comprises 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90% identical to any one of SEQ ID NOs: 1778-1795 -Sequences with 95%, 90-99% or 90-100% sequence identity. An AAV viral genome in which the encoded cotaxin is a human cotaxin may comprise the sequence set forth in any one of SEQ ID NOs: 1796-1810. In some embodiments, the AAV viral genome comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identical to any of SEQ ID NOs: 1796-1810. In some embodiments, the AAV viral genome comprises 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, Sequences with 90-95%, 90-99% or 90-100% sequence identity.

In some embodiments, the AAV viral genome can comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1797. In some embodiments, the AAV viral genome may consist of SEQ ID NO: 1797. In some embodiments, the AAV viral genome can comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1801. In some embodiments, the AAV viral genome may consist of SEQ ID NO: 1801. In some embodiments, the AAV viral genome may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1808. In some embodiments, the AAV viral genome may consist of SEQ ID NO: 1808. In some embodiments, the AAV viral genome may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1809. In some embodiments, the AAV viral genome may consist of SEQ ID NO: 1809.

In some embodiments, the payload region of the AAV vector genome encoding the syntaxin comprises at least 80%, at least 85%, at least 90%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 1822-1824. % or at least 99% identical nucleic acid sequence. In some embodiments, the payload region of the AAV vector genome encoding syntaxin comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 1822-1824. In some embodiments, the nucleic acid sequence encoding syntaxin comprises SEQ ID NO: 1822. In some embodiments, the nucleic acid sequence encoding syntaxin comprises SEQ ID NO: 1823. In some embodiments, the nucleic acid sequence encoding syntaxin comprises SEQ ID NO: 1824. In some embodiments, the nucleic acid sequence encoding syntaxin comprises or has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a fragment of SEQ ID NO: 1728, 1729, or 1730. Variants of sex. In some embodiments, the nucleic acid sequence encoding syntaxin comprises SEQ ID NO: 1728. In some embodiments, the nucleic acid sequence encoding syntaxin comprises nucleotides 221-853 of SEQ ID NO: 1728.

In some embodiments, the payload region of the AAV vector genome comprises a sequence encoding a sequence identical to SEQ ID NO: 1725, 1726, or 1727 that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity. The nucleic acid sequence of the protein polypeptide. In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding the fataxin polypeptide of SEQ ID NO: 1725, 1726, or 1727. In some embodiments, the AAV vector genome comprises a nucleic acid sequence encoding a cotaxin polypeptide comprising SEQ ID NO: 1725. In some embodiments, the payload region of the AAV vector genome comprises a protein encoding a fastaxin polypeptide having at least 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 1731, 1732 or 1733. Nucleic acid sequence. In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding the fataxin polypeptide of SEQ ID NO: 1731, 1732, or 1733.

Viral genomes containing engineered promoters or promoter variants can be incorporated into AAV particles, where the AAV particles comprise the viral genome and capsid. In some embodiments, the capsid comprises a sequence as set forth in Table 1 or is selected from the group consisting of SEQ ID NOs: 1-1724. Non-limiting examples of capsids include AAV9, AAV9 K449R, AAVPHP.B, AAVPHP.N, VOY101 (having the amino acid sequence of SEQ ID NO: 1 and/or having the nucleic acid sequence of SEQ ID NO: 1722) and/or VOY201 (having the amino acid sequence of SEQ ID NO: 1724 and/or having the nucleic acid sequence of SEQ ID NO: 1723). In some embodiments, the capsid is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, 135, 1722, and 1723. In some embodiments, the capsid can have the amino acid sequence set forth in any one of SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 136. In some embodiments, the capsid comprises an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 135. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the capsid comprises an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 4. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the capsid comprises an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1722. In some embodiments, the capsid comprises an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1723. In some embodiments, the capsid comprises the amino acid sequence set forth in SEQ ID NO: 1724.

In some embodiments, the AAV particles described herein can be used in pharmaceutical compositions. In some embodiments, the pharmaceutical composition includes sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188. In some embodiments, the pharmaceutical composition includes 192 mM sodium chloride, 10 mM sodium phosphate, 2.7 mM potassium chloride, 2 mM potassium phosphate, and 0.001% poloxamer 188 (v/v). In some embodiments, the sodium phosphate of the composition is disodium hydrogen phosphate. In some embodiments, the potassium phosphate of the composition is potassium dihydrogen phosphate. In some embodiments, the pH of the pharmaceutical composition is between 7.3 and 7.7. In some embodiments, the pH of the pharmaceutical composition is 7.4.

In some embodiments, the AAV particle may comprise the vector genome set forth in SEQ ID NO: 1797 and the VOY101 capsid. In some embodiments, a pharmaceutical composition comprising the AAV particle comprising the vector genome set forth in SEQ ID NO: 1797 and the VOY101 capsid comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and poloxamer 188; Optionally wherein the pharmaceutical composition contains 192 mM sodium chloride, 10 mM sodium phosphate, 2.7 mM potassium chloride, 2 mM potassium phosphate and 0.001% poloxamer 188 (v/v), and wherein the composition The pH is 7.4.

In some embodiments, the AAV particle may comprise the vector genome set forth in SEQ ID NO: 1801 and the VOY101 capsid. In some embodiments, a pharmaceutical composition comprising the AAV particle comprising the vector genome set forth in SEQ ID NO: 1801 and the VOY101 capsid comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and poloxamer 188; Optionally wherein the pharmaceutical composition contains 192 mM sodium chloride, 10 mM sodium phosphate, 2.7 mM potassium chloride, 2 mM potassium phosphate and 0.001% poloxamer 188 (v/v), and wherein the composition The pH is 7.4.

In some embodiments, the AAV particle may comprise the vector genome set forth in SEQ ID NO: 1808 and the VOY101 capsid. In some embodiments, a pharmaceutical composition comprising the AAV particle comprising the vector genome set forth in SEQ ID NO: 1808 and the VOY101 capsid comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and poloxamer 188; Optionally wherein the pharmaceutical composition contains 192 mM sodium chloride, 10 mM sodium phosphate, 2.7 mM potassium chloride, 2 mM potassium phosphate and 0.001% poloxamer 188 (v/v), and wherein the composition The pH is 7.4.

In some embodiments, the AAV particle may comprise the vector genome set forth in SEQ ID NO: 1809 and the VOY101 capsid. In some embodiments, a pharmaceutical composition comprising the AAV particle comprising the vector genome set forth in SEQ ID NO: 1809 and the VOY101 capsid comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate and poloxamer 188; Optionally wherein the pharmaceutical composition contains 192 mM sodium chloride, 10 mM sodium phosphate, 2.7 mM potassium chloride, 2 mM potassium phosphate and 0.001% poloxamer 188 (v/v), and wherein the composition The pH is 7.4.

The pharmaceutical compositions and/or the AAV particles of the present invention can be used to treat neurological or neuromuscular disorders, such as (but not limited to) Friedrich's ataxia.

In some embodiments, the AAV particles of the invention are used to treat disorders or conditions associated with fataxin expression or reduced protein content. In some embodiments, the disorder or condition associated with fataxin expression or reduced protein content is a neurological or neuromuscular disorder. In some embodiments, the disorder or condition associated with reduced fataxin levels is FA or fataxin deficiency. In some embodiments, administration of AAV particles results in enhanced fastaxin expression in a target cell to the level of fastaxin expression in equivalent target cells in a normal individual not suffering from a disorder associated with reduced fastaxin content. 0.5 to 3× (for example, 0.5 to 1×, 1 to 1.5×, 1.5 to 2×, 2 to 2.5×, 2.5 to 3×). In some embodiments, administration of AAV particles results in expression of syntaxin in target cells at approximately 5.5-32.8 ng/mg protein.

Details of various aspects or embodiments of the invention are set forth below. Other features, objects, and advantages of the present invention will be apparent from the detailed description and patent claims. In the embodiments, the singular forms also include the plural forms unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. In the event of conflict, this instruction manual shall prevail.

Cross-references to related applications

This application claims U.S. Provisional Patent Application No. 62/901,769, filed on September 17, 2019, titled FRATAXIN EXPRESSION CONSTRUCTS HAVING ENGINEERED PROMOTERS AND METHODS OF USE THEREOF, filed on September 17, 2019, and FRATAXIN EXPRESSION CONSTRUCTS, filed on September 27, 2019. HAVING ENGINEERED PROMOTERS AND METHODS OF USE THEREOF's International Patent Application No. PCT/US2019/053681, the contents of each of which are incorporated herein by reference in their entirety. Sequence Listing Reference

This application is submitted together with the sequence listing in electronic format. A sequence listing file named 20571019TWSL.txt was created on January 15, 2020 and its size is 6,687,106 bytes. The information in the electronically formatted sequence listing is incorporated by reference in its entirety. I. Compositions of Adeno-Associated Disease (AAV) Vectors

Parvoviridae viruses are small, non-enveloped icosahedral capsid viruses characterized by a single-stranded DNA genome. Parvoviridae viruses are composed of two subfamilies: Parvovirinae , which infects vertebrates, and Densovirinae , which infects invertebrates. This family of viruses is useful as a biological tool because its relatively simple structure can be manipulated using standard molecular biology techniques. The genome of the virus may be modified to contain minimal components for the assembly of functional recombinant viruses or viral particles loaded with or engineered to express or deliver the desired nucleic acid construct or payload, e.g. Transgenic genes, polynucleotides encoding polypeptides or FXN that can be delivered to target cells, tissues or organisms. In some embodiments, the target cells are CNS cells. In some embodiments, the target tissue is CNS tissue.

Parvoviruses and other members of the family Parvoviridae are generally described in Kenneth I. Berns, " Parvoviridae : The Viruses and Their Replication", Chapter 69, FIELDS VIROLOGY (3rd ed. 1996), the contents of which are incorporated by reference in their entirety. into this article.

The family Parvoviridae contains the Dependovirus genus , which includes adenoviruses capable of replicating in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species. Viruses (AAV).

Adeno-associated virus (AAV) is a dependent parvovirus (like other parvoviruses) that is a single-stranded, non-enveloped DNA virus with a genome of approximately 5000 nucleotides in length and contains a protein encoding the protein responsible for replication (Rep) and two open reading frames of the structural protein of the capsid (Cap). The open reading frame is flanked by two inverted terminal repeat (ITR) sequences that serve as the origin of replication for the viral genome. The wild-type AAV viral genome contains nucleotide sequences for two open reading frames, one for the four nonstructural Rep proteins (Rep78, Rep68, Rep52, Rep40, encoded by the Rep gene) and one for the three capsids Protein or structural protein (VP1, VP2, VP3, encoded by capsid gene or Cap gene). The Rep protein is important for replication and encapsulation, while the capsid protein is assembled to produce the AAV protein shell, or AAV capsid. Alternative splicing and alternating start codons and promoters allow the production of four different Rep proteins from a single open reading frame and three capsid proteins from a single open reading frame. Although it differs by AAV serotype, as a non-limiting example, for AAV9/hu.14 (SEQ ID NO: 123 of US 7,906,111, the contents of which are incorporated by reference in their entirety), VP1 is Refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. Therefore, changes in the sequence in the VP3 region are also changes in VP1 and VP2, however, VP3 will have the greatest percentage difference compared to the parental sequence because it is the shortest sequence of the three. Although described herein with respect to amino acid sequences, nucleic acid sequences encoding such proteins may be similarly described. The three capsid proteins assemble together to produce the AAV capsid protein. While not wishing to be bound by theory, AAV capsid proteins typically comprise VP1:VP2:VP3 in a molar ratio of 1:1:10. As used herein, "AAV serotype" is primarily defined by the AAV capsid. In some cases, ITRs are also specifically described by AAV serotypes (eg, AAV2/9).

AAV vectors often require a co-helper vector (eg, adenovirus) to undergo a toxic infection in infected cells. In the absence of such helper functions, AAV virions essentially enter the host cell but are not integrated into the cell's genome. As used herein, the term "AAV vector" or "AAV particle" includes the capsid and the viral genome containing the polynucleotide payload. As used herein, "payload" or "payload region" refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome, or such polynucleotides or polynucleotide regions. Expression products of nucleotide regions, such as transgenic genes, polynucleotides encoding polypeptides or polypeptides (eg, FXN).

AAV vectors have been studied for delivery due to several unique characteristics. Non-limiting examples of such characteristics include: (i) the ability to infect dividing and non-dividing cells; (ii) a broad infectious host range, including human cells; (iii) wild-type AAV has not been associated with any disease and has not been studied in Replication in infected cells; (iv) lack of cell-mediated immune response against the vector, and (v) non-integrating nature of the host chromosome, thereby reducing the potential for long-term genetic variation. Furthermore, infection with AAV vectors has minimal effect on altering cellular gene expression patterns (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148, the contents of which are incorporated herein by reference in their entirety).

Typically, AAV vectors used for FXN delivery may be recombinant viral vectors that are replication defective due to the lack of sequences encoding functional Rep and Cap proteins in the viral genome. In some cases, a defective AAV vector may lack most or all coding sequences and contain essentially only one or two AAV ITR sequences and a payload sequence. In certain embodiments, the viral genome encodes FXN. For example, the viral genome encodes human FXN.

In one embodiment, the AAV particles of the invention can be introduced into mammalian cells.

AAV vectors can be modified to enhance delivery efficiency. Such modified AAV vectors of the invention can be efficiently encapsulated and used to successfully infect target cells with high frequency and minimal toxicity.

In other embodiments, the AAV particles of the present invention can be used to deliver FXN to the central nervous system (see, eg, U.S. Patent No. 6,180,613; the contents of which are incorporated herein by reference in their entirety). AAV serotypes

The AAV particles of the invention may comprise or be derived from any natural or recombinant AAV serotype. According to the present invention, AAV particles may utilize or be based on a serotype or include a peptide selected from any one of: VOY101, VOY201, AAV9, AAV9 K449R, AAVPHP.B (PHP.B), AAVPHP.A (PHP .A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B- DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN , AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP .B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42- aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61, AAV2- 4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAV4-8/ r11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV16.8/hu. 10. AAV16.12/hu.11, AAV29.3/bb.1, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161. 10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV52/hu.19, AAV52.1/hu. 20. AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh. 45. AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/ hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy. 5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu. 29. AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu. 52. AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/ 9. AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18 , AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh .36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh .51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73 , AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1. 18. AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV- LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV shuffle 100-1, AAV shuffle 100- 3. AAV reorganization 100-7, AAV reorganization 10-2, AAV reorganization 10-6, AAV reorganization 10-8, AAV reorganization 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8/rh.64 , AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46 .2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15 , AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8 and/or AAVF9/HSC9, and their variants or hybrids /chimera/combination.

In some embodiments, the AAV serotypes used in the compositions disclosed herein may be or comprise sequences as described in U.S. Patent Application Publication No. US20030138772, the contents of which are incorporated herein by reference in their entirety, such as (But not limited to) AAV1 (SEQ ID NO: 6 and 64 of US20030138772), AAV2 (SEQ ID NO: 7 and 70 of US20030138772), AAV3 (SEQ ID NO: 8 and 71 of US20030138772), AAV4 (SEQ ID NO. of US20030138772) NO: 63), AAV5 (SEQ ID NO: 114 of US20030138772), AAV6 (SEQ ID NO: 65 of US20030138772), AAV7 (SEQ ID NO: 1-3 of US20030138772), AAV8 (SEQ ID NO: 4 and US20030138772) 95), AAV9 (SEQ ID NO: 5 and 100 of US20030138772), AAV10 (SEQ ID NO: 117 of US20030138772), AAV11 (SEQ ID NO: 118 of US20030138772), AAV12 (SEQ ID NO: 119 of US20030138772), AAVrh10 (Amino acids 1 to 738 of SEQ ID NO: 81 of US20030138772), AAV16.3 (US20030138772 SEQ ID NO: 10), AAV29.3/bb.1 (US20030138772 SEQ ID NO: 11), AAV29.4 (US20030138772 SEQ ID NO: 12), AAV29.5/bb.2 (US20030138772 SEQ ID NO: 13), AAV1.3 (US20030138772 SEQ ID NO: 14), AAV13.3 (US20030138772 SEQ ID NO: 15), AAV24.1 (US20030138772 SEQ ID NO: 16), AAV27.3 (US20030138772 SEQ ID NO: 17), AAV7.2 (US20030138772 SEQ ID NO: 18), AAVC1 (US20030138772 SEQ ID NO: 19), AAVC3 (US20030138772 SEQ ID NO: 20), AAVC5 (US20030138772 SEQ ID NO: 21), AAVF1 (US20030138772 SEQ ID NO: 22), AAVF3 (US20030138772 SEQ ID NO: 23), AAVF5 (US20030138772 SEQ ID NO: 24), AAVH6 (US2003013 8772 SEQ ID NO: A AV42 -1b (US20030138772 SEQ ID NO: 30), AAV42-13 (US20030138772 SEQ ID NO: 31), AAV42-3A (US20030138772 SEQ ID NO: 32), AAV42-4 (US20030138772 SEQ ID N O: 33), AAV42-5A (US20030138772 SEQ ID NO: 34), AAV42-10 (US20030138772 SEQ ID NO: 35), AAV42-3b (US20030138772 SEQ ID NO: 36), AAV42-11 (US20030138772 SEQ ID NO: 37), AAV42-6b (US 20030138772 SEQ ID NO: 38), AAV43-1 (US20030138772 SEQ ID NO: 39), AAV43-5 (US20030138772 SEQ ID NO: 40), AAV43-12 (US20030138772 SEQ ID NO: 41), AAV43-20 (US20030138772 SEQ ID NO: 42), AAV43-21 (US20030138772 SEQ ID NO: 43), AAV43-23 (US20030138772 SEQ ID NO: 44), AAV43-25 (US20030138772 SEQ ID NO: 45), AAV44.1 (US20030138772 SEQ ID NO: 46), AAV44.5 (US20030138772 SEQ ID NO: 47), AAV223.1 (US20030138772 SEQ ID NO: 48), AAV223.2 (US20030138772 SEQ ID NO: 49), AAV223.4 (US20030138772 SEQ ID NO: 50 ) , AAV223.5 (US20030138772 SEQ ID NO: 51), AAV223.6 (US20030138772 SEQ ID NO: 52), AAV223.7 (US20030138772 SEQ ID NO: 53), AAVA3.4 (US20030138772 SEQ ID NO: 54), AAVA 3 .5 (US20030138772 SEQ ID NO: 55), AAVA3.7 (US20030138772 SEQ ID NO: 56), AAVA3.3 (US20030138772 SEQ ID NO: 57), AAV42.12 (US20030138772 SEQ ID NO: 58), AAV44.2 (US20030138772 SEQ ID NO: 59), AAV42-2 (US20030138772 SEQ ID NO: 9) or variants or hybrids/chimeras/combinations thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent Application Publication No. US20150159173, which is incorporated herein by reference in its entirety, such as (but not limited to) the SEQ of AAV2 (US20150159173). ID NO: 7 and 23), rh20 (SEQ ID NO: 1 of US20150159173), rh32/33 (SEQ ID NO: 2 of US20150159173), rh39 (SEQ ID NO: 3, 20 and 36 of US20150159173), rh46 (US20150159173 SEQ ID NO: 4 and 22), rh73 (SEQ ID NO: 5 of US20150159173), rh74 (SEQ ID NO: 6 of US20150159173), AAV6.1 (SEQ ID NO: 29 of US20150159173), rh.8 (SEQ ID NO: 29 of US20150159173) SEQ ID NO: 41), rh.48.1 (SEQ ID NO: 44 of US20150159173), hu.44 (SEQ ID NO: 45 of US20150159173), hu.29 (SEQ ID NO: 42 of US20150159173), hu.48 (SEQ ID NO: 38 of US20150159173), rh54 (SEQ ID NO: 49 of US20150159173), AAV2 (SEQ ID NO: 7 of US20150159173), cy.5 (SEQ ID NO: 8 and 24 of US20150159173), rh.10 (SEQ ID NO: 9 and 25 of US20150159173), rh.13 (SEQ ID NO: 10 and 26 of US20150159173), AAV1 (SEQ ID NO: 11 and 27 of US20150159173), AAV3 (SEQ ID NO: 12 and 27 of US20150159173) 28), AAV6 (SEQ ID NO: 13 and 29 of US20150159173), AAV7 (SEQ ID NO: 14 and 30 of US20150159173), AAV8 (SEQ ID NO: 15 and 31 of US20150159173), hu.13 (SEQ ID of US20150159173 NO: 16 and 32), hu.26 (SEQ ID NO: 17 and 33 of US20150159173), hu.37 (SEQ ID NO: 18 and 34 of US20150159173), hu.53 (SEQ ID NO: 19 and 35 of US20150159173) ), rh.43 (SEQ ID NO: 21 and 37 of US20150159173), rh2 (SEQ ID NO: 39 of US20150159173), rh.37 (SEQ ID NO: 40 of US20150159173), rh.64 (SEQ ID NO of US20150159173 : 43), rh.48 (SEQ ID NO: 44 of US20150159173), ch.5 (SEQ ID NO: 46 of US20150159173), rh.67 (SEQ ID NO: 47 of US20150159173), rh.58 (SEQ ID of US20150159173 NO: 48) or variants thereof, including (but not limited to) Cy5R1, Cy5R2, Cy5R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.48.1, rh.48.2, rh.48.1 .2, hu.44R1, hu.44R2, hu.44R3, hu.29R, ch.5R1, rh64R1, rh64R2, AAV6.2, AAV6.1, AAV6.12, hu.48R1, hu.48R2 or hu.48R3 , or variants or hybrids/chimeras/combinations thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent No. 7,198,951, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV9 (SEQ ID No. 7,198,951). NO: 1-3), AAV2 (SEQ ID NO: 4 of US 7198951), AAV1 (SEQ ID NO: 5 of US 7198951), AAV3 (SEQ ID NO: 6 of US 7198951) and AAV8 (SEQ ID NO of US 7198951 : 7), or variants or hybrids/chimeras/combinations thereof.

In some embodiments, the AAV serotype may be an AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), the contents of which are incorporated by reference in its entirety) or may be Variants thereof, such as (but not limited to) AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68 or AAV9.84 .

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent No. 6,156,303, which is incorporated by reference in its entirety, such as (but not limited to) AAV3B (SEQ ID NO. 6,156,303). NO: 1 and 10), AAV6 (SEQ ID NO: 2, 7 and 11 of US 6156303), AAV2 (SEQ ID NO: 3 and 8 of US 6156303), AAV3A (SEQ ID NO: 4 and 9 of US 6156303) , or derivatives or variants or hybrids/chimeras/combinations thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent Application Publication No. US20140359799, which is incorporated herein by reference in its entirety, such as (but not limited to) the SEQ of AAV8 (US20140359799). ID NO: 1), AAVDJ (SEQ ID NO: 2 and 3 of US20140359799) or variants thereof.

In some embodiments, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008), cited in its entirety) (incorporated into this document). The amino acid sequence of AAVDJ8 may contain two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Patent No. 7,588,772, the contents of which are incorporated herein by reference in its entirety, may contain two mutations: (1) R587Q, where the amine Arginine (R; Arg) at amino acid 587 becomes glutamic acid (Q; Gln); and (2) R590T, in which arginine (R; Arg) at amino acid 590 becomes threonine Acid (T; Thr). As another non-limiting example, the AAV-DJ sequence described in U.S. Patent No. 7,588,772 may contain three mutations: (1) K406R, in which lysine (K; Lys) at amino acid 406 is changed to sperm Amino acid (R; Arg); (2) R587Q, in which arginine (R; Arg) at amino acid 587 becomes glutamic acid (Q; Gln); and (3) R590T, in which amino acid Arginine (R; Arg) at 590 becomes threonine (T; Thr).

In some embodiments, the AAV serotype may be or comprise a sequence of AAV4 as described in International Publication No. WO1998011244, which is incorporated herein by reference in its entirety, such as (but not limited to) the SEQ of AAV4 (WO1998011244 ID NO: 1-20).

In some embodiments, the AAV serotype may be or comprise a mutation in the AAV2 sequence that gave rise to AAV2G9 as described in International Publication No. WO2014144229 and incorporated herein by reference in its entirety.

In some embodiments, an AAV serotype may be or comprise a sequence as described in International Publication No. WO2005033321, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV3-3 (SEQ of WO2005033321 ID NO: 217), AAV1 (SEQ ID NO: 219 and 202 of WO2005033321), AAV106.1/hu.37 (SEQ ID No: 10 of WO2005033321), AAV114.3/hu.40 (SEQ ID No. of WO2005033321: 11), AAV127.2/hu.41 (SEQ ID NO: 6 and 8 of WO2005033321), AAV128.3/hu.44 (SEQ ID No: 81 of WO2005033321), AAV130.4/hu.48 (SEQ of WO2005033321 ID NO: 78), AAV145.1/hu.53 (SEQ ID Nos: 176 and 177 of WO2005033321), AAV145.6/hu.56 (SEQ ID NO: 168 and 192 of WO2005033321), AAV16.12/hu. 11 (SEQ ID NO: 153 and 57 of WO2005033321), AAV16.8/hu.10 (SEQ ID NO: 156 and 56 of WO2005033321), AAV161.10/hu.60 (SEQ ID No: 170 of WO2005033321), AAV161 .6/hu.61 (SEQ ID No: 174 of WO2005033321), AAV1-7/rh.48 (SEQ ID NO: 32 of WO2005033321), AAV1-8/rh.49 (SEQ ID NOs: 103 and 25 of WO2005033321) ), AAV2 (SEQ ID NO: 211 and 221 of WO2005033321), AAV2-15/rh.62 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-3/rh.61 (SEQ ID NO: 21 of WO2005033321) , AAV2-4/rh.50 (SEQ ID No: 23 and 108 of WO2005033321), AAV2-5/rh.51 (SEQ ID NO: 104 and 22 of WO2005033321), AAV3.1/hu.6 (SEQ ID No. of WO2005033321 ID NO: 5 and 84), AAV3.1/hu.9 (SEQ ID NO: 155 and 58 of WO2005033321), AAV3-11/rh.53 (SEQ ID NO: 186 and 176 of WO2005033321), AAV3-3 ( SEQ ID NO: 200 of WO2005033321), AAV33.12/hu.17 (SEQ ID NO: 4 of WO2005033321), AAV33.4/hu.15 (SEQ ID No: 50 of WO2005033321), AAV33.8/hu.16 (SEQ ID NO: 51 of WO2005033321), AAV3-9/rh.52 (SEQ ID NO: 96 and 18 of WO2005033321), AAV4-19/rh.55 (SEQ ID NO: 117 of WO2005033321), AAV4-4 ( SEQ ID NO: 201 and 218 of WO2005033321), AAV4-9/rh.54 (SEQ ID NO: 116 of WO2005033321), AAV5 (SEQ ID NO: 199 and 216 of WO2005033321), AAV52.1/hu.20 (WO2005033321 SEQ ID NO: 63), AAV52/hu.19 (SEQ ID NO: 133 of WO2005033321), AAV5-22/rh.58 (SEQ ID No: 27 of WO2005033321), AAV5-3/rh.57 (SEQ ID NO: 27 of WO2005033321) SEQ ID NO: 105), AAV5-3/rh.57 (SEQ ID No: 26 of WO2005033321), AAV58.2/hu.25 (SEQ ID No: 49 of WO2005033321), AAV6 (SEQ ID NO: 203 of WO2005033321 and 220), AAV7 (SEQ ID NO: 222 and 213 of WO2005033321), AAV7.3/hu.7 (SEQ ID No: 55 of WO2005033321), AAV8 (SEQ ID NO: 223 and 214 of WO2005033321), AAVH-1 /hu.1 (SEQ ID No: 46 of WO2005033321), AAVH-5/hu.3 (SEQ ID No: 44 of WO2005033321), AAVhu.1 (SEQ ID NO: 144 of WO2005033321), AAVhu.10 (SEQ ID No: 144 of WO2005033321) SEQ ID NO: 156), AAVhu.11 (SEQ ID NO: 153 of WO2005033321), AAVhu.12 (SEQ ID NO: 59 of WO2005033321), AAVhu.13 (SEQ ID NO: 129 of WO2005033321), AAVhu.14/AAV9 (SEQ ID NO: 123 and 3 of WO2005033321), AAVhu.15 (SEQ ID NO: 147 of WO2005033321), AAVhu.16 (SEQ ID NO: 148 of WO2005033321), AAVhu.17 (SEQ ID NO: 83 of WO2005033321) , AAVhu.18 (SEQ ID NO: 149 of WO2005033321), AAVhu.19 (SEQ ID NO: 133 of WO2005033321), AAVhu.2 (SEQ ID NO: 143 of WO2005033321), AAVhu.20 (SEQ ID NO: 143 of WO2005033321) 134), AAVhu.21 (SEQ ID NO: 135 of WO2005033321), AAVhu.22 (SEQ ID NO: 138 of WO2005033321), AAVhu.23.2 (SEQ ID NO: 137 of WO2005033321), AAVhu.24 (SEQ ID NO: 137 of WO2005033321) NO: 136), AAVhu.25 (SEQ ID NO: 146 of WO2005033321), AAVhu.27 (SEQ ID NO: 140 of WO2005033321), AAVhu.29 (SEQ ID NO: 132 of WO2005033321), AAVhu.3 (SEQ ID NO: 140 of WO2005033321) SEQ ID NO: 145), AAVhu.31 (SEQ ID NO: 121 of WO2005033321), AAVhu.32 (SEQ ID NO: 122 of WO2005033321), AAVhu.34 (SEQ ID NO: 125 of WO2005033321), AAVhu.35 ( SEQ ID NO: 164 of WO2005033321), AAVhu.37 (SEQ ID NO: 88 of WO2005033321), AAVhu.39 (SEQ ID NO: 102 of WO2005033321), AAVhu.4 (SEQ ID NO: 141 of WO2005033321), AAVhu. 40 (SEQ ID NO: 87 of WO2005033321), AAVhu.41 (SEQ ID NO: 91 of WO2005033321), AAVhu.42 (SEQ ID NO: 85 of WO2005033321), AAVhu.43 (SEQ ID NO: 160 of WO2005033321), AAVhu.44 (SEQ ID NO: 144 of WO2005033321), AAVhu.45 (SEQ ID NO: 127 of WO2005033321), AAVhu.46 (SEQ ID NO: 159 of WO2005033321), AAVhu.47 (SEQ ID NO: 128 of WO2005033321 ), AAVhu.48 (SEQ ID NO: 157 of WO2005033321), AAVhu.49 (SEQ ID NO: 189 of WO2005033321), AAVhu.51 (SEQ ID NO: 190 of WO2005033321), AAVhu.52 (SEQ ID NO of WO2005033321 : 191), AAVhu.53 (SEQ ID NO: 186 of WO2005033321), AAVhu.54 (SEQ ID NO: 188 of WO2005033321), AAVhu.55 (SEQ ID NO: 187 of WO2005033321), AAVhu.56 (SEQ ID NO: 187 of WO2005033321) ID NO: 192), AAVhu.57 (SEQ ID NO: 193 of WO2005033321), AAVhu.58 (SEQ ID NO: 194 of WO2005033321), AAVhu.6 (SEQ ID NO: 84 of WO2005033321), AAVhu.60 (WO2005033321 SEQ ID NO: 184), AAVhu.61 (SEQ ID NO: 185 of WO2005033321), AAVhu.63 (SEQ ID NO: 195 of WO2005033321), AAVhu.64 (SEQ ID NO: 196 of WO2005033321), AAVhu.66 (SEQ ID NO: 197 of WO2005033321), AAVhu.67 (SEQ ID NO: 198 of WO2005033321), AAVhu.7 (SEQ ID NO: 150 of WO2005033321), AAVhu.8 (SEQ ID NO: 12 of WO2005033321), AAVhu. 9 (SEQ ID NO: 155 of WO2005033321), AAVLG-10/rh.40 (SEQ ID No: 14 of WO2005033321), AAVLG-4/rh.38 (SEQ ID NO: 86 of WO2005033321), AAVLG-4/rh .38 (SEQ ID No: 7 of WO2005033321), AAVN721-8/rh.43 (SEQ ID NO: 163 of WO2005033321), AAVN721-8/rh.43 (SEQ ID No: 43 of WO2005033321), AAVpi.1 ( WO2005033321 SEQ ID NO: 28), AAVpi.2 (WO2005033321 SEQ ID NO: 30), AAVpi.3 (WO2005033321 SEQ ID NO: 29), AAVrh.38 (WO2005033321 SEQ ID NO: 86), AAVrh.40 (WO2005033 321 SEQ ID NO: 92), AAVrh.43 (SEQ ID NO: 163 of WO2005033321), AAVrh.44 (SEQ ID NO: 34 of WO2005033321), AAVrh.45 (SEQ ID NO: 41 of WO2005033321), AAVrh.47 (WO2005033321) SEQ ID NO: 38), AAVrh.48 (SEQ ID NO: 115 of WO2005033321), AAVrh.49 (SEQ ID NO: 103 of WO2005033321), AAVrh.50 (SEQ ID NO: 108 of WO2005033321), AAVrh.51 ( WO2005033321 SEQ ID NO: 104), AAVrh.52 (WO2005033321 SEQ ID NO: 96), AAVrh.53 (WO2005033321 SEQ ID NO: 97), AAVrh.55 (WO2005033321 SEQ ID NO: 37), AAVrh.56 (SEQ ID NO: 152 of WO2005033321), AAVrh.57 (SEQ ID NO: 105 of WO2005033321), AAVrh.58 (SEQ ID NO: 106 of WO2005033321), AAVrh.59 (SEQ ID NO: 42 of WO2005033321), AAVrh. 60 (WO2005033321 SEQ ID NO: 31), AAVrh.61 (WO2005033321 SEQ ID NO: 107), AAVrh.62 (WO2005033321 SEQ ID NO: 114), AAVrh.64 (WO2005033321 SEQ ID NO: 99), AAVrh .65 (WO2005033321 SEQ ID NO: 35), AAVrh.68 (WO2005033321 SEQ ID NO: 16), AAVrh.69 (WO2005033321 SEQ ID NO: 39), AAVrh.70 (WO2005033321 SEQ ID NO: 20), AAVrh.72 (WO2005033321 SEQ ID NO: 9) or variants thereof, including (but not limited to) AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.18 , AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.25/42 15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh. 35, AAVrh.36, AAVrh.37 or AAVrh14. Non-limiting examples of variants include SEQ ID NOs: 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51, 52, 53 of WO2005033321, the contents of which are incorporated herein by reference in their entirety. ,54,60,61,62,64,65,66,67,68,69,70,71,72,73,74,75,76,77,79,80,82,89,90,93,94 ,95,98,100,101,109¸110,111,112,113,118,119,120,124,126,131,139,142,151,154,158,161,162,165,166,167 ,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,202,204,205,206,207,208,209,210,211 , 212, 215, 219, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 or 236.

In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2015168666, which is incorporated herein by reference in its entirety, such as (but not limited to) AAVrh8R (SEQ ID NO. of WO2015168666 : 9), AAVrh8R A586R mutant (SEQ ID NO: 10 of WO2015168666), AAVrh8R R533A mutant (SEQ ID NO: 11 of WO2015168666), or variants thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent No. 9,233,131, which is incorporated by reference in its entirety, such as (but not limited to) AAVhE1.1 (SEQ ID NO. 9,233,131). NO:44), AAVhEr1.5 (SEQ ID NO:45 of US9233131), AAVhER1.14 (SEQ ID NO:46 of US9233131), AAVhEr1.8 (SEQ ID NO:47 of US9233131), AAVhEr1.16 (SEQ ID NO:46 of US9233131) SEQ ID NO:48), AAVhEr1.18 (SEQ ID NO:49 of US9233131), AAVhEr1.35 (SEQ ID NO:50 of US9233131), AAVhEr1.7 (SEQ ID NO:51 of US9233131), AAVhEr1.36 ( SEQ ID NO:52 of US9233131), AAVhEr2.29 (SEQ ID NO:53 of US9233131), AAVhEr2.4 (SEQ ID NO:54 of US9233131), AAVhEr2.16 (SEQ ID NO:55 of US9233131), AAVhEr2. 30 (SEQ ID NO:56 of US9233131), AAVhEr2.31 (SEQ ID NO:58 of US9233131), AAVhEr2.36 (SEQ ID NO:57 of US9233131), AAVhER1.23 (SEQ ID NO:53 of US9233131), AAVhEr3.1 (SEQ ID NO:59 of US9233131), AAV2.5T (SEQ ID NO:42 of US9233131) or variants thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in United States Patent Application Publication No. US20150376607, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV-PAEC (US20150376607 SEQ ID NO:1), AAV-LK01 (SEQ ID NO:2 of US20150376607), AAV-LK02 (SEQ ID NO:3 of US20150376607), AAV-LK03 (SEQ ID NO:4 of US20150376607), AAV-LK04 (SEQ ID NO: 5 of US20150376607), AAV-LK05 (SEQ ID NO: 6 of US20150376607), AAV-LK06 (SEQ ID NO: 7 of US20150376607), AAV-LK07 (SEQ ID NO: 8 of US20150376607), AAV -LK08 (SEQ ID NO:9 of US20150376607), AAV-LK09 (SEQ ID NO:10 of US20150376607), AAV-LK10 (SEQ ID NO:11 of US20150376607), AAV-LK11 (SEQ ID NO:12 of US20150376607) , AAV-LK12 (SEQ ID NO:13 of US20150376607), AAV-LK13 (SEQ ID NO:14 of US20150376607), AAV-LK14 (SEQ ID NO:15 of US20150376607), AAV-LK15 (SEQ ID NO:15 of US20150376607) 16), AAV-LK16 (SEQ ID NO:17 of US20150376607), AAV-LK17 (SEQ ID NO:18 of US20150376607), AAV-LK18 (SEQ ID NO:19 of US20150376607), AAV-LK19 (SEQ ID of US20150376607 NO:20), AAV-PAEC2 (SEQ ID NO:21 of US20150376607), AAV-PAEC4 (SEQ ID NO:22 of US20150376607), AAV-PAEC6 (SEQ ID NO:23 of US20150376607), AAV-PAEC7 (SEQ ID NO:23 of US20150376607) SEQ ID NO:24), AAV-PAEC8 (SEQ ID NO:25 of US20150376607), AAV-PAEC11 (SEQ ID NO:26 of US20150376607), AAV-PAEC12 (SEQ ID NO:27 of US20150376607), or variants thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent No. 9,163,261, which is incorporated by reference in its entirety, such as (but not limited to) AAV-2-pre-miRNA- 101 (SEQ ID NO: 1 US9163261) or a variant thereof.

In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. US20150376240, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV-8h (US20150376240 SEQ ID NO: 6), AAV-8b (SEQ ID NO: 5 of US20150376240), AAV-h (SEQ ID NO: 2 of US20150376240), AAV-b (SEQ ID NO: 1 of US20150376240) or variants thereof .

In some embodiments, the AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. US20160017295, which is incorporated by reference in its entirety, such as (but not limited to) AAV SM 10-2 (SEQ ID NO: 22 of US20160017295), AAV reorganization 100-1 (SEQ ID NO: 23 of US20160017295), AAV reorganization 100-3 (SEQ ID NO: 24 of US20160017295), AAV reorganization 100-7 (SEQ ID of US20160017295 NO: 25), AAV reorganization 10-2 (SEQ ID NO: 34 of US20160017295), AAV reorganization 10-6 (SEQ ID NO: 35 of US20160017295), AAV reorganization 10-8 (SEQ ID NO: 36 of US20160017295), AAV reorganization 100-2 (SEQ ID NO: 37 of US20160017295), AAV SM 10-1 (SEQ ID NO: 38 of US20160017295), AAV SM 10-8 (SEQ ID NO: 39 of US20160017295), AAV SM 100-3 (SEQ ID NO: 40 of US20160017295), AAV SM 100-10 (SEQ ID NO: 41 of US20160017295) or variants thereof.

In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Patent Publication No. US20150238550, which is incorporated herein by reference in its entirety, such as (but not limited to) BNP61 AAV (SEQ of US20150238550 ID NO: 1), BNP62 AAV (SEQ ID NO: 3 of US20150238550), BNP63 AAV (SEQ ID NO: 4 of US20150238550) or variants thereof.

In some embodiments, the AAV serotype may be or may include sequences as described in U.S. Patent Publication No. US20150315612, which is incorporated herein by reference in its entirety, such as (but not limited to) AAVrh.50 (US20150315612 SEQ ID NO: 108), AAVrh.43 (SEQ ID NO: 163 of US20150315612), AAVrh.62 (SEQ ID NO: 114 of US20150315612), AAVrh.48 (SEQ ID NO: 115 of US20150315612), AAVhu.19 (SEQ ID NO: 133 of US20150315612), AAVhu.11 (SEQ ID NO: 153 of US20150315612), AAVhu.53 (SEQ ID NO: 186 of US20150315612), AAV4-8/rh.64 (SEQ ID No. of US20150315612) 15), AAVLG-9/hu.39 (SEQ ID No: 24 of US20150315612), AAV54.5/hu.23 (SEQ ID No: 60 of US20150315612), AAV54.2/hu.22 (SEQ ID No of US20150315612 : 67), AAV54.7/hu.24 (SEQ ID No: 66 of US20150315612), AAV54.1/hu.21 (SEQ ID No: 65 of US20150315612), AAV54.4R/hu.27 (SEQ ID of US20150315612 No: 64), AAV46.2/hu.28 (SEQ ID No: 68 of US20150315612), AAV46.6/hu.29 (SEQ ID No: 69 of US20150315612), AAV128.1/hu.43 (SEQ of US20150315612 ID No: 80) or its variants.

In some embodiments, an AAV serotype may be or comprise a sequence as described in International Publication No. WO2015121501, which is incorporated herein by reference in its entirety, such as (but not limited to) true type AAV (ttAAV) ( SEQ ID NO: 2 of WO2015121501, "UPenn AAV10" (SEQ ID NO: 8 of WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501), or variants thereof.

AAV capsid serotypes from various species may be selected or used in accordance with the present invention. In one embodiment, the AAV may be an avian AAV (AAAV). The AAAV serotype may be or have a sequence as described in U.S. Patent No. 9,238,800, which is incorporated herein by reference in its entirety, such as (but not limited to) SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12 or 14) or variants thereof.

In one embodiment, the AAV may be bovine AAV (BAAV). BAAV serotypes may be or have sequences as described in U.S. Patent No. 9,193,769, which is incorporated by reference in its entirety, such as (but not limited to) BAAV (SEQ ID NOs: 1 and 6 of U.S. 9,193,769) or its variants. The BAAV serotype may be or have a sequence as described in U.S. Patent No. 7,427,396, which is incorporated by reference in its entirety, such as (but not limited to) BAAV (SEQ ID NOs: 5 and 6 of U.S. 7,427,396), or Its variants.

In some embodiments, the AAV may be goat AAV. The goat AAV serotype may be or have a sequence as described in US Pat. No. 7,427,396, which is incorporated by reference in its entirety, such as (but not limited to) goat AAV (SEQ ID NO: 3 of US 7,427,396) or its Variants.

In some embodiments, AAV can be engineered as a hybrid AAV from two or more parental serotypes. In one embodiment, the AAV may be AAV2G9 including sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be or have a sequence as described in U.S. Patent Application Publication No. US20160017005, which is incorporated herein by reference in its entirety.

In one embodiment, the AAV can be a serotype generated from an AAV9 capsid library with mutations in amino acids 390-627 (VP1 numbering), such as Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011) ), the contents of which are incorporated herein by reference in their entirety). The serotype and the corresponding nucleotide and amino acid substitutions may be (but are not limited to) AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y) , AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A, G1785T; W595C) , AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S), AAV9.14 (T1340A, T13 62C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9. 34 AAV9.41 (A1348T, T1362C; T450S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V), 9.47 ( G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L 602H), AAV9.53 (G1301A , A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579 A; T492I, H527N) , AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P5 04T ), AAV9.80 (G1441A; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87 (T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G, A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T5 82S, D611V), AAV9.94 (A1675T; M559L) or AAV9.95 (T1605A; F535L).

In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2016049230, which is incorporated herein by reference in its entirety, such as (but not limited to) AAVFl/HSC1 (SEQ of WO2016049230 ID NO: 2 and 20), AAVF2/HSC2 (SEQ ID NO: 3 and 21 of WO2016049230), AAVF3/HSC3 (SEQ ID NO: 5 and 22 of WO2016049230), AAVF4/HSC4 (SEQ ID NO: 6 and 22 of WO2016049230) 23), AAVF5/HSC5 (SEQ ID NO: 11 and 25 of WO2016049230), AAVF6/HSC6 (SEQ ID NO: 7 and 24 of WO2016049230), AAVF7/HSC7 (SEQ ID NO: 8 and 27 of WO2016049230), AAVF8/ HSC8 (SEQ ID NO: 9 and 28 of WO2016049230), AAVF9/HSC9 (SEQ ID NO: 10 and 29 of WO2016049230), AAVF11/HSC11 (SEQ ID NO: 4 and 26 of WO2016049230), AAVF12/HSC12 (SEQ ID NO: WO2016049230) ID NO: 12 and 30), AAVF13/HSC13 (SEQ ID NO: 14 and 31 of WO2016049230), AAVF14/HSC14 (SEQ ID NO: 15 and 32 of WO2016049230), AAVF15/HSC15 (SEQ ID NO: 16 and 32 of WO2016049230) 33), AAVF16/HSC16 (SEQ ID NO: 17 and 34 of WO2016049230), AAVF17/HSC17 (SEQ ID NO: 13 and 35 of WO2016049230), or variants or derivatives thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in U.S. Patent No. 8,734,809, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV CBr-E1 (US Pat. No. 8,734,809). SEQ ID NO: 13 and 87), AAV CBr-E2 (SEQ ID NO: 14 and 88 of US8734809), AAV CBr-E3 (SEQ ID NO: 15 and 89 of US8734809), AAV CBr-E4 (SEQ ID NO: US8734809 NO: 16 and 90), AAV CBr-E5 (SEQ ID NO: 17 and 91 of US8734809), AAV CBr-e5 (SEQ ID NO: 18 and 92 of US8734809), AAV CBr-E6 (SEQ ID NO: US8734809: 19 and 93), AAV CBr-E7 (SEQ ID NO: 20 and 94 of US8734809), AAV CBr-E8 (SEQ ID NO: 21 and 95 of US8734809), AAV CLv-D1 (SEQ ID NO: 22 and 95 of US8734809) 96), AAV CLv-D2 (SEQ ID NO: 23 and 97 of US8734809), AAV CLv-D3 (SEQ ID NO: 24 and 98 of US8734809), AAV CLv-D4 (SEQ ID NO: 25 and 99 of US8734809) , AAV CLv-D5 (SEQ ID NO: 26 and 100 of US8734809), AAV CLv-D6 (SEQ ID NO: 27 and 101 of US8734809), AAV CLv-D7 (SEQ ID NO: 28 and 102 of US8734809), AAV CLv-D8 (SEQ ID NO: 29 and 103 of US8734809), AAV CLv-E1 (SEQ ID NO: 13 and 87 of US8734809), AAV CLv-R1 (SEQ ID NO: 30 and 104 of US8734809), AAV CLv- R2 (SEQ ID NO: 31 and 105 of US8734809), AAV CLv-R3 (SEQ ID NO: 32 and 106 of US8734809), AAV CLv-R4 (SEQ ID NO: 33 and 107 of US8734809), AAV CLv-R5 ( SEQ ID NO: 34 and 108 of US8734809), AAV CLv-R6 (SEQ ID NO: 35 and 109 of US8734809), AAV CLv-R7 (SEQ ID NO: 36 and 110 of US8734809), AAV CLv-R8 (SEQ ID NO: 36 and 110 of US8734809) SEQ ID NO: 37 and 111), AAV CLv-R9 (SEQ ID NO: 38 and 112 of US8734809), AAV CLg-F1 (SEQ ID NO: 39 and 113 of US8734809), AAV CLg-F2 (SEQ ID NO: US8734809 NO: 40 and 114), AAV CLg-F3 (SEQ ID NO: 41 and 115 of US8734809), AAV CLg-F4 (SEQ ID NO: 42 and 116 of US8734809), AAV CLg-F5 (SEQ ID NO of US8734809: 43 and 117), AAV CLg-F6 (SEQ ID NO: 43 and 117 of US8734809), AAV CLg-F7 (SEQ ID NO: 44 and 118 of US8734809), AAV CLg-F8 (SEQ ID NO: 43 and 118 of US8734809) 117), AAV CSp-1 (SEQ ID NO: 45 and 119 of US8734809), AAV CSp-10 (SEQ ID NO: 46 and 120 of US8734809), AAV CSp-11 (SEQ ID NO: 47 and 121 of US8734809) , AAV CSp-2 (SEQ ID NO: 48 and 122 of US8734809), AAV CSp-3 (SEQ ID NO: 49 and 123 of US8734809), AAV CSp-4 (SEQ ID NO: 50 and 124 of US8734809), AAV CSp-6 (SEQ ID NO: 51 and 125 of US8734809), AAV CSp-7 (SEQ ID NO: 52 and 126 of US8734809), AAV CSp-8 (SEQ ID NO: 53 and 127 of US8734809), AAV CSp- 9 (SEQ ID NO: 54 and 128 of US8734809), AAV CHt-2 (SEQ ID NO: 55 and 129 of US8734809), AAV CHt-3 (SEQ ID NO: 56 and 130 of US8734809), AAV CKd-1 ( SEQ ID NO: 57 and 131 of US8734809), AAV CKd-10 (SEQ ID NO: 58 and 132 of US8734809), AAV CKd-2 (SEQ ID NO: 59 and 133 of US8734809), AAV CKd-3 (SEQ ID NO: 59 and 133 of US8734809) SEQ ID NO: 60 and 134), AAV CKd-4 (SEQ ID NO: 61 and 135 of US8734809), AAV CKd-6 (SEQ ID NO: 62 and 136 of US8734809), AAV CKd-7 (SEQ ID NO of US8734809 NO: 63 and 137), AAV CKd-8 (SEQ ID NO: 64 and 138 of US8734809), AAV CLv-1 (SEQ ID NO: 35 and 139 of US8734809), AAV CLv-12 (SEQ ID NO: US8734809) 66 and 140), AAV CLv-13 (SEQ ID NO: 67 and 141 of US8734809), AAV CLv-2 (SEQ ID NO: 68 and 142 of US8734809), AAV CLv-3 (SEQ ID NO: 69 and 142 of US8734809) 143), AAV CLv-4 (SEQ ID NO: 70 and 144 of US8734809), AAV CLv-6 (SEQ ID NO: 71 and 145 of US8734809), AAV CLv-8 (SEQ ID NO: 72 and 146 of US8734809) , AAV CKd-B1 (SEQ ID NO: 73 and 147 of US8734809), AAV CKd-B2 (SEQ ID NO: 74 and 148 of US8734809), AAV CKd-B3 (SEQ ID NO: 75 and 149 of US8734809), AAV CKd-B4 (SEQ ID NO: 76 and 150 of US8734809), AAV CKd-B5 (SEQ ID NO: 77 and 151 of US8734809), AAV CKd-B6 (SEQ ID NO: 78 and 152 of US8734809), AAV CKd- B7 (SEQ ID NO: 79 and 153 of US8734809), AAV CKd-B8 (SEQ ID NO: 80 and 154 of US8734809), AAV CKd-H1 (SEQ ID NO: 81 and 155 of US8734809), AAV CKd-H2 ( SEQ ID NO: 82 and 156 of US8734809), AAV CKd-H3 (SEQ ID NO: 83 and 157 of US8734809), AAV CKd-H4 (SEQ ID NO: 84 and 158 of US8734809), AAV CKd-H5 (SEQ ID NO: 84 and 158 of US8734809) SEQ ID NO: 85 and 159), AAV CKd-H6 (SEQ ID NO: 77 and 151 of US8734809), AAV CHt-1 (SEQ ID NO: 86 and 160 of US8734809), AAV CLv1-1 (SEQ ID NO: US8734809 NO: 171), AAV CLv1-2 (SEQ ID NO: 172 of US8734809), AAV CLv1-3 (SEQ ID NO: 173 of US8734809), AAV CLv1-4 (SEQ ID NO: 174 of US8734809), AAV Clv1- 7 (SEQ ID NO: 175 of US8734809), AAV Clv1-8 (SEQ ID NO: 176 of US8734809), AAV Clv1-9 (SEQ ID NO: 177 of US8734809), AAV Clv1-10 (SEQ ID NO: 177 of US8734809) 178), AAV.VR-355 (SEQ ID NO: 181 of US8734809), AAV.hu.48R3 (SEQ ID NO: 183 of US8734809), or variants or derivatives thereof.

In some embodiments, an AAV serotype may be or comprise a sequence as described in International Publication No. WO2016065001, which is incorporated by reference in its entirety, such as (but not limited to) AAV CHt-P2 (WO2016065001). SEQ ID NO: 1 and 51), AAV CHt-P5 (SEQ ID NO: 2 and 52 of WO2016065001), AAV CHt-P9 (SEQ ID NO: 3 and 53 of WO2016065001), AAV CBr-7.1 (SEQ ID NO of WO2016065001 NO: 4 and 54), AAV CBr-7.2 (SEQ ID NO: 5 and 55 of WO2016065001), AAV CBr-7.3 (SEQ ID NO: 6 and 56 of WO2016065001), AAV CBr-7.4 (SEQ ID NO: WO2016065001) 7 and 57), AAV CBr-7.5 (SEQ ID NO: 8 and 58 of WO2016065001), AAV CBr-7.7 (SEQ ID NO: 9 and 59 of WO2016065001), AAV CBr-7.8 (SEQ ID NO: 10 and 59 of WO2016065001) 60), AAV CBr-7.10 (SEQ ID NO: 11 and 61 of WO2016065001), AAV CKd-N3 (SEQ ID NO: 12 and 62 of WO2016065001), AAV CKd-N4 (SEQ ID NO: 13 and 63 of WO2016065001) , AAV CKd-N9 (SEQ ID NO: 14 and 64 of WO2016065001), AAV CLv-L4 (SEQ ID NO: 15 and 65 of WO2016065001), AAV CLv-L5 (SEQ ID NO: 16 and 66 of WO2016065001), AAV CLv-L6 (SEQ ID NO: 17 and 67 of WO2016065001), AAV CLv-K1 (SEQ ID NO: 18 and 68 of WO2016065001), AAV CLv-K3 (SEQ ID NO: 19 and 69 of WO2016065001), AAV CLv- K6 (SEQ ID NO: 20 and 70 of WO2016065001), AAV CLv-M1 (SEQ ID NO: 21 and 71 of WO2016065001), AAV CLv-M11 (SEQ ID NO: 22 and 72 of WO2016065001), AAV CLv-M2 ( SEQ ID NO: 23 and 73 of WO2016065001), AAV CLv-M5 (SEQ ID NO: 24 and 74 of WO2016065001), AAV CLv-M6 (SEQ ID NO: 25 and 75 of WO2016065001), AAV CLv-M7 (SEQ ID NO: 25 and 75 of WO2016065001) SEQ ID NO: 26 and 76), AAV CLv-M8 (SEQ ID NO: 27 and 77 of WO2016065001), AAV CLv-M9 (SEQ ID NO: 28 and 78 of WO2016065001), AAV CHt-P1 (SEQ ID NO: WO2016065001 NO: 29 and 79), AAV CHt-P6 (SEQ ID NO: 30 and 80 of WO2016065001), AAV CHt-P8 (SEQ ID NO: 31 and 81 of WO2016065001), AAV CHt-6.1 (SEQ ID NO: WO2016065001) 32 and 82), AAV CHt-6.10 (SEQ ID NO: 33 and 83 of WO2016065001), AAV CHt-6.5 (SEQ ID NO: 34 and 84 of WO2016065001), AAV CHt-6.6 (SEQ ID NO: 35 and 84 of WO2016065001) 85), AAV CHt-6.7 (SEQ ID NO: 36 and 86 of WO2016065001), AAV CHt-6.8 (SEQ ID NO: 37 and 87 of WO2016065001), AAV CSp-8.10 (SEQ ID NO: 38 and 88 of WO2016065001) , AAV CSp-8.2 (SEQ ID NO: 39 and 89 of WO2016065001), AAV CSp-8.4 (SEQ ID NO: 40 and 90 of WO2016065001), AAV CSp-8.5 (SEQ ID NO: 41 and 91 of WO2016065001), AAV CSp-8.6 (SEQ ID NO: 42 and 92 of WO2016065001), AAV CSp-8.7 (SEQ ID NO: 43 and 93 of WO2016065001), AAV CSp-8.8 (SEQ ID NO: 44 and 94 of WO2016065001), AAV CSp- 8.9 (SEQ ID NO: 45 and 95 of WO2016065001), AAV CBr-B7.3 (SEQ ID NO: 46 and 96 of WO2016065001), AAV CBr-B7.4 (SEQ ID NO: 47 and 97 of WO2016065001), AAV3B (SEQ ID NO: 48 and 98 of WO2016065001), AAV4 (SEQ ID NO: 49 and 99 of WO2016065001), AAV5 (SEQ ID NO: 50 and 100 of WO2016065001), or variants or derivatives thereof.

In some embodiments, the AAV particle may be or comprise a serotype selected from any of those serotypes found in Table 1.

In some embodiments, AAV particles may comprise sequences, fragments or variants of any of the sequences in Table 1.

In some embodiments, AAV particles may be encoded by sequences, fragments, or variants of any of the sequences in Table 1.

In the DNA and RNA sequences mentioned and/or described herein, the single-letter symbols are as follows: A represents adenine; C represents cytosine; G represents guanine; T represents thymine; U represents uracil; W represents weak Bases, such as adenine or thymine; S represents strong nucleotides, such as cytosine and guanine; M represents amino nucleotides, such as adenine and cytosine; K represents keto nucleotides, such as guanine and thymine; R represents purines: adenine and guanine; Y represents pyrimidines: cytosine and thymine; B represents any non-A base (for example, cytosine, guanine, and thymine); D represents any non-C base base (e.g., adenine, guanine, and thymine); H represents any non-G base (e.g., adenine, cytosine, and thymine); V represents any non-T base (e.g., adenine, cytosine, and guanine); N represents any nucleotide (which is not a gap); and Z represents zero.

In any amino acid sequence mentioned and/or described herein, the single-letter symbols are described as follows: G (Gly) represents glycine; A (Ala) represents alanine; L (Leu) represents leucine; M (Met) represents methionine; F (Phe) represents phenylalanine; W (Trp) represents tryptophan; K (Lys) represents lysine; Q (Gln) represents glutamine; E (Glu) Represents glutamic acid; S (Ser) represents serine; P (Pro) represents proline; V (Val) represents valine; I (Ile) represents isoleucine; C (Cys) represents cysteamine Acid; Y (Tyr) represents tyrosine; H (His) represents histine; R (Arg) represents arginine; N (Asn) represents asparagine; D (Asp) represents aspartic acid; T (Thr) represents threonine; B (Asx) represents aspartic acid or asparagine; J (Xle) represents leucine or isoleucine; O (Pyl) represents pyrrolidine; U (Sec ) represents selenocysteine; X (Xaa) represents any amino acid; and Z (Glx) represents glutamic acid or glutamic acid. Table 1. Representative AAV serotypes serotype SEQ ID NO Reference information VOY101 1 or 1722 - VOY201 1723 or 1724 - PHP.N/PHP.B-DGT 2 WO2017100671 SEQ ID NO: 46 AAVPHP.B or G2B-26 3 WO2015038958 SEQ ID NO: 8 and 13 AAVPHP.B 4 WO2015038958 SEQ ID NO: 9 AAVG2B-13 5 WO2015038958 SEQ ID NO: 12 AAVTH1.1-32 6 WO2015038958 SEQ ID NO: 14 AAVTH1.1-35 7 WO2015038958 SEQ ID NO: 15 PHP.S/G2A12 8 WO2017100671 SEQ ID NO: 47 AAV9/hu.14 K449R 9 WO2017100671 SEQ ID NO: 45 AAV1 10 US20150159173 SEQ ID NO: 11, US20150315612 SEQ ID NO: 202 AAV1 11 US20160017295 SEQ ID NO: 1, US20030138772 SEQ ID NO: 64, US20150159173 SEQ ID NO: 27, US20150315612 SEQ ID NO: 219, US7198951 SEQ ID NO: 5 AAV1 12 US20030138772 SEQ ID NO: 6 AAV1.3 13 US20030138772 SEQ ID NO: 14 AAV10 14 US20030138772 SEQ ID NO: 117 AAV10 15 WO2015121501 SEQ ID NO: 9 AAV10 16 WO2015121501 SEQ ID NO: 8 AAV11 17 US20030138772 SEQ ID NO: 118 AAV12 18 US20030138772 SEQ ID NO: 119 AAV2 19 US20150159173 SEQ ID NO: 7, US20150315612 SEQ ID NO: 211 AAV2 20 US20030138772 SEQ ID NO: 70, US20150159173 SEQ ID NO: 23, US20150315612 SEQ ID NO: 221, US20160017295 SEQ ID NO: 2, US6156303 SEQ ID NO: 4, US7198951 SEQ ID NO: 4, WO2015 121501 SEQ ID NO: 1 AAV2 twenty one US6156303 SEQ ID NO: 8 AAV2 twenty two US20030138772 SEQ ID NO: 7 AAV2 twenty three US6156303 SEQ ID NO: 3 AAV2.5T twenty four US9233131 SEQ ID NO: 42 AAV223.10 25 US20030138772 SEQ ID NO: 75 AAV223.2 26 US20030138772 SEQ ID NO: 49 AAV223.2 27 US20030138772 SEQ ID NO: 76 AAV223.4 28 US20030138772 SEQ ID NO: 50 AAV223.4 29 US20030138772 SEQ ID NO: 73 AAV223.5 30 US20030138772 SEQ ID NO: 51 AAV223.5 31 US20030138772 SEQ ID NO: 74 AAV223.6 32 US20030138772 SEQ ID NO: 52 AAV223.6 33 US20030138772 SEQ ID NO: 78 AAV223.7 34 US20030138772 SEQ ID NO: 53 AAV223.7 35 US20030138772 SEQ ID NO: 77 AAV29.3 36 US20030138772 SEQ ID NO: 82 AAV29.4 37 US20030138772 SEQ ID NO: 12 AAV29.5 38 US20030138772 SEQ ID NO: 83 AAV29.5 (AAVbb.2) 39 US20030138772 SEQ ID NO: 13 AAV3 40 US20150159173 SEQ ID NO: 12 AAV3 41 US20030138772 SEQ ID NO: 71, US20150159173 SEQ ID NO: 28, US20160017295 SEQ ID NO: 3, US7198951 SEQ ID NO: 6 AAV3 42 US20030138772 SEQ ID NO: 8 AAV3.3b 43 US20030138772 SEQ ID NO: 72 AAV3-3 44 US20150315612 SEQ ID NO: 200 AAV3-3 45 US20150315612 SEQ ID NO: 217 AAV3a 46 US6156303 SEQ ID NO: 5 AAV3a 47 US6156303 SEQ ID NO: 9 AAV3b 48 US6156303 SEQ ID NO: 6 AAV3b 49 US6156303 SEQ ID NO: 10 AAV3b 50 US6156303 SEQ ID NO: 1 AAV4 51 US20140348794 SEQ ID NO: 17 AAV4 52 US20140348794 SEQ ID NO: 5 AAV4 53 US20140348794 SEQ ID NO: 3 AAV4 54 US20140348794 SEQ ID NO: 14 AAV4 55 US20140348794 SEQ ID NO: 15 AAV4 56 US20140348794 SEQ ID NO: 19 AAV4 57 US20140348794 SEQ ID NO: 12 AAV4 58 US20140348794 SEQ ID NO: 13 AAV4 59 US20140348794 SEQ ID NO: 7 AAV4 60 US20140348794 SEQ ID NO: 8 AAV4 61 US20140348794 SEQ ID NO: 9 AAV4 62 US20140348794 SEQ ID NO: 2 AAV4 63 US20140348794 SEQ ID NO: 10 AAV4 64 US20140348794 SEQ ID NO: 11 AAV4 65 US20140348794 SEQ ID NO: 18 AAV4 66 US20030138772 SEQ ID NO: 63, US20160017295 SEQ ID NO: 4, US20140348794 SEQ ID NO: 4 AAV4 67 US20140348794 SEQ ID NO: 16 AAV4 68 US20140348794 SEQ ID NO: 20 AAV4 69 US20140348794 SEQ ID NO: 6 AAV4 70 US20140348794 SEQ ID NO: 1 AAV42.2 71 US20030138772 SEQ ID NO: 9 AAV42.2 72 US20030138772 SEQ ID NO: 102 AAV42.3b 73 US20030138772 SEQ ID NO: 36 AAV42.3B 74 US20030138772 SEQ ID NO: 107 AAV42.4 75 US20030138772 SEQ ID NO: 33 AAV42.4 76 US20030138772 SEQ ID NO: 88 AAV42.8 77 US20030138772 SEQ ID NO: 27 AAV42.8 78 US20030138772 SEQ ID NO: 85 AAV43.1 79 US20030138772 SEQ ID NO: 39 AAV43.1 80 US20030138772 SEQ ID NO: 92 AAV43.12 81 US20030138772 SEQ ID NO: 41 AAV43.12 82 US20030138772 SEQ ID NO: 93 AAV43.20 83 US20030138772 SEQ ID NO: 42 AAV43.20 84 US20030138772 SEQ ID NO: 99 AAV43.21 85 US20030138772 SEQ ID NO: 43 AAV43.21 86 US20030138772 SEQ ID NO: 96 AAV43.23 87 US20030138772 SEQ ID NO: 44 AAV43.23 88 US20030138772 SEQ ID NO: 98 AAV43.25 89 US20030138772 SEQ ID NO: 45 AAV43.25 90 US20030138772 SEQ ID NO: 97 AAV43.5 91 US20030138772 SEQ ID NO: 40 AAV43.5 92 US20030138772 SEQ ID NO: 94 AAV4-4 93 US20150315612 SEQ ID NO: 201 AAV4-4 94 US20150315612 SEQ ID NO: 218 AAV44.1 95 US20030138772 SEQ ID NO: 46 AAV44.1 96 US20030138772 SEQ ID NO: 79 AAV44.5 97 US20030138772 SEQ ID NO: 47 AAV44.5 98 US20030138772 SEQ ID NO: 80 AAV4407 99 US20150315612 SEQ ID NO: 90 AAV5 100 US7427396 SEQ ID NO: 1 AAV5 101 US20030138772 SEQ ID NO: 114 AAV5 102 US20160017295 SEQ ID NO: 5, US7427396 SEQ ID NO: 2, US20150315612 SEQ ID NO: 216 AAV5 103 US20150315612 SEQ ID NO: 199 AAV6 104 US20150159173 SEQ ID NO: 13 AAV6 105 US20030138772 SEQ ID NO: 65, US20150159173 SEQ ID NO: 29, US20160017295 SEQ ID NO: 6, US6156303 SEQ ID NO: 7 AAV6 106 US6156303 SEQ ID NO: 11 AAV6 107 US6156303 SEQ ID NO: 2 AAV6 108 US20150315612 SEQ ID NO: 203 AAV6 109 US20150315612 SEQ ID NO: 220 AAV6.1 110 US20150159173 AAV6.12 111 US20150159173 AAV6.2 112 US20150159173 AAV7 113 US20150159173 SEQ ID NO: 14 AAV7 114 US20150315612 SEQ ID NO: 183 AAV7 115 US20030138772 SEQ ID NO: 2, US20150159173 SEQ ID NO: 30, US20150315612 SEQ ID NO: 181, US20160017295 SEQ ID NO: 7 AAV7 116 US20030138772 SEQ ID NO: 3 AAV7 117 US20030138772 SEQ ID NO: 1, US20150315612 SEQ ID NO: 180 AAV7 118 US20150315612 SEQ ID NO: 213 AAV7 119 US20150315612 SEQ ID NO: 222 AAV8 120 US20150159173 SEQ ID NO: 15 AAV8 121 US20150376240 SEQ ID NO: 7 AAV8 122 US20030138772 SEQ ID NO: 4, US20150315612 SEQ ID NO: 182 AAV8 123 US20030138772 SEQ ID NO: 95, US20140359799 SEQ ID NO: 1, US20150159173 SEQ ID NO: 31, US20160017295 SEQ ID NO: 8, US7198951 SEQ ID NO: 7, US20150315612 SEQ ID NO: 223 AAV8 124 US20150376240 SEQ ID NO: 8 AAV8 125 US20150315612 SEQ ID NO: 214 AAV-8b 126 US20150376240 SEQ ID NO: 5 AAV-8b 127 US20150376240 SEQ ID NO: 3 AAV-8h 128 US20150376240 SEQ ID NO: 6 AAV-8h 129 US20150376240 SEQ ID NO: 4 AAV9 130 US20030138772 SEQ ID NO: 5 AAV9 131 US7198951 SEQ ID NO: 1 AAV9 132 US20160017295 SEQ ID NO: 9 AAV9 133 US20030138772 SEQ ID NO: 100, US7198951 SEQ ID NO: 2 AAV9 134 US7198951 SEQ ID NO: 3 AAV9 (AAVhu.14) 135 US7906111 SEQ ID NO: 3; WO2015038958 SEQ ID NO: 11 AAV9 (AAVhu.14) 136 US7906111 SEQ ID NO: 123; WO2015038958 SEQ ID NO: 2 AAVA3.1 137 US20030138772 SEQ ID NO: 120 AAVA3.3 138 US20030138772 SEQ ID NO: 57 AAVA3.3 139 US20030138772 SEQ ID NO: 66 AAVA3.4 140 US20030138772 SEQ ID NO: 54 AAVA3.4 141 US20030138772 SEQ ID NO: 68 AAVA3.5 142 US20030138772 SEQ ID NO: 55 AAVA3.5 143 US20030138772 SEQ ID NO: 69 AAVA3.7 144 US20030138772 SEQ ID NO: 56 AAVA3.7 145 US20030138772 SEQ ID NO: 67 AAV29.3 (AAVbb.1) 146 US20030138772 SEQ ID NO: 11 AAVC2 147 US20030138772 SEQ ID NO: 61 AAVCh.5 148 US20150159173 SEQ ID NO: 46, US20150315612 SEQ ID NO: 234 AAVcy.2 (AAV13.3) 149 US20030138772 SEQ ID NO: 15 AAV24.1 150 US20030138772 SEQ ID NO: 101 AAVcy.3 (AAV24.1) 151 US20030138772 SEQ ID NO: 16 AAV27.3 152 US20030138772 SEQ ID NO: 104 AAVcy.4 (AAV27.3) 153 US20030138772 SEQ ID NO: 17 AAVcy.5 154 US20150315612 SEQ ID NO: 227 AAV7.2 155 US20030138772 SEQ ID NO: 103 AAVcy.5 (AAV7.2) 156 US20030138772 SEQ ID NO: 18 AAV16.3 157 US20030138772 SEQ ID NO: 105 AAVcy.6 (AAV16.3) 158 US20030138772 SEQ ID NO: 10 AAVcy.5 159 US20150159173 SEQ ID NO: 8 AAVcy.5 160 US20150159173 SEQ ID NO: 24 AAVCy.5R1 161 US20150159173 AAVCy.5R2 162 US20150159173 AAVCy.5R3 163 US20150159173 AAVCy.5R4 164 US20150159173 AAVDJ 165 US20140359799 SEQ ID NO: 3, US7588772 SEQ ID NO: 2 AAVDJ 166 US20140359799 SEQ ID NO: 2, US7588772 SEQ ID NO: 1 AAVDJ-8 167 US7588772; Grimm et al. 2008 AAVDJ-8 168 US7588772; Grimm et al. 2008 AAVF5 169 US20030138772 SEQ ID NO: 110 AAVH2 170 US20030138772 SEQ ID NO: 26 AAVH6 171 US20030138772 SEQ ID NO: 25 AAVhE1.1 172 US9233131 SEQ ID NO: 44 AAVhEr1.14 173 US9233131 SEQ ID NO: 46 AAVhEr1.16 174 US9233131 SEQ ID NO: 48 AAVhEr1.18 175 US9233131 SEQ ID NO: 49 AAVhEr1.23 (AAVhEr2.29) 176 US9233131 SEQ ID NO: 53 AAVhEr1.35 177 US9233131 SEQ ID NO: 50 AAVhEr1.36 178 US9233131 SEQ ID NO: 52 AAVhEr1.5 179 US9233131 SEQ ID NO: 45 AAVhEr1.7 180 US9233131 SEQ ID NO: 51 AAVhEr1.8 181 US9233131 SEQ ID NO: 47 AAVhEr2.16 182 US9233131 SEQ ID NO: 55 AAVhEr2.30 183 US9233131 SEQ ID NO: 56 AAVhEr2.31 184 US9233131 SEQ ID NO: 58 AAVhEr2.36 185 US9233131 SEQ ID NO: 57 AAVhEr2.4 186 US9233131 SEQ ID NO: 54 AAVhEr3.1 187 US9233131 SEQ ID NO: 59 AAVhu.1 188 US20150315612 SEQ ID NO: 46 AAVhu.1 189 US20150315612 SEQ ID NO: 144 AAVhu.10 (AAV16.8) 190 US20150315612 SEQ ID NO: 56 AAVhu.10 (AAV16.8) 191 US20150315612 SEQ ID NO: 156 AAVhu.11 (AAV16.12) 192 US20150315612 SEQ ID NO: 57 AAVhu.11 (AAV16.12) 193 US20150315612 SEQ ID NO: 153 AAVhu.12 194 US20150315612 SEQ ID NO: 59 AAVhu.12 195 US20150315612 SEQ ID NO: 154 AAVhu.13 196 US20150159173 SEQ ID NO: 16, US20150315612 SEQ ID NO: 71 AAVhu.13 197 US20150159173 SEQ ID NO: 32, US20150315612 SEQ ID NO: 129 AAVhu.136.1 198 US20150315612 SEQ ID NO: 165 AAVhu.140.1 199 US20150315612 SEQ ID NO: 166 AAVhu.140.2 200 US20150315612 SEQ ID NO: 167 AAVhu.145.6 201 US20150315612 SEQ ID No: 178 AAVhu.15 202 US20150315612 SEQ ID NO: 147 AAVhu.15 (AAV33.4) 203 US20150315612 SEQ ID NO: 50 AAVhu.156.1 204 US20150315612 SEQ ID No: 179 AAVhu.16 205 US20150315612 SEQ ID NO: 148 AAVhu.16 (AAV33.8) 206 US20150315612 SEQ ID NO: 51 AAVhu.17 207 US20150315612 SEQ ID NO: 83 AAVhu.17 (AAV33.12) 208 US20150315612 SEQ ID NO: 4 AAVhu.172.1 209 US20150315612 SEQ ID NO: 171 AAVhu.172.2 210 US20150315612 SEQ ID NO: 172 AAVhu.173.4 211 US20150315612 SEQ ID NO: 173 AAVhu.173.8 212 US20150315612 SEQ ID NO: 175 AAVhu.18 213 US20150315612 SEQ ID NO: 52 AAVhu.18 214 US20150315612 SEQ ID NO: 149 AAVhu.19 215 US20150315612 SEQ ID NO: 62 AAVhu.19 216 US20150315612 SEQ ID NO: 133 AAVhu.2 217 US20150315612 SEQ ID NO: 48 AAVhu.2 218 US20150315612 SEQ ID NO: 143 AAVhu.20 219 US20150315612 SEQ ID NO: 63 AAVhu.20 220 US20150315612 SEQ ID NO: 134 AAVhu.21 221 US20150315612 SEQ ID NO: 65 AAVhu.21 222 US20150315612 SEQ ID NO: 135 AAVhu.22 223 US20150315612 SEQ ID NO: 67 AAVhu.22 224 US20150315612 SEQ ID NO: 138 AAVhu.23 225 US20150315612 SEQ ID NO: 60 AAVhu.23.2 226 US20150315612 SEQ ID NO: 137 AAVhu.24 227 US20150315612 SEQ ID NO: 66 AAVhu.24 228 US20150315612 SEQ ID NO: 136 AAVhu.25 229 US20150315612 SEQ ID NO: 49 AAVhu.25 230 US20150315612 SEQ ID NO: 146 AAVhu.26 231 US20150159173 SEQ ID NO: 17, US20150315612 SEQ ID NO: 61 AAVhu.26 232 US20150159173 SEQ ID NO: 33, US20150315612 SEQ ID NO: 139 AAVhu.27 233 US20150315612 SEQ ID NO: 64 AAVhu.27 234 US20150315612 SEQ ID NO: 140 AAVhu.28 235 US20150315612 SEQ ID NO: 68 AAVhu.28 236 US20150315612 SEQ ID NO: 130 AAVhu.29 237 US20150315612 SEQ ID NO: 69 AAVhu.29 238 US20150159173 SEQ ID NO: 42, US20150315612 SEQ ID NO: 132 AAVhu.29 239 US20150315612 SEQ ID NO: 225 AAVhu.29R 240 US20150159173 AAVhu.3 241 US20150315612 SEQ ID NO: 44 AAVhu.3 242 US20150315612 SEQ ID NO: 145 AAVhu.30 243 US20150315612 SEQ ID NO: 70 AAVhu.30 244 US20150315612 SEQ ID NO: 131 AAVhu.31 245 US20150315612 SEQ ID NO: 1 AAVhu.31 246 US20150315612 SEQ ID NO: 121 AAVhu.32 247 US20150315612 SEQ ID NO: 2 AAVhu.32 248 US20150315612 SEQ ID NO: 122 AAVhu.33 249 US20150315612 SEQ ID NO: 75 AAVhu.33 250 US20150315612 SEQ ID NO: 124 AAVhu.34 251 US20150315612 SEQ ID NO: 72 AAVhu.34 252 US20150315612 SEQ ID NO: 125 AAVhu.35 253 US20150315612 SEQ ID NO: 73 AAVhu.35 254 US20150315612 SEQ ID NO: 164 AAVhu.36 255 US20150315612 SEQ ID NO: 74 AAVhu.36 256 US20150315612 SEQ ID NO: 126 AAVhu.37 257 US20150159173 SEQ ID NO: 34, US20150315612 SEQ ID NO: 88 AAVhu.37 (AAV106.1) 258 US20150315612 SEQ ID NO: 10, US20150159173 SEQ ID NO: 18 AAVhu.38 259 US20150315612 SEQ ID NO: 161 AAVhu.39 260 US20150315612 SEQ ID NO: 102 AAVhu.39 (AAVLG-9) 261 US20150315612 SEQ ID NO: 24 AAVhu.4 262 US20150315612 SEQ ID NO: 47 AAVhu.4 263 US20150315612 SEQ ID NO: 141 AAVhu.40 264 US20150315612 SEQ ID NO: 87 AAVhu.40 (AAV114.3) 265 US20150315612 SEQ ID No: 11 AAVhu.41 266 US20150315612 SEQ ID NO: 91 AAVhu.41 (AAV127.2) 267 US20150315612 SEQ ID NO: 6 AAVhu.42 268 US20150315612 SEQ ID NO: 85 AAVhu.42 (AAV127.5) 269 US20150315612 SEQ ID NO: 8 AAVhu.43 270 US20150315612 SEQ ID NO: 160 AAVhu.43 271 US20150315612 SEQ ID NO: 236 AAVhu.43 (AAV128.1) 272 US20150315612 SEQ ID NO: 80 AAVhu.44 273 US20150159173 SEQ ID NO: 45, US20150315612 SEQ ID NO: 158 AAVhu.44 (AAV128.3) 274 US20150315612 SEQ ID NO: 81 AAVhu.44R1 275 US20150159173 AAVhu.44R2 276 US20150159173 AAVhu.44R3 277 US20150159173 AAVhu.45 278 US20150315612 SEQ ID NO: 76 AAVhu.45 279 US20150315612 SEQ ID NO: 127 AAVhu.46 280 US20150315612 SEQ ID NO: 82 AAVhu.46 281 US20150315612 SEQ ID NO: 159 AAVhu.46 282 US20150315612 SEQ ID NO: 224 AAVhu.47 283 US20150315612 SEQ ID NO: 77 AAVhu.47 284 US20150315612 SEQ ID NO: 128 AAVhu.48 285 US20150159173 SEQ ID NO: 38 AAVhu.48 286 US20150315612 SEQ ID NO: 157 AAVhu.48 (AAV130.4) 287 US20150315612 SEQ ID NO: 78 AAVhu.48R1 288 US20150159173 AAVhu.48R2 289 US20150159173 AAVhu.48R3 290 US20150159173 AAVhu.49 291 US20150315612 SEQ ID NO: 209 AAVhu.49 292 US20150315612 SEQ ID NO: 189 AAVhu.5 293 US20150315612 SEQ ID NO: 45 AAVhu.5 294 US20150315612 SEQ ID NO: 142 AAVhu.51 295 US20150315612 SEQ ID NO: 208 AAVhu.51 296 US20150315612 SEQ ID NO: 190 AAVhu.52 297 US20150315612 SEQ ID NO: 210 AAVhu.52 298 US20150315612 SEQ ID NO: 191 AAVhu.53 299 US20150159173 SEQ ID NO: 19 AAVhu.53 300 US20150159173 SEQ ID NO: 35 AAVhu.53 (AAV145.1) 301 US20150315612 SEQ ID NO: 176 AAVhu.54 302 US20150315612 SEQ ID NO: 188 AAVhu.54 (AAV145.5) 303 US20150315612 SEQ ID No: 177 AAVhu.55 304 US20150315612 SEQ ID NO: 187 AAVhu.56 305 US20150315612 SEQ ID NO: 205 AAVhu.56 (AAV145.6) 306 US20150315612 SEQ ID NO: 168 AAVhu.56 (AAV145.6) 307 US20150315612 SEQ ID NO: 192 AAVhu.57 308 US20150315612 SEQ ID NO: 206 AAVhu.57 309 US20150315612 SEQ ID NO: 169 AAVhu.57 310 US20150315612 SEQ ID NO: 193 AAVhu.58 311 US20150315612 SEQ ID NO: 207 AAVhu.58 312 US20150315612 SEQ ID NO: 194 AAVhu.6 (AAV3.1) 313 US20150315612 SEQ ID NO: 5 AAVhu.6 (AAV3.1) 314 US20150315612 SEQ ID NO: 84 AAVhu.60 315 US20150315612 SEQ ID NO: 184 AAVhu.60 (AAV161.10) 316 US20150315612 SEQ ID NO: 170 AAVhu.61 317 US20150315612 SEQ ID NO: 185 AAVhu.61 (AAV161.6) 318 US20150315612 SEQ ID NO: 174 AAVhu.63 319 US20150315612 SEQ ID NO: 204 AAVhu.63 320 US20150315612 SEQ ID NO: 195 AAVhu.64 321 US20150315612 SEQ ID NO: 212 AAVhu.64 322 US20150315612 SEQ ID NO: 196 AAVhu.66 323 US20150315612 SEQ ID NO: 197 AAVhu.67 324 US20150315612 SEQ ID NO: 215 AAVhu.67 325 US20150315612 SEQ ID NO: 198 AAVhu.7 326 US20150315612 SEQ ID NO: 226 AAVhu.7 327 US20150315612 SEQ ID NO: 150 AAVhu.7 (AAV7.3) 328 US20150315612 SEQ ID NO: 55 AAVhu.71 329 US20150315612 SEQ ID NO: 79 AAVhu.8 330 US20150315612 SEQ ID NO: 53 AAVhu.8 331 US20150315612 SEQ ID NO: 12 AAVhu.8 332 US20150315612 SEQ ID NO: 151 AAVhu.9 (AAV3.1) 333 US20150315612 SEQ ID NO: 58 AAVhu.9 (AAV3.1) 334 US20150315612 SEQ ID NO: 155 AAV-LK01 335 US20150376607 SEQ ID NO: 2 AAV-LK01 336 US20150376607 SEQ ID NO: 29 AAV-LK02 337 US20150376607 SEQ ID NO: 3 AAV-LK02 338 US20150376607 SEQ ID NO: 30 AAV-LK03 339 US20150376607 SEQ ID NO: 4 AAV-LK03 340 WO2015121501 SEQ ID NO: 12, US20150376607 SEQ ID NO: 31 AAV-LK04 341 US20150376607 SEQ ID NO: 5 AAV-LK04 342 US20150376607 SEQ ID NO: 32 AAV-LK05 343 US20150376607 SEQ ID NO: 6 AAV-LK05 344 US20150376607 SEQ ID NO: 33 AAV-LK06 345 US20150376607 SEQ ID NO: 7 AAV-LK06 346 US20150376607 SEQ ID NO: 34 AAV-LK07 347 US20150376607 SEQ ID NO: 8 AAV-LK07 348 US20150376607 SEQ ID NO: 35 AAV-LK08 349 US20150376607 SEQ ID NO: 9 AAV-LK08 350 US20150376607 SEQ ID NO: 36 AAV-LK09 351 US20150376607 SEQ ID NO: 10 AAV-LK09 352 US20150376607 SEQ ID NO: 37 AAV-LK10 353 US20150376607 SEQ ID NO: 11 AAV-LK10 354 US20150376607 SEQ ID NO: 38 AAV-LK11 355 US20150376607 SEQ ID NO: 12 AAV-LK11 356 US20150376607 SEQ ID NO: 39 AAV-LK12 357 US20150376607 SEQ ID NO: 13 AAV-LK12 358 US20150376607 SEQ ID NO: 40 AAV-LK13 359 US20150376607 SEQ ID NO: 14 AAV-LK13 360 US20150376607 SEQ ID NO: 41 AAV-LK14 361 US20150376607 SEQ ID NO: 15 AAV-LK14 362 US20150376607 SEQ ID NO: 42 AAV-LK15 363 US20150376607 SEQ ID NO: 16 AAV-LK15 364 US20150376607 SEQ ID NO: 43 AAV-LK16 365 US20150376607 SEQ ID NO: 17 AAV-LK16 366 US20150376607 SEQ ID NO: 44 AAV-LK17 367 US20150376607 SEQ ID NO: 18 AAV-LK17 368 US20150376607 SEQ ID NO: 45 AAV-LK18 369 US20150376607 SEQ ID NO: 19 AAV-LK18 370 US20150376607 SEQ ID NO: 46 AAV-LK19 371 US20150376607 SEQ ID NO: 20 AAV-LK19 372 US20150376607 SEQ ID NO: 47 AAV-PAEC 373 US20150376607 SEQ ID NO: 1 AAV-PAEC 374 US20150376607 SEQ ID NO: 48 AAV-PAEC11 375 US20150376607 SEQ ID NO: 26 AAV-PAEC11 376 US20150376607 SEQ ID NO: 54 AAV-PAEC12 377 US20150376607 SEQ ID NO: 27 AAV-PAEC12 378 US20150376607 SEQ ID NO: 51 AAV-PAEC13 379 US20150376607 SEQ ID NO: 28 AAV-PAEC13 380 US20150376607 SEQ ID NO: 49 AAV-PAEC2 381 US20150376607 SEQ ID NO: 21 AAV-PAEC2 382 US20150376607 SEQ ID NO: 56 AAV-PAEC4 383 US20150376607 SEQ ID NO: 22 AAV-PAEC4 384 US20150376607 SEQ ID NO: 55 AAV-PAEC6 385 US20150376607 SEQ ID NO: 23 AAV-PAEC6 386 US20150376607 SEQ ID NO: 52 AAV-PAEC7 387 US20150376607 SEQ ID NO: 24 AAV-PAEC7 388 US20150376607 SEQ ID NO: 53 AAV-PAEC8 389 US20150376607 SEQ ID NO: 25 AAV-PAEC8 390 US20150376607 SEQ ID NO: 50 AAVpi.1 391 US20150315612 SEQ ID NO: 28 AAVpi.1 392 US20150315612 SEQ ID NO: 93 AAVpi.2 393 US20150315612 SEQ ID NO: 30 AAVpi.2 394 US20150315612 SEQ ID NO: 95 AAVpi.3 395 US20150315612 SEQ ID NO: 29 AAVpi.3 396 US20150315612 SEQ ID NO: 94 AAVrh.10 397 US20150159173 SEQ ID NO: 9 AAVrh.10 398 US20150159173 SEQ ID NO: 25 AAV44.2 399 US20030138772 SEQ ID NO: 59 AAVrh.10 (AAV44.2) 400 US20030138772 SEQ ID NO: 81 AAV42.1B 401 US20030138772 SEQ ID NO: 90 AAVrh.12 (AAV42.1b) 402 US20030138772 SEQ ID NO: 30 AAVrh.13 403 US20150159173 SEQ ID NO: 10 AAVrh.13 404 US20150159173 SEQ ID NO: 26 AAVrh.13 405 US20150315612 SEQ ID NO: 228 AAVrh.13R 406 US20150159173 AAV42.3A 407 US20030138772 SEQ ID NO: 87 AAVrh.14 (AAV42.3a) 408 US20030138772 SEQ ID NO: 32 AAV42.5A 409 US20030138772 SEQ ID NO: 89 AAVrh.17 (AAV42.5a) 410 US20030138772 SEQ ID NO: 34 AAV42.5B 411 US20030138772 SEQ ID NO: 91 AAVrh.18 (AAV42.5b) 412 US20030138772 SEQ ID NO: 29 AAV42.6B 413 US20030138772 SEQ ID NO: 112 AAVrh.19 (AAV42.6b) 414 US20030138772 SEQ ID NO: 38 AAVrh.2 415 US20150159173 SEQ ID NO: 39 AAVrh.2 416 US20150315612 SEQ ID NO: 231 AAVrh.20 417 US20150159173 SEQ ID NO: 1 AAV42.10 418 US20030138772 SEQ ID NO: 106 AAVrh.21 (AAV42.10) 419 US20030138772 SEQ ID NO: 35 AAV42.11 420 US20030138772 SEQ ID NO: 108 AAVrh.22 (AAV42.11) 421 US20030138772 SEQ ID NO: 37 AAV42.12 422 US20030138772 SEQ ID NO: 113 AAVrh.23 (AAV42.12) 423 US20030138772 SEQ ID NO: 58 AAV42.13 424 US20030138772 SEQ ID NO: 86 AAVrh.24 (AAV42.13) 425 US20030138772 SEQ ID NO: 31 AAV42.15 426 US20030138772 SEQ ID NO: 84 AAVrh.25 (AAV42.15) 427 US20030138772 SEQ ID NO: 28 AAVrh.2R 428 US20150159173 AAVrh.31 (AAV223.1) 429 US20030138772 SEQ ID NO: 48 AAVC1 430 US20030138772 SEQ ID NO: 60 AAVrh.32 (AAVC1) 431 US20030138772 SEQ ID NO: 19 AAVrh.32/33 432 US20150159173 SEQ ID NO: 2 AAVrh.33 (AAVC3) 433 US20030138772 SEQ ID NO: 20 AAVC5 434 US20030138772 SEQ ID NO: 62 AAVrh.34 (AAVC5) 435 US20030138772 SEQ ID NO: 21 AAVF1 436 US20030138772 SEQ ID NO: 109 AAVrh.35 (AAVF1) 437 US20030138772 SEQ ID NO: 22 AAVF3 438 US20030138772 SEQ ID NO: 111 AAVrh.36 (AAVF3) 439 US20030138772 SEQ ID NO: 23 AAVrh.37 440 US20030138772 SEQ ID NO: 24 AAVrh.37 441 US20150159173 SEQ ID NO: 40 AAVrh.37 442 US20150315612 SEQ ID NO: 229 AAVrh.37R2 443 US20150159173 AAVrh.38 (AAVLG-4) 444 US20150315612 SEQ ID NO: 7 AAVrh.38 (AAVLG-4) 445 US20150315612 SEQ ID NO: 86 AAVrh.39 446 US20150159173 SEQ ID NO: 20, US20150315612 SEQ ID NO: 13 AAVrh.39 447 US20150159173 SEQ ID NO: 3, US20150159173 SEQ ID NO: 36, US20150315612 SEQ ID NO: 89 AAVrh.40 448 US20150315612 SEQ ID NO: 92 AAVrh.40 (AAVLG-10) 449 US20150315612 SEQ ID No: 14 AAVrh.43 (AAVN721-8) 450 US20150315612 SEQ ID NO: 43, US20150159173 SEQ ID NO: 21 AAVrh.43 (AAVN721-8) 451 US20150315612 SEQ ID NO: 163, US20150159173 SEQ ID NO: 37 AAVrh.44 452 US20150315612 SEQ ID NO: 34 AAVrh.44 453 US20150315612 SEQ ID NO: 111 AAVrh.45 454 US20150315612 SEQ ID NO: 41 AAVrh.45 455 US20150315612 SEQ ID NO: 109 AAVrh.46 456 US20150159173 SEQ ID NO: 22, US20150315612 SEQ ID NO: 19 AAVrh.46 457 US20150159173 SEQ ID NO: 4, US20150315612 SEQ ID NO: 101 AAVrh.47 458 US20150315612 SEQ ID NO: 38 AAVrh.47 459 US20150315612 SEQ ID NO: 118 AAVrh.48 460 US20150159173 SEQ ID NO: 44, US20150315612 SEQ ID NO: 115 AAVrh.48.1 461 US20150159173 AAVrh.48.1.2 462 US20150159173 AAVrh.48.2 463 US20150159173 AAVrh.48 (AAV1-7) 464 US20150315612 SEQ ID NO: 32 AAVrh.49 (AAV1-8) 465 US20150315612 SEQ ID NO: 25 AAVrh.49 (AAV1-8) 466 US20150315612 SEQ ID NO: 103 AAVrh.50 (AAV2-4) 467 US20150315612 SEQ ID NO: 23 AAVrh.50 (AAV2-4) 468 US20150315612 SEQ ID NO: 108 AAVrh.51 (AAV2-5) 469 US20150315612 SEQ ID No: 22 AAVrh.51 (AAV2-5) 470 US20150315612 SEQ ID NO: 104 AAVrh.52 (AAV3-9) 471 US20150315612 SEQ ID NO: 18 AAVrh.52 (AAV3-9) 472 US20150315612 SEQ ID NO: 96 AAVrh.53 473 US20150315612 SEQ ID NO: 97 AAVrh.53 (AAV3-11) 474 US20150315612 SEQ ID NO: 17 AAVrh.53 (AAV3-11) 475 US20150315612 SEQ ID NO: 186 AAVrh.54 476 US20150315612 SEQ ID NO: 40 AAVrh.54 477 US20150159173 SEQ ID NO: 49, US20150315612 SEQ ID NO: 116 AAVrh.55 478 US20150315612 SEQ ID NO: 37 AAVrh.55 (AAV4-19) 479 US20150315612 SEQ ID NO: 117 AAVrh.56 480 US20150315612 SEQ ID NO: 54 AAVrh.56 481 US20150315612 SEQ ID NO: 152 AAVrh.57 482 US20150315612 SEQ ID NO: 26 AAVrh.57 483 US20150315612 SEQ ID NO: 105 AAVrh.58 484 US20150315612 SEQ ID NO: 27 AAVrh.58 485 US20150159173 SEQ ID NO: 48, US20150315612 SEQ ID NO: 106 AAVrh.58 486 US20150315612 SEQ ID NO: 232 AAVrh.59 487 US20150315612 SEQ ID NO: 42 AAVrh.59 488 US20150315612 SEQ ID NO: 110 AAVrh.60 489 US20150315612 SEQ ID NO: 31 AAVrh.60 490 US20150315612 SEQ ID NO: 120 AAVrh.61 491 US20150315612 SEQ ID NO: 107 AAVrh.61 (AAV2-3) 492 US20150315612 SEQ ID NO: 21 AAVrh.62 (AAV2-15) 493 US20150315612 SEQ ID No: 33 AAVrh.62 (AAV2-15) 494 US20150315612 SEQ ID NO: 114 AAVrh.64 495 US20150315612 SEQ ID No: 15 AAVrh.64 496 US20150159173 SEQ ID NO: 43, US20150315612 SEQ ID NO: 99 AAVrh.64 497 US20150315612 SEQ ID NO: 233 AAVRh.64R1 498 US20150159173 AAVRh.64R2 499 US20150159173 AAVrh.65 500 US20150315612 SEQ ID NO: 35 AAVrh.65 501 US20150315612 SEQ ID NO: 112 AAVrh.67 502 US20150315612 SEQ ID NO: 36 AAVrh.67 503 US20150315612 SEQ ID NO: 230 AAVrh.67 504 US20150159173 SEQ ID NO: 47, US20150315612 SEQ ID NO: 113 AAVrh.68 505 US20150315612 SEQ ID NO: 16 AAVrh.68 506 US20150315612 SEQ ID NO: 100 AAVrh.69 507 US20150315612 SEQ ID NO: 39 AAVrh.69 508 US20150315612 SEQ ID NO: 119 AAVrh.70 509 US20150315612 SEQ ID NO: 20 AAVrh.70 510 US20150315612 SEQ ID NO: 98 AAVrh.71 511 US20150315612 SEQ ID NO: 162 AAVrh.72 512 US20150315612 SEQ ID NO: 9 AAVrh.73 513 US20150159173 SEQ ID NO: 5 AAVrh.74 514 US20150159173 SEQ ID NO: 6 AAVrh.8 515 US20150159173 SEQ ID NO: 41 AAVrh.8 516 US20150315612 SEQ ID NO: 235 AAVrh.8R 517 US20150159173, WO2015168666 SEQ ID NO: 9 AAVrh.8R A586R mutant 518 WO2015168666 SEQ ID NO: 10 AAVrh.8R R533A mutant 519 WO2015168666 SEQ ID NO: 11 BAAV (bovine AAV) 520 US9193769 SEQ ID NO: 8 BAAV (bovine AAV) 521 US9193769 SEQ ID NO: 10 BAAV (bovine AAV) 522 US9193769 SEQ ID NO: 4 BAAV (bovine AAV) 523 US9193769 SEQ ID NO: 2 BAAV (bovine AAV) 524 US9193769 SEQ ID NO: 6 BAAV (bovine AAV) 525 US9193769 SEQ ID NO: 1 BAAV (bovine AAV) 526 US9193769 SEQ ID NO: 5 BAAV (bovine AAV) 527 US9193769 SEQ ID NO: 3 BAAV (bovine AAV) 528 US9193769 SEQ ID NO: 11 BAAV (bovine AAV) 529 US7427396 SEQ ID NO: 5 BAAV (bovine AAV) 530 US7427396 SEQ ID NO: 6 BAAV (bovine AAV) 531 US9193769 SEQ ID NO: 7 BAAV (bovine AAV) 532 US9193769 SEQ ID NO: 9 BNP61 AAV 533 US20150238550 SEQ ID NO: 1 BNP61 AAV 534 US20150238550 SEQ ID NO: 2 BNP62 AAV 535 US20150238550 SEQ ID NO: 3 BNP63 AAV 536 US20150238550 SEQ ID NO: 4 Goat AAV 537 US7427396 SEQ ID NO: 3 Goat AAV 538 US7427396 SEQ ID NO: 4 True AAV (ttAAV) 539 WO2015121501 SEQ ID NO: 2 AAAV (Avian AAV) 540 US9238800 SEQ ID NO: 12 AAAV (Avian AAV) 541 US9238800 SEQ ID NO: 2 AAAV (Avian AAV) 542 US9238800 SEQ ID NO: 6 AAAV (Avian AAV) 543 US9238800 SEQ ID NO: 4 AAAV (Avian AAV) 544 US9238800 SEQ ID NO: 8 AAAV (Avian AAV) 545 US9238800 SEQ ID NO: 14 AAAV (Avian AAV) 546 US9238800 SEQ ID NO: 10 AAAV (Avian AAV) 547 US9238800 SEQ ID NO: 15 AAAV (Avian AAV) 548 US9238800 SEQ ID NO: 5 AAAV (Avian AAV) 549 US9238800 SEQ ID NO: 9 AAAV (Avian AAV) 550 US9238800 SEQ ID NO: 3 AAAV (Avian AAV) 551 US9238800 SEQ ID NO: 7 AAAV (Avian AAV) 552 US9238800 SEQ ID NO: 11 AAAV (Avian AAV) 553 US9238800 SEQ ID NO: 13 AAAV (Avian AAV) 554 US9238800 SEQ ID NO: 1 AAV revamp 100-1 555 US20160017295 SEQ ID NO: 23 AAV revamp 100-1 556 US20160017295 SEQ ID NO: 11 AAV reorganization 100-2 557 US20160017295 SEQ ID NO: 37 AAV reorganization 100-2 558 US20160017295 SEQ ID NO: 29 AAV reorganization 100-3 559 US20160017295 SEQ ID NO: 24 AAV reorganization 100-3 560 US20160017295 SEQ ID NO: 12 AAV reshuffle 100-7 561 US20160017295 SEQ ID NO: 25 AAV reshuffle 100-7 562 US20160017295 SEQ ID NO: 13 AAV reshuffles 10-2 563 US20160017295 SEQ ID NO: 34 AAV reshuffles 10-2 564 US20160017295 SEQ ID NO: 26 AAV restructures 10-6 565 US20160017295 SEQ ID NO: 35 AAV restructures 10-6 566 US20160017295 SEQ ID NO: 27 AAV restructures 10-8 567 US20160017295 SEQ ID NO: 36 AAV restructures 10-8 568 US20160017295 SEQ ID NO: 28 AAV SM 100-10 569 US20160017295 SEQ ID NO: 41 AAV SM 100-10 570 US20160017295 SEQ ID NO: 33 AAV SM 100-3 571 US20160017295 SEQ ID NO: 40 AAV SM 100-3 572 US20160017295 SEQ ID NO: 32 AAV SM 10-1 573 US20160017295 SEQ ID NO: 38 AAV SM 10-1 574 US20160017295 SEQ ID NO: 30 AAV SM 10-2 575 US20160017295 SEQ ID NO: 10 AAV SM 10-2 576 US20160017295 SEQ ID NO: 22 AAV SM 10-8 577 US20160017295 SEQ ID NO: 39 AAV SM 10-8 578 US20160017295 SEQ ID NO: 31 AAVF1/HSC1 579 WO2016049230 SEQ ID NO: 20 AAVF2/HSC2 580 WO2016049230 SEQ ID NO: 21 AAVF3/HSC3 581 WO2016049230 SEQ ID NO: 22 AAVF4/HSC4 582 WO2016049230 SEQ ID NO: 23 AAVF5/HSC5 583 WO2016049230 SEQ ID NO: 25 AAVF6/HSC6 584 WO2016049230 SEQ ID NO: 24 AAVF7/HSC7 585 WO2016049230 SEQ ID NO: 27 AAVF8/HSC8 586 WO2016049230 SEQ ID NO: 28 AAVF9/HSC9 587 WO2016049230 SEQ ID NO: 29 AAVF11/HSC11 588 WO2016049230 SEQ ID NO: 26 AAVF12/HSC12 589 WO2016049230 SEQ ID NO: 30 AAVF13/HSC13 590 WO2016049230 SEQ ID NO: 31 AAVF14/HSC14 591 WO2016049230 SEQ ID NO: 32 AAVF15/HSC15 592 WO2016049230 SEQ ID NO: 33 AAVF16/HSC16 593 WO2016049230 SEQ ID NO: 34 AAVF17/HSC17 594 WO2016049230 SEQ ID NO: 35 AAVF1/HSC1 595 WO2016049230 SEQ ID NO: 2 AAVF2/HSC2 596 WO2016049230 SEQ ID NO: 3 AAVF3/HSC3 597 WO2016049230 SEQ ID NO: 5 AAVF4/HSC4 598 WO2016049230 SEQ ID NO: 6 AAVF5/HSC5 599 WO2016049230 SEQ ID NO: 11 AAVF6/HSC6 600 WO2016049230 SEQ ID NO: 7 AAVF7/HSC7 601 WO2016049230 SEQ ID NO: 8 AAVF8/HSC8 602 WO2016049230 SEQ ID NO: 9 AAVF9/HSC9 603 WO2016049230 SEQ ID NO: 10 AAVF11/HSC11 604 WO2016049230 SEQ ID NO: 4 AAVF12/HSC12 605 WO2016049230 SEQ ID NO: 12 AAVF13/HSC13 606 WO2016049230 SEQ ID NO: 14 AAVF14/HSC14 607 WO2016049230 SEQ ID NO: 15 AAVF15/HSC15 608 WO2016049230 SEQ ID NO: 16 AAVF16/HSC16 609 WO2016049230 SEQ ID NO: 17 AAVF17/HSC17 610 WO2016049230 SEQ ID NO: 13 AAV CBr-E1 611 US8734809 SEQ ID NO: 13 AAV CBr-E2 612 US8734809 SEQ ID NO: 14 AAV CBr-E3 613 US8734809 SEQ ID NO: 15 AAV CBr-E4 614 US8734809 SEQ ID NO: 16 AAV CBr-E5 615 US8734809 SEQ ID NO: 17 AAV CBr-e5 616 US8734809 SEQ ID NO: 18 AAV CBr-E6 617 US8734809 SEQ ID NO: 19 AAV CBr-E7 618 US8734809 SEQ ID NO: 20 AAV CBr-E8 619 US8734809 SEQ ID NO: 21 AAV CLv-D1 620 US8734809 SEQ ID NO: 22 AAV CLv-D2 621 US8734809 SEQ ID NO: 23 AAV CLv-D3 622 US8734809 SEQ ID NO: 24 AAV CLv-D4 623 US8734809 SEQ ID NO: 25 AAV CLv-D5 624 US8734809 SEQ ID NO: 26 AAV CLv-D6 625 US8734809 SEQ ID NO: 27 AAV CLv-D7 626 US8734809 SEQ ID NO: 28 AAV CLv-D8 627 US8734809 SEQ ID NO: 29 AAV CLv-E1 628 US8734809 SEQ ID NO: 13 AAV CLv-R1 629 US8734809 SEQ ID NO: 30 AAV CLv-R2 630 US8734809 SEQ ID NO: 31 AAV CLv-R3 631 US8734809 SEQ ID NO: 32 AAV CLv-R4 632 US8734809 SEQ ID NO: 33 AAV CLv-R5 633 US8734809 SEQ ID NO: 34 AAV CLv-R6 634 US8734809 SEQ ID NO: 35 AAV CLv-R7 635 US8734809 SEQ ID NO: 36 AAV CLv-R8 636 US8734809 SEQ ID NO: 37 AAV CLv-R9 637 US8734809 SEQ ID NO: 38 AAV CLg-F1 638 US8734809 SEQ ID NO: 39 AAV CLg-F2 639 US8734809 SEQ ID NO: 40 AAV CLg-F3 640 US8734809 SEQ ID NO: 41 AAV CLg-F4 641 US8734809 SEQ ID NO: 42 AAV CLg-F5 642 US8734809 SEQ ID NO: 43 AAV CLg-F6 643 US8734809 SEQ ID NO: 43 AAV CLg-F7 644 US8734809 SEQ ID NO: 44 AAV CLg-F8 645 US8734809 SEQ ID NO: 43 AAV CSp-1 646 US8734809 SEQ ID NO: 45 AAV CSp-10 647 US8734809 SEQ ID NO: 46 AAV CSp-11 648 US8734809 SEQ ID NO: 47 AAV CSp-2 649 US8734809 SEQ ID NO: 48 AAV CSp-3 650 US8734809 SEQ ID NO: 49 AAV CSp-4 651 US8734809 SEQ ID NO: 50 AAV CSp-6 652 US8734809 SEQ ID NO: 51 AAV CSp-7 653 US8734809 SEQ ID NO: 52 AAV CSp-8 654 US8734809 SEQ ID NO: 53 AAV CSp-9 655 US8734809 SEQ ID NO: 54 AAV CHt-2 656 US8734809 SEQ ID NO: 55 AAV CHt-3 657 US8734809 SEQ ID NO: 56 AAV CKd-1 658 US8734809 SEQ ID NO: 57 AAV CKd-10 659 US8734809 SEQ ID NO: 58 AAV CKd-2 660 US8734809 SEQ ID NO: 59 AAV CKd-3 661 US8734809 SEQ ID NO: 60 AAV CKd-4 662 US8734809 SEQ ID NO: 61 AAV CKd-6 663 US8734809 SEQ ID NO: 62 AAV CKd-7 664 US8734809 SEQ ID NO: 63 AAV CKd-8 665 US8734809 SEQ ID NO: 64 AAVCLv-1 666 US8734809 SEQ ID NO: 65 AAV CLv-12 667 US8734809 SEQ ID NO: 66 AAV CLv-13 668 US8734809 SEQ ID NO: 67 AAVCLv-2 669 US8734809 SEQ ID NO: 68 AAVCLv-3 670 US8734809 SEQ ID NO: 69 AAVCLv-4 671 US8734809 SEQ ID NO: 70 AAVCLv-6 672 US8734809 SEQ ID NO: 71 AAVCLv-8 673 US8734809 SEQ ID NO: 72 AAV CKd-B1 674 US8734809 SEQ ID NO: 73 AAV CKd-B2 675 US8734809 SEQ ID NO: 74 AAV CKd-B3 676 US8734809 SEQ ID NO: 75 AAV CKd-B4 677 US8734809 SEQ ID NO: 76 AAV CKd-B5 678 US8734809 SEQ ID NO: 77 AAV CKd-B6 679 US8734809 SEQ ID NO: 78 AAV CKd-B7 680 US8734809 SEQ ID NO: 79 AAV CKd-B8 681 US8734809 SEQ ID NO: 80 AAV CKd-H1 682 US8734809 SEQ ID NO: 81 AAV CKd-H2 683 US8734809 SEQ ID NO: 82 AAV CKd-H3 684 US8734809 SEQ ID NO: 83 AAV CKd-H4 685 US8734809 SEQ ID NO: 84 AAV CKd-H5 686 US8734809 SEQ ID NO: 85 AAV CKd-H6 687 US8734809 SEQ ID NO: 77 AAV CHt-1 688 US8734809 SEQ ID NO: 86 AAVCLv1-1 689 US8734809 SEQ ID NO: 171 AAVCLv1-2 690 US8734809 SEQ ID NO: 172 AAVCLv1-3 691 US8734809 SEQ ID NO: 173 AAVCLv1-4 692 US8734809 SEQ ID NO: 174 AAV Clv1-7 693 US8734809 SEQ ID NO: 175 AAV Clv1-8 694 US8734809 SEQ ID NO: 176 AAV Clv1-9 695 US8734809 SEQ ID NO: 177 AAV Clv1-10 696 US8734809 SEQ ID NO: 178 AAV.VR-355 697 US8734809 SEQ ID NO: 181 AAV.hu.48R3 698 US8734809 SEQ ID NO: 183 AAV CBr-E1 699 US8734809 SEQ ID NO: 87 AAV CBr-E2 700 US8734809 SEQ ID NO: 88 AAV CBr-E3 701 US8734809 SEQ ID NO: 89 AAV CBr-E4 702 US8734809 SEQ ID NO: 90 AAV CBr-E5 703 US8734809 SEQ ID NO: 91 AAV CBr-e5 704 US8734809 SEQ ID NO: 92 AAV CBr-E6 705 US8734809 SEQ ID NO: 93 AAV CBr-E7 706 US8734809 SEQ ID NO: 94 AAV CBr-E8 707 US8734809 SEQ ID NO: 95 AAV CLv-D1 708 US8734809 SEQ ID NO: 96 AAV CLv-D2 709 US8734809 SEQ ID NO: 97 AAV CLv-D3 710 US8734809 SEQ ID NO: 98 AAV CLv-D4 711 US8734809 SEQ ID NO: 99 AAV CLv-D5 712 US8734809 SEQ ID NO: 100 AAV CLv-D6 713 US8734809 SEQ ID NO: 101 AAV CLv-D7 714 US8734809 SEQ ID NO: 102 AAV CLv-D8 715 US8734809 SEQ ID NO: 103 AAV CLv-E1 716 US8734809 SEQ ID NO: 87 AAV CLv-R1 717 US8734809 SEQ ID NO: 104 AAV CLv-R2 718 US8734809 SEQ ID NO: 105 AAV CLv-R3 719 US8734809 SEQ ID NO: 106 AAV CLv-R4 720 US8734809 SEQ ID NO: 107 AAV CLv-R5 721 US8734809 SEQ ID NO: 108 AAV CLv-R6 722 US8734809 SEQ ID NO: 109 AAV CLv-R7 723 US8734809 SEQ ID NO: 110 AAV CLv-R8 724 US8734809 SEQ ID NO: 111 AAV CLv-R9 725 US8734809 SEQ ID NO: 112 AAV CLg-F1 726 US8734809 SEQ ID NO: 113 AAV CLg-F2 727 US8734809 SEQ ID NO: 114 AAV CLg-F3 728 US8734809 SEQ ID NO: 115 AAV CLg-F4 729 US8734809 SEQ ID NO: 116 AAV CLg-F5 730 US8734809 SEQ ID NO: 117 AAV CLg-F6 731 US8734809 SEQ ID NO: 117 AAV CLg-F7 732 US8734809 SEQ ID NO: 118 AAV CLg-F8 733 US8734809 SEQ ID NO: 117 AAV CSp-1 734 US8734809 SEQ ID NO: 119 AAV CSp-10 735 US8734809 SEQ ID NO: 120 AAV CSp-11 736 US8734809 SEQ ID NO: 121 AAV CSp-2 737 US8734809 SEQ ID NO: 122 AAV CSp-3 738 US8734809 SEQ ID NO: 123 AAV CSp-4 739 US8734809 SEQ ID NO: 124 AAV CSp-6 740 US8734809 SEQ ID NO: 125 AAV CSp-7 741 US8734809 SEQ ID NO: 126 AAV CSp-8 742 US8734809 SEQ ID NO: 127 AAV CSp-9 743 US8734809 SEQ ID NO: 128 AAV CHt-2 744 US8734809 SEQ ID NO: 129 AAV CHt-3 745 US8734809 SEQ ID NO: 130 AAV CKd-1 746 US8734809 SEQ ID NO: 131 AAV CKd-10 747 US8734809 SEQ ID NO: 132 AAV CKd-2 748 US8734809 SEQ ID NO: 133 AAV CKd-3 749 US8734809 SEQ ID NO: 134 AAV CKd-4 750 US8734809 SEQ ID NO: 135 AAV CKd-6 751 US8734809 SEQ ID NO: 136 AAV CKd-7 752 US8734809 SEQ ID NO: 137 AAV CKd-8 753 US8734809 SEQ ID NO: 138 AAVCLv-1 754 US8734809 SEQ ID NO: 139 AAV CLv-12 755 US8734809 SEQ ID NO: 140 AAV CLv-13 756 US8734809 SEQ ID NO: 141 AAVCLv-2 757 US8734809 SEQ ID NO: 142 AAVCLv-3 758 US8734809 SEQ ID NO: 143 AAVCLv-4 759 US8734809 SEQ ID NO: 144 AAVCLv-6 760 US8734809 SEQ ID NO: 145 AAVCLv-8 761 US8734809 SEQ ID NO: 146 AAV CKd-B1 762 US8734809 SEQ ID NO: 147 AAV CKd-B2 763 US8734809 SEQ ID NO: 148 AAV CKd-B3 764 US8734809 SEQ ID NO: 149 AAV CKd-B4 765 US8734809 SEQ ID NO: 150 AAV CKd-B5 766 US8734809 SEQ ID NO: 151 AAV CKd-B6 767 US8734809 SEQ ID NO: 152 AAV CKd-B7 768 US8734809 SEQ ID NO: 153 AAV CKd-B8 769 US8734809 SEQ ID NO: 154 AAV CKd-H1 770 US8734809 SEQ ID NO: 155 AAV CKd-H2 771 US8734809 SEQ ID NO: 156 AAV CKd-H3 772 US8734809 SEQ ID NO: 157 AAV CKd-H4 773 US8734809 SEQ ID NO: 158 AAV CKd-H5 774 US8734809 SEQ ID NO: 159 AAV CKd-H6 775 US8734809 SEQ ID NO: 151 AAV CHt-1 776 US8734809 SEQ ID NO: 160 AAV CHt-P2 777 WO2016065001 SEQ ID NO: 1 AAV CHt-P5 778 WO2016065001 SEQ ID NO: 2 AAV CHt-P9 779 WO2016065001 SEQ ID NO: 3 AAV CBr-7.1 780 WO2016065001 SEQ ID NO: 4 AAV CBr-7.2 781 WO2016065001 SEQ ID NO: 5 AAV CBr-7.3 782 WO2016065001 SEQ ID NO: 6 AAV CBr-7.4 783 WO2016065001 SEQ ID NO: 7 AAV CBr-7.5 784 WO2016065001 SEQ ID NO: 8 AAV CBr-7.7 785 WO2016065001 SEQ ID NO: 9 AAV CBr-7.8 786 WO2016065001 SEQ ID NO: 10 AAV CBr-7.10 787 WO2016065001 SEQ ID NO: 11 AAV CKd-N3 788 WO2016065001 SEQ ID NO: 12 AAV CKd-N4 789 WO2016065001 SEQ ID NO: 13 AAV CKd-N9 790 WO2016065001 SEQ ID NO: 14 AAV CLv-L4 791 WO2016065001 SEQ ID NO: 15 AAV CLv-L5 792 WO2016065001 SEQ ID NO: 16 AAV CLv-L6 793 WO2016065001 SEQ ID NO: 17 AAV CLv-K1 794 WO2016065001 SEQ ID NO: 18 AAV CLv-K3 795 WO2016065001 SEQ ID NO: 19 AAV CLv-K6 796 WO2016065001 SEQ ID NO: 20 AAV CLv-M1 797 WO2016065001 SEQ ID NO: 21 AAV CLv-M11 798 WO2016065001 SEQ ID NO: 22 AAV CLv-M2 799 WO2016065001 SEQ ID NO: 23 AAV CLv-M5 800 WO2016065001 SEQ ID NO: 24 AAV CLv-M6 801 WO2016065001 SEQ ID NO: 25 AAV CLv-M7 802 WO2016065001 SEQ ID NO: 26 AAV CLv-M8 803 WO2016065001 SEQ ID NO: 27 AAV CLv-M9 804 WO2016065001 SEQ ID NO: 28 AAV CHt-P1 805 WO2016065001 SEQ ID NO: 29 AAV CHt-P6 806 WO2016065001 SEQ ID NO: 30 AAV CHt-P8 807 WO2016065001 SEQ ID NO: 31 AAV CHt-6.1 808 WO2016065001 SEQ ID NO: 32 AAV CHt-6.10 809 WO2016065001 SEQ ID NO: 33 AAV CHt-6.5 810 WO2016065001 SEQ ID NO: 34 AAV CHt-6.6 811 WO2016065001 SEQ ID NO: 35 AAV CHt-6.7 812 WO2016065001 SEQ ID NO: 36 AAV CHt-6.8 813 WO2016065001 SEQ ID NO: 37 AAV CSp-8.10 814 WO2016065001 SEQ ID NO: 38 AAV CSp-8.2 815 WO2016065001 SEQ ID NO: 39 AAV CSp-8.4 816 WO2016065001 SEQ ID NO: 40 AAV CSp-8.5 817 WO2016065001 SEQ ID NO: 41 AAV CSp-8.6 818 WO2016065001 SEQ ID NO: 42 AAV CSp-8.7 819 WO2016065001 SEQ ID NO: 43 AAV CSp-8.8 820 WO2016065001 SEQ ID NO: 44 AAV CSp-8.9 821 WO2016065001 SEQ ID NO: 45 AAV CBr-B7.3 822 WO2016065001 SEQ ID NO: 46 AAV CBr-B7.4 823 WO2016065001 SEQ ID NO: 47 AAV3B 824 WO2016065001 SEQ ID NO: 48 AAV4 825 WO2016065001 SEQ ID NO: 49 AAV5 826 WO2016065001 SEQ ID NO: 50 AAV CHt-P2 827 WO2016065001 SEQ ID NO: 51 AAV CHt-P5 828 WO2016065001 SEQ ID NO: 52 AAV CHt-P9 829 WO2016065001 SEQ ID NO: 53 AAV CBr-7.1 830 WO2016065001 SEQ ID NO: 54 AAV CBr-7.2 831 WO2016065001 SEQ ID NO: 55 AAV CBr-7.3 832 WO2016065001 SEQ ID NO: 56 AAV CBr-7.4 833 WO2016065001 SEQ ID NO: 57 AAV CBr-7.5 834 WO2016065001 SEQ ID NO: 58 AAV CBr-7.7 835 WO2016065001 SEQ ID NO: 59 AAV CBr-7.8 836 WO2016065001 SEQ ID NO: 60 AAV CBr-7.10 837 WO2016065001 SEQ ID NO: 61 AAV CKd-N3 838 WO2016065001 SEQ ID NO: 62 AAV CKd-N4 839 WO2016065001 SEQ ID NO: 63 AAV CKd-N9 840 WO2016065001 SEQ ID NO: 64 AAV CLv-L4 841 WO2016065001 SEQ ID NO: 65 AAV CLv-L5 842 WO2016065001 SEQ ID NO: 66 AAV CLv-L6 843 WO2016065001 SEQ ID NO: 67 AAV CLv-K1 844 WO2016065001 SEQ ID NO: 68 AAV CLv-K3 845 WO2016065001 SEQ ID NO: 69 AAV CLv-K6 846 WO2016065001 SEQ ID NO: 70 AAV CLv-M1 847 WO2016065001 SEQ ID NO: 71 AAV CLv-M11 848 WO2016065001 SEQ ID NO: 72 AAV CLv-M2 849 WO2016065001 SEQ ID NO: 73 AAV CLv-M5 850 WO2016065001 SEQ ID NO: 74 AAV CLv-M6 851 WO2016065001 SEQ ID NO: 75 AAV CLv-M7 852 WO2016065001 SEQ ID NO: 76 AAV CLv-M8 853 WO2016065001 SEQ ID NO: 77 AAV CLv-M9 854 WO2016065001 SEQ ID NO: 78 AAV CHt-P1 855 WO2016065001 SEQ ID NO: 79 AAV CHt-P6 856 WO2016065001 SEQ ID NO: 80 AAV CHt-P8 857 WO2016065001 SEQ ID NO: 81 AAV CHt-6.1 858 WO2016065001 SEQ ID NO: 82 AAV CHt-6.10 859 WO2016065001 SEQ ID NO: 83 AAV CHt-6.5 860 WO2016065001 SEQ ID NO: 84 AAV CHt-6.6 861 WO2016065001 SEQ ID NO: 85 AAV CHt-6.7 862 WO2016065001 SEQ ID NO: 86 AAV CHt-6.8 863 WO2016065001 SEQ ID NO: 87 AAV CSp-8.10 864 WO2016065001 SEQ ID NO: 88 AAV CSp-8.2 865 WO2016065001 SEQ ID NO: 89 AAV CSp-8.4 866 WO2016065001 SEQ ID NO: 90 AAV CSp-8.5 867 WO2016065001 SEQ ID NO: 91 AAV CSp-8.6 868 WO2016065001 SEQ ID NO: 92 AAV CSp-8.7 869 WO2016065001 SEQ ID NO: 93 AAV CSp-8.8 870 WO2016065001 SEQ ID NO: 94 AAV CSp-8.9 871 WO2016065001 SEQ ID NO: 95 AAV CBr-B7.3 872 WO2016065001 SEQ ID NO: 96 AAV CBr-B7.4 873 WO2016065001 SEQ ID NO: 97 AAV3B 874 WO2016065001 SEQ ID NO: 98 AAV4 875 WO2016065001 SEQ ID NO: 99 AAV5 876 WO2016065001 SEQ ID NO: 100 GPV 877 US9624274B2 SEQ ID NO: 192 B19 878 US9624274B2 SEQ ID NO: 193 MVM 879 US9624274B2 SEQ ID NO: 194 FPV 880 US9624274B2 SEQ ID NO: 195 CPV 881 US9624274B2 SEQ ID NO: 196 AAV6 882 US9546112B2 SEQ ID NO: 5 AAV6 883 US9457103B2 SEQ ID NO: 1 AAV2 884 US9457103B2 SEQ ID NO: 2 ShH10 885 US9457103B2 SEQ ID NO: 3 ShH13 886 US9457103B2 SEQ ID NO: 4 ShH10 887 US9457103B2 SEQ ID NO: 5 ShH10 888 US9457103B2 SEQ ID NO: 6 ShH10 889 US9457103B2 SEQ ID NO: 7 ShH10 890 US9457103B2 SEQ ID NO: 8 ShH10 891 US9457103B2 SEQ ID NO: 9 rh74 892 US9434928B2 SEQ ID NO: 1, US2015023924A1 SEQ ID NO: 2 rh74 893 US9434928B2 SEQ ID NO: 2, US2015023924A1 SEQ ID NO: 1 AAV8 894 US9434928B2 SEQ ID NO: 4 rh74 895 US9434928B2 SEQ ID NO: 5 rh74 (RHM4-1) 896 US2015023924A1 SEQ ID NO: 5, US20160375110A1 SEQ ID NO: 4 rh74 (RHM15-1) 897 US2015023924A1 SEQ ID NO: 6, US20160375110A1 SEQ ID NO: 5 rh74 (RHM15-2) 898 US2015023924A1 SEQ ID NO: 7, US20160375110A1 SEQ ID NO: 6 rh74 (RHM15-3/RHM15-5) 899 US2015023924A1 SEQ ID NO: 8, US20160375110A1 SEQ ID NO: 7 rh74 (RHM15-4) 900 US2015023924A1 SEQ ID NO: 9, US20160375110A1 SEQ ID NO: 8 rh74 (RHM15-6) 901 US2015023924A1 SEQ ID NO: 10, US20160375110A1 SEQ ID NO: 9 rh74 (RHM4-1) 902 US2015023924A1 SEQ ID NO: 11 rh74 (RHM15-1) 903 US2015023924A1 SEQ ID NO: 12 rh74 (RHM15-2) 904 US2015023924A1 SEQ ID NO: 13 rh74 (RHM15-3/RHM15-5) 905 US2015023924A1 SEQ ID NO: 14 rh74 (RHM15-4) 906 US2015023924A1 SEQ ID NO: 15 rh74 (RHM15-6) 907 US2015023924A1 SEQ ID NO: 16 AAV2 (contains lung-specific peptides) 908 US20160175389A1 SEQ ID NO: 9 AAV2 (contains lung-specific peptides) 909 US20160175389A1 SEQ ID NO: 10 Anc80 910 US20170051257A1 SEQ ID NO: 1 Anc80 911 US20170051257A1 SEQ ID NO: 2 Anc81 912 US20170051257A1 SEQ ID NO: 3 Anc80 913 US20170051257A1 SEQ ID NO: 4 Anc82 914 US20170051257A1 SEQ ID NO: 5 Anc82 915 US20170051257A1 SEQ ID NO: 6 Anc83 916 US20170051257A1 SEQ ID NO: 7 Anc83 917 US20170051257A1 SEQ ID NO: 8 Anc84 918 US20170051257A1 SEQ ID NO: 9 Anc84 919 US20170051257A1 SEQ ID NO: 10 Anc94 920 US20170051257A1 SEQ ID NO: 11 Anc94 921 US20170051257A1 SEQ ID NO: 12 Anc113 922 US20170051257A1 SEQ ID NO: 13 Anc113 923 US20170051257A1 SEQ ID NO: 14 Anc126 924 US20170051257A1 SEQ ID NO: 15 Anc126 925 US20170051257A1 SEQ ID NO: 16 Anc127 926 US20170051257A1 SEQ ID NO: 17 Anc127 927 US20170051257A1 SEQ ID NO: 18 Anc80L27 928 US20170051257A1 SEQ ID NO: 19 Anc80L59 929 US20170051257A1 SEQ ID NO: 20 Anc80L60 930 US20170051257A1 SEQ ID NO: 21 Anc80L62 931 US20170051257A1 SEQ ID NO: 22 Anc80L65 932 US20170051257A1 SEQ ID NO: 23 Anc80L33 933 US20170051257A1 SEQ ID NO: 24 Anc80L36 934 US20170051257A1 SEQ ID NO: 25 Anc80L44 935 US20170051257A1 SEQ ID NO: 26 Anc80L1 936 US20170051257A1 SEQ ID NO: 35 Anc80L1 937 US20170051257A1 SEQ ID NO: 36 AAV-X1 938 US8283151B2 SEQ ID NO: 11 AAV-X1b 939 US8283151B2 SEQ ID NO: 12 AAV-X5 940 US8283151B2 SEQ ID NO: 13 AAV-X19 941 US8283151B2 SEQ ID NO: 14 AAV-X21 942 US8283151B2 SEQ ID NO: 15 AAV-X22 943 US8283151B2 SEQ ID NO: 16 AAV-X23 944 US8283151B2 SEQ ID NO: 17 AAV-X24 945 US8283151B2 SEQ ID NO: 18 AAV-X25 946 US8283151B2 SEQ ID NO: 19 AAV-X26 947 US8283151B2 SEQ ID NO: 20 AAV-X1 948 US8283151B2 SEQ ID NO: 21 AAV-X1b 949 US8283151B2 SEQ ID NO: 22 AAV-X5 950 US8283151B2 SEQ ID NO: 23 AAV-X19 951 US8283151B2 SEQ ID NO: 24 AAV-X21 952 US8283151B2 SEQ ID NO: 25 AAV-X22 953 US8283151B2 SEQ ID NO: 26 AAV-X23 954 US8283151B2 SEQ ID NO: 27 AAV-X24 955 US8283151B2 SEQ ID NO: 28 AAV-X25 956 US8283151B2 SEQ ID NO: 29 AAV-X26 957 US8283151B2 SEQ ID NO: 30 AAVrh8 958 WO2016054554A1 SEQ ID NO: 8 AAVrh8VP2FC5 959 WO2016054554A1 SEQ ID NO: 9 AAVrh8VP2FC44 960 WO2016054554A1 SEQ ID NO: 10 AAVrh8VP2ApoB100 961 WO2016054554A1 SEQ ID NO: 11 AAVrh8VP2RVG 962 WO2016054554A1 SEQ ID NO: 12 AAVrh8VP2Angiopep-2 VP2 963 WO2016054554A1 SEQ ID NO: 13 AAV9.47VP1.3 964 WO2016054554A1 SEQ ID NO: 14 AAV9.47VP2ICAMg3 965 WO2016054554A1 SEQ ID NO: 15 AAV9.47VP2RVG 966 WO2016054554A1 SEQ ID NO: 16 AAV9.47VP2Angiopep-2 967 WO2016054554A1 SEQ ID NO: 17 AAV9.47VP2A-String 968 WO2016054554A1 SEQ ID NO: 18 AAVrh8VP2FC5 VP2 969 WO2016054554A1 SEQ ID NO: 19 AAVrh8VP2FC44 VP2 970 WO2016054554A1 SEQ ID NO: 20 AAVrh8VP2ApoB100 VP2 971 WO2016054554A1 SEQ ID NO: 21 AAVrh8VP2RVG VP2 972 WO2016054554A1 SEQ ID NO: 22 AAVrh8VP2Angiopep-2 VP2 973 WO2016054554A1 SEQ ID NO: 23 AAV9.47VP2ICAMg3 VP2 974 WO2016054554A1 SEQ ID NO: 24 AAV9.47VP2RVG VP2 975 WO2016054554A1 SEQ ID NO: 25 AAV9.47VP2Angiopep-2 VP2 976 WO2016054554A1 SEQ ID NO: 26 AAV9.47VP2A-String VP2 977 WO2016054554A1 SEQ ID NO: 27 rAAV-B1 978 WO2016054557A1 SEQ ID NO: 1 rAAV-B2 979 WO2016054557A1 SEQ ID NO: 2 rAAV-B3 980 WO2016054557A1 SEQ ID NO: 3 rAAV-B4 981 WO2016054557A1 SEQ ID NO: 4 rAAV-B1 982 WO2016054557A1 SEQ ID NO: 5 rAAV-B2 983 WO2016054557A1 SEQ ID NO: 6 rAAV-B3 984 WO2016054557A1 SEQ ID NO: 7 rAAV-B4 985 WO2016054557A1 SEQ ID NO: 8 rAAV-L1 986 WO2016054557A1 SEQ ID NO: 9 rAAV-L2 987 WO2016054557A1 SEQ ID NO: 10 rAAV-L3 988 WO2016054557A1 SEQ ID NO: 11 rAAV-L4 989 WO2016054557A1 SEQ ID NO: 12 rAAV-L1 990 WO2016054557A1 SEQ ID NO: 13 rAAV-L2 991 WO2016054557A1 SEQ ID NO: 14 rAAV-L3 992 WO2016054557A1 SEQ ID NO: 15 rAAV-L4 993 WO2016054557A1 SEQ ID NO: 16 AAV9 994 WO2016073739A1 SEQ ID NO: 3 rAAV 995 WO2016081811A1 SEQ ID NO: 1 rAAV 996 WO2016081811A1 SEQ ID NO: 2 rAAV 997 WO2016081811A1 SEQ ID NO: 3 rAAV 998 WO2016081811A1 SEQ ID NO: 4 rAAV 999 WO2016081811A1 SEQ ID NO: 5 rAAV 1000 WO2016081811A1 SEQ ID NO: 6 rAAV 1001 WO2016081811A1 SEQ ID NO: 7 rAAV 1002 WO2016081811A1 SEQ ID NO: 8 rAAV 1003 WO2016081811A1 SEQ ID NO: 9 rAAV 1004 WO2016081811A1 SEQ ID NO: 10 rAAV 1005 WO2016081811A1 SEQ ID NO: 11 rAAV 1006 WO2016081811A1 SEQ ID NO: 12 rAAV 1007 WO2016081811A1 SEQ ID NO: 13 rAAV 1008 WO2016081811A1 SEQ ID NO: 14 rAAV 1009 WO2016081811A1 SEQ ID NO: 15 rAAV 1010 WO2016081811A1 SEQ ID NO: 16 rAAV 1011 WO2016081811A1 SEQ ID NO: 17 rAAV 1012 WO2016081811A1 SEQ ID NO: 18 rAAV 1013 WO2016081811A1 SEQ ID NO: 19 rAAV 1014 WO2016081811A1 SEQ ID NO: 20 rAAV 1015 WO2016081811A1 SEQ ID NO: 21 rAAV 1016 WO2016081811A1 SEQ ID NO: 22 rAAV 1017 WO2016081811A1 SEQ ID NO: 23 rAAV 1018 WO2016081811A1 SEQ ID NO: 24 rAAV 1019 WO2016081811A1 SEQ ID NO: 25 rAAV 1020 WO2016081811A1 SEQ ID NO: 26 rAAV 1021 WO2016081811A1 SEQ ID NO: 27 rAAV 1022 WO2016081811A1 SEQ ID NO: 28 rAAV 1023 WO2016081811A1 SEQ ID NO: 29 rAAV 1024 WO2016081811A1 SEQ ID NO: 30 rAAV 1025 WO2016081811A1 SEQ ID NO: 31 rAAV 1026 WO2016081811A1 SEQ ID NO: 32 rAAV 1027 WO2016081811A1 SEQ ID NO: 33 rAAV 1028 WO2016081811A1 SEQ ID NO: 34 rAAV 1029 WO2016081811A1 SEQ ID NO: 35 rAAV 1030 WO2016081811A1 SEQ ID NO: 36 rAAV 1031 WO2016081811A1 SEQ ID NO: 37 rAAV 1032 WO2016081811A1 SEQ ID NO: 38 rAAV 1033 WO2016081811A1 SEQ ID NO: 39 rAAV 1034 WO2016081811A1 SEQ ID NO: 40 rAAV 1035 WO2016081811A1 SEQ ID NO: 41 rAAV 1036 WO2016081811A1 SEQ ID NO: 42 rAAV 1037 WO2016081811A1 SEQ ID NO: 43 rAAV 1038 WO2016081811A1 SEQ ID NO: 44 rAAV 1039 WO2016081811A1 SEQ ID NO: 45 rAAV 1040 WO2016081811A1 SEQ ID NO: 46 rAAV 1041 WO2016081811A1 SEQ ID NO: 47 rAAV 1042 WO2016081811A1 SEQ ID NO: 48 rAAV 1043 WO2016081811A1 SEQ ID NO: 49 rAAV 1044 WO2016081811A1 SEQ ID NO: 50 rAAV 1045 WO2016081811A1 SEQ ID NO: 51 rAAV 1046 WO2016081811A1 SEQ ID NO: 52 rAAV 1047 WO2016081811A1 SEQ ID NO: 53 rAAV 1048 WO2016081811A1 SEQ ID NO: 54 rAAV 1049 WO2016081811A1 SEQ ID NO: 55 rAAV 1050 WO2016081811A1 SEQ ID NO: 56 rAAV 1051 WO2016081811A1 SEQ ID NO: 57 rAAV 1052 WO2016081811A1 SEQ ID NO: 58 rAAV 1053 WO2016081811A1 SEQ ID NO: 59 rAAV 1054 WO2016081811A1 SEQ ID NO: 60 rAAV 1055 WO2016081811A1 SEQ ID NO: 61 rAAV 1056 WO2016081811A1 SEQ ID NO: 62 rAAV 1057 WO2016081811A1 SEQ ID NO: 63 rAAV 1058 WO2016081811A1 SEQ ID NO: 64 rAAV 1059 WO2016081811A1 SEQ ID NO: 65 rAAV 1060 WO2016081811A1 SEQ ID NO: 66 rAAV 1061 WO2016081811A1 SEQ ID NO: 67 rAAV 1062 WO2016081811A1 SEQ ID NO: 68 rAAV 1063 WO2016081811A1 SEQ ID NO: 69 rAAV 1064 WO2016081811A1 SEQ ID NO: 70 rAAV 1065 WO2016081811A1 SEQ ID NO: 71 rAAV 1066 WO2016081811A1 SEQ ID NO: 72 rAAV 1067 WO2016081811A1 SEQ ID NO: 73 rAAV 1068 WO2016081811A1 SEQ ID NO: 74 rAAV 1069 WO2016081811A1 SEQ ID NO: 75 rAAV 1070 WO2016081811A1 SEQ ID NO: 76 rAAV 1071 WO2016081811A1 SEQ ID NO: 77 rAAV 1072 WO2016081811A1 SEQ ID NO: 78 rAAV 1073 WO2016081811A1 SEQ ID NO: 79 rAAV 1074 WO2016081811A1 SEQ ID NO: 80 rAAV 1075 WO2016081811A1 SEQ ID NO: 81 rAAV 1076 WO2016081811A1 SEQ ID NO: 82 rAAV 1077 WO2016081811A1 SEQ ID NO: 83 rAAV 1078 WO2016081811A1 SEQ ID NO: 84 rAAV 1079 WO2016081811A1 SEQ ID NO: 85 rAAV 1080 WO2016081811A1 SEQ ID NO: 86 rAAV 1081 WO2016081811A1 SEQ ID NO: 87 rAAV 1082 WO2016081811A1 SEQ ID NO: 88 rAAV 1083 WO2016081811A1 SEQ ID NO: 89 rAAV 1084 WO2016081811A1 SEQ ID NO: 90 rAAV 1085 WO2016081811A1 SEQ ID NO: 91 rAAV 1086 WO2016081811A1 SEQ ID NO: 92 rAAV 1087 WO2016081811A1 SEQ ID NO: 93 rAAV 1088 WO2016081811A1 SEQ ID NO: 94 rAAV 1089 WO2016081811A1 SEQ ID NO: 95 rAAV 1090 WO2016081811A1 SEQ ID NO: 96 rAAV 1091 WO2016081811A1 SEQ ID NO: 97 rAAV 1092 WO2016081811A1 SEQ ID NO: 98 rAAV 1093 WO2016081811A1 SEQ ID NO: 99 rAAV 1094 WO2016081811A1 SEQ ID NO: 100 rAAV 1095 WO2016081811A1 SEQ ID NO: 101 rAAV 1096 WO2016081811A1 SEQ ID NO: 102 rAAV 1097 WO2016081811A1 SEQ ID NO: 103 rAAV 1098 WO2016081811A1 SEQ ID NO: 104 rAAV 1099 WO2016081811A1 SEQ ID NO: 105 rAAV 1100 WO2016081811A1 SEQ ID NO: 106 rAAV 1101 WO2016081811A1 SEQ ID NO: 107 rAAV 1102 WO2016081811A1 SEQ ID NO: 108 rAAV 1103 WO2016081811A1 SEQ ID NO: 109 rAAV 1104 WO2016081811A1 SEQ ID NO: 110 rAAV 1105 WO2016081811A1 SEQ ID NO: 111 rAAV 1106 WO2016081811A1 SEQ ID NO: 112 rAAV 1107 WO2016081811A1 SEQ ID NO: 113 rAAV 1108 WO2016081811A1 SEQ ID NO: 114 rAAV 1109 WO2016081811A1 SEQ ID NO: 115 rAAV 1110 WO2016081811A1 SEQ ID NO: 116 rAAV 1111 WO2016081811A1 SEQ ID NO: 117 rAAV 1112 WO2016081811A1 SEQ ID NO: 118 rAAV 1113 WO2016081811A1 SEQ ID NO: 119 rAAV 1114 WO2016081811A1 SEQ ID NO: 120 rAAV 1115 WO2016081811A1 SEQ ID NO: 121 rAAV 1116 WO2016081811A1 SEQ ID NO: 122 rAAV 1117 WO2016081811A1 SEQ ID NO: 123 rAAV 1118 WO2016081811A1 SEQ ID NO: 124 rAAV 1119 WO2016081811A1 SEQ ID NO: 125 rAAV 1120 WO2016081811A1 SEQ ID NO: 126 rAAV 1121 WO2016081811A1 SEQ ID NO: 127 rAAV 1122 WO2016081811A1 SEQ ID NO: 128 AAV8 E532K 1123 WO2016081811A1 SEQ ID NO: 133 AAV8 E532K 1124 WO2016081811A1 SEQ ID NO: 134 rAAV4 1125 WO2016115382A1 SEQ ID NO: 2 rAAV4 1126 WO2016115382A1 SEQ ID NO: 3 rAAV4 1127 WO2016115382A1 SEQ ID NO: 4 rAAV4 1128 WO2016115382A1 SEQ ID NO: 5 rAAV4 1129 WO2016115382A1 SEQ ID NO: 6 rAAV4 1130 WO2016115382A1 SEQ ID NO: 7 rAAV4 1131 WO2016115382A1 SEQ ID NO: 8 rAAV4 1132 WO2016115382A1 SEQ ID NO: 9 rAAV4 1133 WO2016115382A1 SEQ ID NO: 10 rAAV4 1134 WO2016115382A1 SEQ ID NO: 11 rAAV4 1135 WO2016115382A1 SEQ ID NO: 12 rAAV4 1136 WO2016115382A1 SEQ ID NO: 13 rAAV4 1137 WO2016115382A1 SEQ ID NO: 14 rAAV4 1138 WO2016115382A1 SEQ ID NO: 15 rAAV4 1139 WO2016115382A1 SEQ ID NO: 16 rAAV4 1140 WO2016115382A1 SEQ ID NO: 17 rAAV4 1141 WO2016115382A1 SEQ ID NO: 18 rAAV4 1142 WO2016115382A1 SEQ ID NO: 19 rAAV4 1143 WO2016115382A1 SEQ ID NO: 20 rAAV4 1144 WO2016115382A1 SEQ ID NO: 21 AAV11 1145 WO2016115382A1 SEQ ID NO: 22 AAV12 1146 WO2016115382A1 SEQ ID NO: 23 rh32 1147 WO2016115382A1 SEQ ID NO: 25 rh33 1148 WO2016115382A1 SEQ ID NO: 26 rh34 1149 WO2016115382A1 SEQ ID NO: 27 rAAV4 1150 WO2016115382A1 SEQ ID NO: 28 rAAV4 1151 WO2016115382A1 SEQ ID NO: 29 rAAV4 1152 WO2016115382A1 SEQ ID NO: 30 rAAV4 1153 WO2016115382A1 SEQ ID NO: 31 rAAV4 1154 WO2016115382A1 SEQ ID NO: 32 rAAV4 1155 WO2016115382A1 SEQ ID NO: 33 AAV2/8 1156 WO2016131981A1 SEQ ID NO: 47 AAV2/8 1157 WO2016131981A1 SEQ ID NO: 48 Previous generation AAV 1158 WO2016154344A1 SEQ ID NO: 7 Previous generation AAV variant C4 1159 WO2016154344A1 SEQ ID NO: 13 Previous generation AAV variant C7 1160 WO2016154344A1 SEQ ID NO: 14 Previous generation AAV variant G4 1161 WO2016154344A1 SEQ ID NO: 15 Common amino acid sequences of previous generation AAV variants C4, C7 and G4 1162 WO2016154344A1 SEQ ID NO: 16 Common amino acid sequences of previous generation AAV variants C4 and C7 1163 WO2016154344A1 SEQ ID NO: 17 AAV8 (has AAV2 phospholipase domain) 1164 WO2016150403A1 SEQ ID NO: 13 AAV VR-942n 1165 US20160289275A1 SEQ ID NO: 10 AAV5-A (M569V) 1166 US20160289275A1 SEQ ID NO: 13 AAV5-A (M569V) 1167 US20160289275A1 SEQ ID NO: 14 AAV5-A (Y585V) 1168 US20160289275A1 SEQ ID NO: 16 AAV5-A (Y585V) 1169 US20160289275A1 SEQ ID NO: 17 AAV5-A (L587T) 1170 US20160289275A1 SEQ ID NO: 19 AAV5-A (L587T) 1171 US20160289275A1 SEQ ID NO: 20 AAV5-A (Y585V/L587T) 1172 US20160289275A1 SEQ ID NO: 22 AAV5-A (Y585V/L587T) 1173 US20160289275A1 SEQ ID NO: 23 AAV5-B (D652A) 1174 US20160289275A1 SEQ ID NO: 25 AAV5-B (D652A) 1175 US20160289275A1 SEQ ID NO: 26 AAV5-B (T362M) 1176 US20160289275A1 SEQ ID NO: 28 AAV5-B (T362M) 1177 US20160289275A1 SEQ ID NO: 29 AAV5-B (Q359D) 1178 US20160289275A1 SEQ ID NO: 31 AAV5-B (Q359D) 1179 US20160289275A1 SEQ ID NO: 32 AAV5-B (E350Q) 1180 US20160289275A1 SEQ ID NO: 34 AAV5-B (E350Q) 1181 US20160289275A1 SEQ ID NO: 35 AAV5-B (P533S) 1182 US20160289275A1 SEQ ID NO: 37 AAV5-B (P533S) 1183 US20160289275A1 SEQ ID NO: 38 AAV5-B (P533G) 1184 US20160289275A1 SEQ ID NO: 40 AAV5-B (P533G) 1185 US20160289275A1 SEQ ID NO: 41 AAV5 mutations in ring VII 1186 US20160289275A1 SEQ ID NO: 43 AAV5 mutations in ring VII 1187 US20160289275A1 SEQ ID NO: 44 AAV8 1188 US20160289275A1 SEQ ID NO: 47 Mut A (LK03/AAV8) 1189 WO2016181123A1 SEQ ID NO: 1 Mut B (LK03/AAV5) 1190 WO2016181123A1 SEQ ID NO: 2 Mut C (AAV8/AAV3B) 1191 WO2016181123A1 SEQ ID NO: 3 Mut D (AAV5/AAV3B) 1192 WO2016181123A1 SEQ ID NO: 4 Mut E (AAV8/AAV3B) 1193 WO2016181123A1 SEQ ID NO: 5 Mut F (AAV3B/AAV8) 1194 WO2016181123A1 SEQ ID NO: 6 AAV44.9 1195 WO2016183297A1 SEQ ID NO: 4 AAV44.9 1196 WO2016183297A1 SEQ ID NO: 5 AAVrh8 1197 WO2016183297A1 SEQ ID NO: 6 AAV44.9 (S470N) 1198 WO2016183297A1 SEQ ID NO: 9 rh74 VP1 1199 US20160375110A1 SEQ ID NO: 1 AAV-LK03 (L125I) 1200 WO2017015102A1 SEQ ID NO: 5 AAV3B (S663V+T492V) 1201 WO2017015102A1 SEQ ID NO: 6 Anc80 1202 WO2017019994A2 SEQ ID NO: 1 Anc80 1203 WO2017019994A2 SEQ ID NO: 2 Anc81 1204 WO2017019994A2 SEQ ID NO: 3 Anc81 1205 WO2017019994A2 SEQ ID NO: 4 Anc82 1206 WO2017019994A2 SEQ ID NO: 5 Anc82 1207 WO2017019994A2 SEQ ID NO: 6 Anc83 1208 WO2017019994A2 SEQ ID NO: 7 Anc83 1209 WO2017019994A2 SEQ ID NO: 8 Anc84 1210 WO2017019994A2 SEQ ID NO: 9 Anc84 1211 WO2017019994A2 SEQ ID NO: 10 Anc94 1212 WO2017019994A2 SEQ ID NO: 11 Anc94 1213 WO2017019994A2 SEQ ID NO: 12 Anc113 1214 WO2017019994A2 SEQ ID NO: 13 Anc113 1215 WO2017019994A2 SEQ ID NO: 14 Anc126 1216 WO2017019994A2 SEQ ID NO: 15 Anc126 1217 WO2017019994A2 SEQ ID NO: 16 Anc127 1218 WO2017019994A2 SEQ ID NO: 17 Anc127 1219 WO2017019994A2 SEQ ID NO: 18 Anc80L27 1220 WO2017019994A2 SEQ ID NO: 19 Anc80L59 1221 WO2017019994A2 SEQ ID NO: 20 Anc80L60 1222 WO2017019994A2 SEQ ID NO: 21 Anc80L62 1223 WO2017019994A2 SEQ ID NO: 22 Anc80L65 1224 WO2017019994A2 SEQ ID NO: 23 Anc80L33 1225 WO2017019994A2 SEQ ID NO: 24 Anc80L36 1226 WO2017019994A2 SEQ ID NO: 25 Anc80L44 1227 WO2017019994A2 SEQ ID NO: 26 Anc80L1 1228 WO2017019994A2 SEQ ID NO: 35 Anc80L1 1229 WO2017019994A2 SEQ ID NO: 36 AAVrh10 1230 WO2017019994A2 SEQ ID NO: 41 Anc110 1231 WO2017019994A2 SEQ ID NO: 42 Anc110 1232 WO2017019994A2 SEQ ID NO: 43 AAVrh32.33 1233 WO2017019994A2 SEQ ID NO: 45 AAVrh74 1234 WO2017049031A1 SEQ ID NO: 1 AAV2 1235 WO2017053629A2 SEQ ID NO: 49 AAV2 1236 WO2017053629A2 SEQ ID NO: 50 AAV2 1237 WO2017053629A2 SEQ ID NO: 82 parvovirus 1238 WO2017070476A2 SEQ ID NO: 1 parvovirus 1239 WO2017070476A2 SEQ ID NO: 2 parvovirus 1240 WO2017070476A2 SEQ ID NO: 3 parvovirus 1241 WO2017070476A2 SEQ ID NO: 4 parvovirus 1242 WO2017070476A2 SEQ ID NO: 5 parvovirus 1243 WO2017070476A2 SEQ ID NO: 6 AAVrh.10 1244 WO2017070516A1 SEQ ID NO: 7 AAVrh.10 1245 WO2017070516A1 SEQ ID NO: 14 AAV2tYF 1246 WO2017070491A1 SEQ ID NO: 1 AAV-SPK 1247 WO2017075619A1 SEQ ID NO:28 AAV2.5 1248 US20170128528A1 SEQ ID NO: 13 AAV1.1 1249 US20170128528A1 SEQ ID NO: 15 AAV6.1 1250 US20170128528A1 SEQ ID NO: 17 AAV6.3.1 1251 US20170128528A1 SEQ ID NO: 18 AAV2i8 1252 US20170128528A1 SEQ ID NO: 28 AAV2i8 1253 US20170128528A1 SEQ ID NO: 29 ttAAV 1254 US20170128528A1 SEQ ID NO: 30 ttAAV-S312N 1255 US20170128528A1 SEQ ID NO: 32 ttAAV-S312N 1256 US20170128528A1 SEQ ID NO: 33 AAV6 (Y705, Y731 and T492) 1257 WO2016134337A1 SEQ ID NO: 24 AAV2 1258 WO2016134375A1 SEQ ID NO: 9 AAV2 1259 WO2016134375A1 SEQ ID NO: 10

The contents of each of the patents, applications, and/or publications listed in Table 1 are incorporated herein by reference in their entirety.

In some embodiments, an AAV serotype may be or may comprise a sequence as described in International Patent Publication WO2015038958, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV9 (SEQ ID NO. of WO2015038958 : 2 and 11 or SEQ ID NO: 135 and 136 in this article), PHP.B (SEQ ID NO: 8 and 9 of WO2015038958, SEQ ID NO: 3 and 4 in this article), G2B-13 (SEQ ID of WO2015038958 NO: 12, SEQ ID NO: 5 in this article, G2B-26 (SEQ ID NO: 13 of WO2015038958, SEQ ID NO: 3 in this article), TH1.1-32 (SEQ ID NO: 14 of WO2015038958, This article is SEQ ID NO: 6), TH1.1-35 (SEQ ID NO: 15 of WO2015038958, this article is SEQ ID NO: 7) or variants thereof. Additionally, any of the "targeting peptides" or "amino acid insertion sequences" described in WO2015038958 (used interchangeably herein to mean that can be inserted into AAV capsid sequences to facilitate delivery to CNS tissues sequence) can be inserted into any parent AAV serotype, such as (but not limited to) AAV9 (DNA sequence SEQ ID NO: 135 and amino acid sequence SEQ ID NO: 136). In some embodiments, the amino acid insertion sequence is inserted between amino acids 586-592 of the parent AAV (eg, AAV9). In some embodiments, the amino acid insertion sequence is inserted between amino acids 588-589 of the parental AAV sequence. The amino acid insertion sequence may be (but is not limited to) any of the following amino acid sequences: TLAVPFK (SEQ ID NO: 1 of WO2015038958; SEQ ID NO: 1260 in this article), KFPVALT (SEQ ID NO of WO2015038958) : 3; in this article, it is SEQ ID NO: 1261), LAVPFK (SEQ ID NO: 31 of WO2015038958; in this article, it is SEQ ID NO: 1262), AVPFK (SEQ ID NO: 32 of WO2015038958; in this article, it is SEQ ID NO: 1263), VPFK (SEQ ID NO: 33 of WO2015038958; SEQ ID NO: 1264 in this article), TLAVPF (SEQ ID NO: 34 of WO2015038958; SEQ ID NO: 1265 in this article), TLAVP (SEQ ID NO of WO2015038958) : 35; SEQ ID NO: 1266 in this article), TLAV (SEQ ID NO: 36 of WO2015038958; SEQ ID NO: 1267 in this article), SVSKPFL (SEQ ID NO: 28 of WO2015038958; SEQ ID NO: 28 in this article) 1268), FLTLTPK (SEQ ID NO: 29 of WO2015038958; SEQ ID NO: 1269 in this article), MNATKNV (SEQ ID NO: 30 of WO2015038958; SEQ ID NO: 1270 in this article), QSSQTPR (SEQ ID NO of WO2015038958) : 54; in this article, it is SEQ ID NO: 1271), ILGTGTS (SEQ ID NO: 55 of WO2015038958; in this article, it is SEQ ID NO: 1272), TRTNPEA (SEQ ID NO: 56 of WO2015038958; in this article, it is SEQ ID NO: 1273), NGGTSSS (SEQ ID NO: 58 of WO2015038958; herein SEQ ID NO: 1274) or YTLSQGW (SEQ ID NO: 60 of WO2015038958; herein SEQ ID NO: 1275). Non-limiting examples of nucleotide sequences that may encode amino acid inserts include, but are not limited to, the following: AAGTTTCCTGTGGCGTTGACT (SEQ ID NO: 3 of WO2015038958; herein SEQ ID NO: 1276), ACTTTGGCGGTGCCTTTTAAG (SEQ of WO2015038958) ID NO: 24 and 49; herein referred to as SEQ ID NO: 1277), AGTGTGAGTAAGCCTTTTTTTG (SEQ ID NO: 25 of WO2015038958; herein referred to as SEQ ID NO: 1278), TTTACTTGACGACGACGCCTAAG (SEQ ID NO: 26 of WO2015038958; herein referred to as SEQ ID NO: 1279), ATGAATGCTACGAAGAATGTG (SEQ ID NO: 27 of WO2015038958; SEQ ID NO: 1280 in this article), CAGTCGTCCGCAGACGCCTAGG (SEQ ID NO: 48 of WO2015038958; SEQ ID NO: 1281 in this article), ATTCTGGGGACTGGTACTTCG (WO20 15038958 SEQ ID NO: 50 and 52; in this article, SEQ ID NO: 1282), ACGCGGACTAATCCTGAGGCT (SEQ ID NO: 51 in WO2015038958; in this article, SEQ ID NO: 1283), AATGGGGGGACTAGTAGTTCT (SEQ ID NO: 53 in WO2015038958; this article) SEQ ID NO: 1284) or TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 59 of WO2015038958; SEQ ID NO: 1285 in this article).

In some embodiments, the AAV serotype may be or may include sequences as described in International Patent Publication WO2017100671, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV9 K449R (SEQ ID NO. WO2017100671 NO: 45, SEQ ID NO: 9 in this article, PHP.N (SEQ ID NO: 46 of WO2017100671, SEQ ID NO: 2 in this article), PHP.S (SEQ ID NO: 47 of WO2017100671, SEQ ID NO: 47 in this article) Is SEQ ID NO: 8) or a variant thereof. Furthermore, any of the targeting peptide or amino acid insertion sequences described in WO2017100671 can be inserted into any parent AAV serotype, such as (but not limited to) AAV9 (SEQ ID NO: 9 or SEQ ID NO: 136). In some embodiments, the amino acid insertion sequence is inserted between amino acids 586-592 of the parent AAV (eg, AAV9). In some embodiments, the amino acid insertion sequence is inserted between amino acids 588-589 of the parental AAV sequence. The amino acid insertion sequence may be (but is not limited to) any of the following amino acid sequences: AQTLAVPFKAQ (SEQ ID NO: 1 of WO2017100671; SEQ ID NO: 1286 in this article), AQSVSKPFLAQ (SEQ ID NO of WO2017100671) : 2; SEQ ID NO: 1287 in this article), AQFTLTTPKAQ (SEQ ID NO: 3 in the sequence listing in WO2017100671; SEQ ID NO: 1288 in this article), DGTLAVPFKAQ (SEQ ID NO in the sequence listing in WO2017100671 : 4; SEQ ID NO: 1289 in this article), ESTLAVPFKAQ (SEQ ID NO: 5 of WO2017100671; SEQ ID NO: 1290 in this article), GGTLAVPFKAQ (SEQ ID NO: 6 of WO2017100671; SEQ ID NO: 6 in this article) 1291), AQTLATPFKAQ (SEQ ID NO: 7 and 33 of WO2017100671; SEQ ID NO: 1292 in this article), ATTLATPFKAQ (SEQ ID NO: 8 of WO2017100671; SEQ ID NO: 1293 in this article), DGTLATPFKAQ (SEQ ID NO: 1293 of WO2017100671) ID NO: 9; SEQ ID NO: 1294 in this article), GGTLATPFKAQ (SEQ ID NO: 10 of WO2017100671; SEQ ID NO: 1295 in this article), SGSLAVPFKAQ (SEQ ID NO: 11 of WO2017100671; SEQ ID in this article) NO: 1296), AQTLAQPFKAQ (SEQ ID NO: 12 of WO2017100671; SEQ ID NO: 1297 in this article), AQTLQQPFKAQ (SEQ ID NO: 13 of WO2017100671; SEQ ID NO: 1298 in this article), AQTLSNPFKAQ (SEQ ID NO: 1298 of WO2017100671) ID NO: 14; SEQ ID NO: 1299 in this article), AQTLAVPFSNP (SEQ ID NO: 15 in WO2017100671; SEQ ID NO: 1300 in this article), QGTLAVPFKAQ (SEQ ID NO: 16 in WO2017100671; SEQ ID in this article) NO: 1301), NQTLAVPFKAQ (SEQ ID NO: 17 of WO2017100671; SEQ ID NO: 1302 in this article), EGSLAVPFKAQ (SEQ ID NO: 18 of WO2017100671; SEQ ID NO: 1303 in this article), SGNLAVPFKAQ (SEQ ID NO: 1303 of WO2017100671) ID NO: 19; SEQ ID NO: 1304 in this article), EGTLAVPFKAQ (SEQ ID NO: 20 in WO2017100671; SEQ ID NO: 1305 in this article), DSTLAVPFKAQ (SEQ ID NO: 21 in Table 1 of WO2017100671; this article Among them are SEQ ID NO: 1306), AVTLAVPFKAQ (SEQ ID NO: 22 of WO2017100671; SEQ ID NO: 1307 in this article), AQTLSTPFKAQ (SEQ ID NO: 23 of WO2017100671; SEQ ID NO: 1308 in this article), AQTLPQPFKAQ (SEQ ID NO: 24 and 32 of WO2017100671; SEQ ID NO: 1309 in this article), AQTLSQPFKAQ (SEQ ID NO: 25 of WO2017100671; SEQ ID NO: 1310 in this article), AQTLQLPFKAQ (SEQ ID NO: 26 of WO2017100671) ; This article is SEQ ID NO: 1311), AQTLTMPFKAQ (SEQ ID NO: 27 and 34 of WO2017100671 and SEQ ID NO: 35 in the sequence listing of WO2017100671; this article is SEQ ID NO: 1312), AQTLTTPFKAQ (SEQ ID NO: WO2017100671) ID NO: 28; SEQ ID NO: 1313 in this article), AQYTLSQGWAQ (SEQ ID NO: 29 of WO2017100671; SEQ ID NO: 1314 in this article), AQMNATKNVAQ (SEQ ID NO: 30 of WO2017100671; SEQ ID in this article) NO: 1315), AQVSGGHHSAQ (SEQ ID NO: 31 of WO2017100671; SEQ ID NO: 1316 in this article), AQTLTAPFKAQ (SEQ ID NO: 35 in Table 1 of WO2017100671; SEQ ID NO: 1317 in this article), AQTLSKPFKAQ (SEQ ID NO: 36 of WO2017100671; SEQ ID NO: 1318 in this article), QAVRTSL (SEQ ID NO: 37 of WO2017100671; SEQ ID NO: 1319 in this article), YTLSQGW (SEQ ID NO: 38 of WO2017100671; SEQ ID NO: 1319 in this article) In the middle, it is SEQ ID NO: 1275), LAKERLS (SEQ ID NO: 39 in WO2017100671; in this article, it is SEQ ID NO: 1320), TLAVPFK (SEQ ID NO: 40 in the sequence listing of WO2017100671; in this article, it is SEQ ID NO: 1260), SVSKPFL (SEQ ID NO: 41 of WO2017100671; SEQ ID NO: 1268 in this article), FLTLTPK (SEQ ID NO: 42 of WO2017100671; SEQ ID NO: 1269 in this article), MNSTKNV (SEQ ID NO of WO2017100671) : 43; SEQ ID NO: 1321 in this article), VSGGHHS (SEQ ID NO: 44 of WO2017100671; SEQ ID NO: 1322 in this article), SAQTLAVPFKAQAQ (SEQ ID NO: 48 of WO2017100671; SEQ ID NO: 48 in this article) 1323), SXXXLAVPFKAQAQ (SEQ ID NO: 49 of WO2017100671, where ; In this article, it is SEQ ID NO: 1325), SAQTLXXXFKAQAQ (SEQ ID NO: 51 of WO2017100671, where Wherein SEQ ID NO: 65 of WO2017100671; SEQ ID NO: 1329 in this article), AQAQTGW (SEQ ID NO: 66 of WO2017100671; SEQ ID NO: 1330 in this article), DGTLATPFK (SEQ ID NO: 67 of WO2017100671; SEQ ID NO: 1330 in this article) It is SEQ ID NO: 1331), DGTLATPFKXX (SEQ ID NO: 68 of WO2017100671, where SEQ ID NO: 1333), VPFKAQ (SEQ ID NO: 81 of WO2017100671; SEQ ID NO: 1334 in this article), FKAQ (SEQ ID NO: 82 of WO2017100671; SEQ ID NO: 1335 in this article), AQTLAV (WO2017100671 SEQ ID NO: 83; SEQ ID NO: 1336 in this article), AQTLAVPF (SEQ ID NO: 84 in WO2017100671; SEQ ID NO: 1337 in this article), QAVR (SEQ ID NO: 85 in WO2017100671; SEQ ID NO: 85 in this article) SEQ ID NO: 1338), AVRT (SEQ ID NO: 86 of WO2017100671; SEQ ID NO: 1339 in this article), VRTS (SEQ ID NO: 87 of WO2017100671; SEQ ID NO: 1340 in this article), RTSL (WO2017100671 SEQ ID NO: 88; this article is SEQ ID NO: 1341), QAVRT (WO2017100671 SEQ ID NO: 89; this article is SEQ ID NO: 1342), AVRTS (WO2017100671 SEQ ID NO: 90; this article is SEQ ID NO: 90) SEQ ID NO: 1343), VRTSL (SEQ ID NO: 91 of WO2017100671; herein SEQ ID NO: 1344), QAVRTS (SEQ ID NO: 92 of WO2017100671; herein SEQ ID NO: 1345) or AVRTSL (WO2017100671 (SEQ ID NO: 93; in this article, SEQ ID NO: 1346).

Non-limiting examples of nucleotide sequences that may encode amino acid inserts include the following: GATGGGACTTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 54 of WO2017100671; herein SEQ ID NO: 1347), GATGGGACGTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 55 of WO2017100671; This article is SEQ ID NO: 1348), CAGGCGGTTAGGACGTCTTTG (WO2017100671's SEQ ID NO: 56; this article is SEQ ID NO: 1349), CAGGTCTTCACGGACTCAGACTATCAG (WO2017100671's SEQ ID NO: 57 and 78; this article is SEQ ID NO: 1350 ), CAAGTAAAACCTCTACAAATGTGGTAAAATCG (SEQ ID NO: 58 of WO2017100671; SEQ ID NO: 1351 in this article), ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC (SEQ ID NO: 59 of WO2017100671; SEQ ID NO: 1352 in this article), GGAAGTATTCCTTGGTTTTGAACCCA (WO2 017100671 SEQ ID NO: 60; SEQ ID NO: 1353 in this article), GGTCGCGGTTTCTTGTTTGTGGAT (SEQ ID NO: 61 in WO2017100671; SEQ ID NO: 1354 in this article), CGACCTTGAAGCGCATGAACTCCT (SEQ ID NO: 62 in WO2017100671; SEQ ID NO: 1355 in this article) ), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNMMNNMNNMNNTTGGGCACTCTGGTGGGTTTGTC (SEQ ID NO: 63 of WO2017100671, where N can be A, C, T or G; in this article, SEQ ID NO: 1356), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCMNNMNNMNNAAAAGGCACCGCCAAAGTTTG (WO2017100671) SEQ ID NO: 69 of 2017100671, where N can be A, C, T or G; in this article, it is SEQ ID NO: 1357), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNCACCGCCAAAGTTTGGGCACT (SEQ ID NO: 70 of WO2017100671, where N can be A, C, T or G; in this article, it is SEQ ID NO: 1358), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCC TTAAAMNNMNNMNNCAAAGTTTGGGCACTCTGGTGG ( SEQ ID NO: 71 of WO2017100671, wherein N can be A, C, T or G; in this article, it is SEQ ID NO: 1359), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAAGGCACMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG (SEQ ID NO: 72 of WO2017100671, where N can be A, C, T or G; SEQ ID NO: 1360 in this article), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NO: 74 in WO2017100671; SEQ ID NO: 1277 in this article), AGTGTGAGTAAGCCTTTTTG (SEQ ID NO: 75 in WO2017100671; SEQ ID NO: 1278 in this article) ), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 76 of WO2017100671; SEQ ID NO: 1279 in this article), TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 77 of WO2017100671; SEQ ID NO: 1285 in this article) or CTTGCGAAGGAGCGGCTTTCG (SEQ ID NO: 1285 of WO2017100671) SEQ ID NO: 79; herein SEQ ID NO: 1361).

In some embodiments, an AAV serotype may be or may include a sequence as described in U.S. Patent No. 9,624,274, which is incorporated herein by reference in its entirety, such as (but not limited to) SEQ ID NO. NO: 181), AAV6 (SEQ ID NO: 182 of US9624274), AAV2 (SEQ ID NO: 183 of US9624274), AAV3b (SEQ ID NO: 184 of US9624274), AAV7 (SEQ ID NO: 185 of US9624274), AAV8 (SEQ ID NO: 186 of US9624274), AAV10 (SEQ ID NO: 187 of US9624274), AAV4 (SEQ ID NO: 188 of US9624274), AAV11 (SEQ ID NO: 189 of US9624274), bAAV (SEQ ID NO of US9624274 : 190), AAV5 (SEQ ID NO: 191 of US9624274), GPV (SEQ ID NO: 192 of US9624274; SEQ ID NO: 992 in this article), B19 (SEQ ID NO: 193 of US9624274; SEQ ID in this article) NO: 993), MVM (SEQ ID NO: 194 of US9624274; SEQ ID NO: 994 in this article), FPV (SEQ ID NO: 195 of US9624274; SEQ ID NO: 995 in this article), CPV (SEQ ID NO: 995 of US9624274) ID NO: 196; herein SEQ ID NO: 996) or a variant thereof. In addition, any of the structural protein insertion sequences described in US Pat. No. 9,624,274 can be inserted into (but not limited to) I-453 and I-587 of any parent AAV serotype, such as (but not limited to) AAV2 (SEQ ID NO: 183 of US9624274). The amino acid insertion sequence may be (but is not limited to) any of the following amino acid sequences: VNLTWSRASG (SEQ ID NO: 50 of US9624274; herein SEQ ID NO: 1362), EFCINHRGYWVCGD (SEQ ID NO of US9624274) :55; SEQ ID NO: 1363 in this article), EDGQVMDVDLS (SEQ ID NO: 85 of US9624274; SEQ ID NO: 1364 in this article), EKQRNGTLT (SEQ ID NO: 86 of US9624274; SEQ ID NO: 86 in this article): 1365), TYQCRVTHPHLPRALMR (SEQ ID NO: 87 of US9624274; SEQ ID NO: 1366 in this article), RHSTTQPRKTKGSG (SEQ ID NO: 88 of US9624274; SEQ ID NO: 1367 in this article), DSNPRGVSAYLSR (SEQ ID NO of US9624274) : 89; SEQ ID NO: 1368 in this article), TITCLWDLAPSK (SEQ ID NO: 90 in US9624274; SEQ ID NO: 1369 in this article), KTKGSGFFVF (SEQ ID NO: 91 in US9624274; SEQ ID NO: 91 in this article) 1370), THPHLPRALMRS (SEQ ID NO: 92 of US9624274; SEQ ID NO: 1371 in this article), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 93 of US9624274; SEQ ID NO: 1372 in this article), LPRALMRS (SEQ ID NO of US9624274) : 94; SEQ ID NO: 1373 in this article), INHRGYWV (SEQ ID NO: 95 of US9624274; SEQ ID NO: 1374 in this article), CDAGSVRTNAPD (SEQ ID NO: 60 of US9624274; SEQ ID NO: 60 in this article) 1375), AKAVSNLTESRSSESLQS (SEQ ID NO: 96 of US9624274; SEQ ID NO: 1376 in this article), SLTGDEFKKVLET (SEQ ID NO: 97 of US9624274; SEQ ID NO: 1377 in this article), REAVAYRFEED (SEQ ID NO of US9624274) : 98; SEQ ID NO: 1378 in this article), INPEIITLDG (SEQ ID NO: 99 of US9624274; SEQ ID NO: 1379 in this article), DISVTGAPVITATYL (SEQ ID NO: 100 of US9624274; SEQ ID NO: 100 in this article) 1380), DISVTGAPVITA (SEQ ID NO: 101 of US9624274; SEQ ID NO: 1381 in this article), PKTVSNLTESSSESVQS (SEQ ID NO: 102 of US9624274; SEQ ID NO: 1382 in this article), SLMGDEFKAVLET (SEQ ID NO of US9624274) : 103; in this article, it is SEQ ID NO: 1383), QHSVAYTFEED (SEQ ID NO: 104 of US9624274; in this article, it is SEQ ID NO: 1384), INPEIITRDG (SEQ ID NO: 105 of US9624274; in this article, it is SEQ ID NO: 1385), DISLTGDPVITASYL (SEQ ID NO: 106 of US9624274; SEQ ID NO: 1386 in this article), DISLTGDPVITA (SEQ ID NO: 107 of US9624274; SEQ ID NO: 1387 in this article), DQSIDFEIDSA (SEQ ID NO of US9624274) : 108; SEQ ID NO: 1388 in this article), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 109 of US9624274; SEQ ID NO: 1389 in this article), KNVSEDLPLPT (SEQ ID NO: 110 of US9624274; SEQ ID NO: 110 in this article) 1390), CDSGRVRTDAPD (SEQ ID NO: 111 of US9624274; SEQ ID NO: 1391 in this article), FPEHLLVDFLQSLS (SEQ ID NO: 112 of US9624274; SEQ ID NO: 1392 in this article), DAEFRHSG (SEQ ID NO of US9624274) : 65; SEQ ID NO: 1393 in this article), HYAAAQWDFGNTMCQL (SEQ ID NO: 113 of US9624274; SEQ ID NO: 1394 in this article), YAAQWDFGNTMCQ (SEQ ID NO: 114 of US9624274; SEQ ID NO: 114 in this article) 1395), RSQKEGLHYT (SEQ ID NO: 115 of US9624274; SEQ ID NO: 1396 in this article), SSRTPSDKPVAHWANPQAE (SEQ ID NO: 116 of US9624274; SEQ ID NO: 1397 in this article), SRTPSDKPVAHWANP (SEQ ID NO of US9624274) : 117; SEQ ID NO: 1398 in this article), SSRTPSDKP (SEQ ID NO: 118 in US9624274; SEQ ID NO: 1399 in this article), NADGNVDYHMNSVP (SEQ ID NO: 119 in US9624274; SEQ ID NO: 119 in this article) 1400), DGNVDYHMNSV (SEQ ID NO: 120 of US9624274; SEQ ID NO: 1401 in this article), RSFKEFLQSSLRALRQ (SEQ ID NO: 121 of US9624274; SEQ ID NO: 1402 in this article); FKEFLQSSLRA (SEQ ID NO of US9624274) : 122; herein SEQ ID NO: 1403) or QMWAPQWGPD (SEQ ID NO: 123 of US9624274; herein SEQ ID NO: 1404).

In some embodiments, the AAV serotype may be or may have sequences as described in U.S. Patent No. 9,475,845, which is incorporated herein by reference in its entirety, such as (but not limited to) contained in native AAV2 capsids. AAV capsid protein modified with one or more amino acids at amino acid positions 585 to 590 of the protein. In addition, the modification can produce, but is not limited to, the amino acid sequence RGNRQA (SEQ ID NO: 3 of US9475845; herein SEQ ID NO: 1405), SSSTDP (SEQ ID NO: 4 of US9475845; herein SEQ ID NO : 1406), SSNTAP (SEQ ID NO: 5 of US9475845; SEQ ID NO: 1407 in this article), SNSNLP (SEQ ID NO: 6 of US9475845; SEQ ID NO: 1408 in this article), SSTTAP (SEQ ID NO: 1408 of US9475845) NO: 7; SEQ ID NO: 1409 in this article), AANTAA (SEQ ID NO: 8 of US9475845; SEQ ID NO: 1410 in this article), QQNTAP (SEQ ID NO: 9 of US9475845; SEQ ID NO in this article) : 1411), SAQAQA (SEQ ID NO: 10 of US9475845; SEQ ID NO: 1412 in this article), QANTGP (SEQ ID NO: 11 of US9475845; SEQ ID NO: 1413 in this article), NATTAP (SEQ ID NO: 1413 of US9475845) NO: 12; SEQ ID NO: 1414 in this article), SSTAGP (SEQ ID NO: 13 and 20 of US9475845; SEQ ID NO: 1415 in this article), QQNTAA (SEQ ID NO: 14 of US9475845; SEQ in this article) ID NO: 1416), PSTAGP (SEQ ID NO: 15 of US9475845; SEQ ID NO: 1417 in this article), NQNTAP (SEQ ID NO: 16 of US9475845; SEQ ID NO: 1418 in this article), QAANAP (SEQ ID NO: 1418 of US9475845) SEQ ID NO: 17; herein SEQ ID NO: 1419), SIVGLP (SEQ ID NO: 18 of US9475845; herein SEQ ID NO: 1420), AASTAA (SEQ ID NO: 19 and 27 of US9475845; herein It is SEQ ID NO: 1421), SQNTTA (SEQ ID NO: 21 of US9475845; SEQ ID NO: 1422 in this article), QQDTAP (SEQ ID NO: 22 of US9475845; SEQ ID NO: 1423 in this article), QTNTGP ( SEQ ID NO: 23 of US9475845; SEQ ID NO: 1424 in this article), QTNGAP (SEQ ID NO: 24 of US9475845; SEQ ID NO: 1425 in this article), QQNAAP (SEQ ID NO: 25 of US9475845; SEQ ID NO: 1425 in this article) is SEQ ID NO: 1426) or AANTQA (SEQ ID NO: 26 of US9475845; SEQ ID NO: 1427 in this article). In some embodiments, the amino acid modification is a substitution at amino acid positions 262 to 265 in the native AAV2 capsid protein or at the corresponding position in the capsid protein of another AAV with the targeting sequence. The targeting sequence may be (but is not limited to) any of the following amino acid sequences: NGRAHA (SEQ ID NO: 38 of US9475845; here SEQ ID NO: 1428), QPEHSST (SEQ ID NO: 39 of US9475845) and 50; herein SEQ ID NO: 1429), VNTANST (SEQ ID NO: 40 of US9475845; herein SEQ ID NO: 1430), HGPMQKS (SEQ ID NO: 41 of US9475845; herein SEQ ID NO: 1431), PHKPPLA (SEQ ID NO: 42 of US9475845; SEQ ID NO: 1432 in this article), IKNNEMW (SEQ ID NO: 43 of US9475845; SEQ ID NO: 1433 in this article), RNLDTPM (SEQ ID NO of US9475845) : 44; in this article, it is SEQ ID NO: 1434), VDSHRQS (SEQ ID NO: 45 of US9475845; in this article, it is SEQ ID NO: 1435), YDSKTKT (SEQ ID NO: 46 of US9475845; in this article, it is SEQ ID NO: 1436), SQLPHQK (SEQ ID NO: 47 of US9475845; SEQ ID NO: 1437 in this article), STMQQNT (SEQ ID NO: 48 of US9475845; SEQ ID NO: 1438 in this article), TERYMTQ (SEQ ID NO of US9475845) : 49; this article is SEQ ID NO: 1439), DASLSTS (US9475845's SEQ ID NO: 51; this article is SEQ ID NO: 1440), DLPNKKT (US9475845's SEQ ID NO: 52; this article is SEQ ID NO: 1441), DLTAARL (SEQ ID NO: 53 of US9475845; SEQ ID NO: 1442 in this article), EPHQFNY (SEQ ID NO: 54 of US9475845; SEQ ID NO: 1443 in this article), EPQSNHT (SEQ ID NO of US9475845) : 55; in this article, it is SEQ ID NO: 1444), MSSWPSQ (SEQ ID NO: 56 of US9475845; in this article, it is SEQ ID NO: 1445), NPKHNAT (SEQ ID NO: 57 of US9475845; in this article, it is SEQ ID NO: 1446), PDGMRTT (SEQ ID NO: 58 of US9475845; SEQ ID NO: 1447 in this article), PNNNKTT (SEQ ID NO: 59 of US9475845; SEQ ID NO: 1448 in this article), QSTTHDS (SEQ ID NO of US9475845) : 60; in this article, it is SEQ ID NO: 1449), TGSKQKQ (SEQ ID NO: 61 of US9475845; in this article, it is SEQ ID NO: 1450), SLKHQAL (SEQ ID NO: 62 of US9475845; in this article, it is SEQ ID NO: 1451), SPIDGEQ (SEQ ID NO: 63 of US9475845; SEQ ID NO: 1452 in this article), WIFPWIQL (SEQ ID NO: 64 and 112 of US9475845; SEQ ID NO: 1453 in this article), CDCRGDCFC (SEQ ID NO: 1453 of US9475845) ID NO: 65; SEQ ID NO: 1454 in this article), CNGRC (SEQ ID NO: 66 of US9475845; SEQ ID NO: 1455 in this article), CPRECES (SEQ ID NO: 67 of US9475845; SEQ ID in this article) NO: 1456), CTTHWGFTLC (SEQ ID NO: 68 and 123 of US9475845; SEQ ID NO: 1457 in this article), CGRRAGGSC (SEQ ID NO: 69 of US9475845; SEQ ID NO: 1458 in this article), CKGGRAKDC (US9475845 SEQ ID NO: 70; this article is SEQ ID NO: 1459), CVPELGHEC (US9475845's SEQ ID NO: 71 and 115; this article is SEQ ID NO: 1460), CRRETAWAK (US9475845's SEQ ID NO: 72; this article) Among them are SEQ ID NO: 1461), VSWFSHRYSPFAVS (SEQ ID NO: 73 of US9475845; SEQ ID NO: 1462 in this article), GYRDGYAGPILYN (SEQ ID NO: 74 of US9475845; SEQ ID NO: 1463 in this article), XXXYXXX (SEQ ID NO: 75 of US9475845; SEQ ID NO: 1464 in this article), YXNW (SEQ ID NO: 76 of US9475845; SEQ ID NO: 1465 in this article), RPLPPLP (SEQ ID NO: 77 of US9475845; this article Among them are SEQ ID NO: 1466), APPLPPR (SEQ ID NO: 78 of US9475845; SEQ ID NO: 1467 in this article), DVFYPYPYASGS (SEQ ID NO: 79 of US9475845; SEQ ID NO: 1468 in this article), MYWYPY (SEQ ID NO: 80 of US9475845; SEQ ID NO: 1469 in this article), DITWDQLWDLMK (SEQ ID NO: 81 of US9475845; SEQ ID NO: 1470 in this article), CWDDXWLC (SEQ ID NO: 82 of US9475845; SEQ ID NO: 1470 in this article) Among them are SEQ ID NO: 1471), EWCEYLGGYLRCYA (SEQ ID NO: 83 of US9475845; SEQ ID NO: 1472 in this article), YXCXXGPXTWXCXP (SEQ ID NO: 84 of US9475845; SEQ ID NO: 1473 in this article), IEGPTLRQWLAARA (SEQ ID NO: 85 of US9475845; SEQ ID NO: 1474 in this article), LWXXX (SEQ ID NO: 86 of US9475845; SEQ ID NO: 1475 in this article), XFXXYLW (SEQ ID NO: 87 of US9475845; SEQ ID NO: 1475 in this article) SEQ ID NO: 1476), SSIISHFRWGLCD (SEQ ID NO: 88 of US9475845; SEQ ID NO: 1477 in this article), MSRPACPPNDKYE (SEQ ID NO: 89 of US9475845; SEQ ID NO: 1478 in this article), CLRSGRGC (SEQ ID NO: 90 of US9475845; SEQ ID NO: 1479 in this article), CHWMFSPWC (SEQ ID NO: 91 of US9475845; SEQ ID NO: 1480 in this article), WXXF (SEQ ID NO: 92 of US9475845; SEQ ID NO: 1480 in this article) Among them are SEQ ID NO: 1481), CSSRLDAC (SEQ ID NO: 93 of US9475845; SEQ ID NO: 1482 in this article), CLPVASC (SEQ ID NO: 94 of US9475845; SEQ ID NO: 1483 in this article), CGFECVRQCPERC (SEQ ID NO: 95 of US9475845; SEQ ID NO: 1484 in this article), CVALCREACGEGC (SEQ ID NO: 96 of US9475845; SEQ ID NO: 1485 in this article), SWCPGWCR (SEQ ID NO: 97 of US9475845; this article Among them are SEQ ID NO: 1486), YSGKWGW (SEQ ID NO: 98 of US9475845; SEQ ID NO: 1487 in this article), GLSGGRS (SEQ ID NO: 99 of US9475845; SEQ ID NO: 1488 in this article), LMLPRAD (SEQ ID NO: 100 of US9475845; SEQ ID NO: 1489 in this article), CSCFRDVCC (SEQ ID NO: 101 of US9475845; SEQ ID NO: 1490 in this article), CRDVVSVIC (SEQ ID NO: 102 of US9475845; SEQ ID NO: 1490 in this article) Among them are SEQ ID NO: 1491), MARSGL (SEQ ID NO: 103 of US9475845; SEQ ID NO: 1492 in this article), MARAKE (SEQ ID NO: 104 of US9475845; SEQ ID NO: 1493 in this article), MSRTMS (SEQ ID NO: 105 of US9475845; SEQ ID NO: 1494 in this article), KCCYSL (SEQ ID NO: 106 of US9475845; SEQ ID NO: 1495 in this article), MYWGDSHWLQYWYE (SEQ ID NO: 107 of US9475845; SEQ ID NO: 1495 in this article) Among them are SEQ ID NO: 1496), MQLPLAT (SEQ ID NO: 108 of US9475845; SEQ ID NO: 1497 in this article), EWLS (SEQ ID NO: 109 of US9475845; SEQ ID NO: 1498 in this article), SNEW (SEQ ID NO: 110 of US9475845; SEQ ID NO: 1499 in this article), TNYL (SEQ ID NO: 111 of US9475845; SEQ ID NO: 1500 in this article), WDLAWMFLPVG (SEQ ID NO: 113 of US9475845; SEQ ID NO: 113 in this article) Among them are SEQ ID NO: 1501), CTVALPGGYVRVC (SEQ ID NO: 114 of US9475845; SEQ ID NO: 1502 in this article), CVAYCIEHHCWTC (SEQ ID NO: 116 of US9475845; SEQ ID NO: 1503 in this article), CVFAHNYDYLVC (SEQ ID NO: 117 of US9475845; SEQ ID NO: 1504 in this article), CVFTSNYAFC (SEQ ID NO: 118 of US9475845; SEQ ID NO: 1505 in this article), VHSPNKK (SEQ ID NO: 119 of US9475845; this article Among them are SEQ ID NO: 1506), CRGDGWC (SEQ ID NO: 120 of US9475845; SEQ ID NO: 1507 in this article), XRGCDX (SEQ ID NO: 121 of US9475845; SEQ ID NO: 1508 in this article), PXXX (SEQ ID NO: 122 of US9475845; SEQ ID NO: 1509 in this article), SGKGPRQITAL (SEQ ID NO: 124 of US9475845; SEQ ID NO: 1510 in this article), AAAAAAAAAXXXXX (SEQ ID NO: 125 of US9475845; SEQ ID NO: 1510 in this article) Among them are SEQ ID NO: 1511), VYMSPF (SEQ ID NO: 126 of US9475845; SEQ ID NO: 1512 in this article), ATWLPPR (SEQ ID NO: 127 of US9475845; SEQ ID NO: 1513 in this article), HTMYYHHYQHHL (SEQ ID NO: 128 of US9475845; SEQ ID NO: 1514 in this article), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 129 of US9475845; SEQ ID NO: 1515 in this article), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 130 of US9475845; this article) Among them are SEQ ID NO: 1516), CKGQCDRFKGLPWEC (SEQ ID NO: 131 of US9475845; SEQ ID NO: 1517 in this article), SGRSA (SEQ ID NO: 132 of US9475845; SEQ ID NO: 1518 in this article), WGFP (SEQ ID NO: 133 of US9475845; SEQ ID NO: 1519 in this article), AEPMPHSLNFSQYLWYT (SEQ ID NO: 134 of US9475845; SEQ ID NO: 1520 in this article), WAYXSP (SEQ ID NO: 135 of US9475845; SEQ ID NO: 1520 in this article) Among them are SEQ ID NO: 1521), IELLQAR (SEQ ID NO: 136 of US9475845; SEQ ID NO: 1522 in this article), AYTKCSRQWRTCMTTH (SEQ ID NO: 137 of US9475845; SEQ ID NO: 1523 in this article), PQNSKIPGPTFLDPH (SEQ ID NO: 138 of US9475845; SEQ ID NO: 1524 in this article), SMEPALPDWWWKMFK (SEQ ID NO: 139 of US9475845; SEQ ID NO: 1525 in this article), ANTPCGPYTHDCPVKR (SEQ ID NO: 140 of US9475845; this article) Among them are SEQ ID NO: 1526), TACHQHVRMVRP (SEQ ID NO: 141 of US9475845; SEQ ID NO: 1527 in this article), VPWMEPAYQRFL (SEQ ID NO: 142 of US9475845; SEQ ID NO: 1528 in this article), DPRATPGS (SEQ ID NO: 143 of US9475845; SEQ ID NO: 1529 in this article), FRPNRAQDYNTN (SEQ ID NO: 144 of US9475845; SEQ ID NO: 1530 in this article), CTKNSYLMC (SEQ ID NO: 145 of US9475845; SEQ ID NO: 1530 in this article) Among them are SEQ ID NO: 1531), CXXTXXXGXGC (SEQ ID NO: 146 of US9475845; SEQ ID NO: 1532 in this article), CPIEDRPMC (SEQ ID NO: 147 of US9475845; SEQ ID NO: 1533 in this article), HEWSYLAPYPWF (SEQ ID NO: 148 of US9475845; SEQ ID NO: 1534 in this article), MCPKHPLGC (SEQ ID NO: 149 of US9475845; SEQ ID NO: 1535 in this article), RMWPSSTVNLSAGRR (SEQ ID NO: 150 of US9475845; SEQ ID NO: 153 in this article) Among them are SEQ ID NO: 1536), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 151 of US9475845; SEQ ID NO: 1537 in this article), KSREHVNNSACPSKRITAAL (SEQ ID NO: 152 of US9475845; SEQ ID NO: 1538 in this article), EGFR (SEQ ID NO: 153 of US9475845; SEQ ID NO: 1539 in this article), AGLGVR (SEQ ID NO: 154 of US9475845; SEQ ID NO: 1540 in this article), GTRQGHTMRLGVSDG (SEQ ID NO: 155 of US9475845; SEQ ID NO: 154 in this article) Among them are SEQ ID NO: 1541), IAGLATPGWSHWLAL (SEQ ID NO: 156 of US9475845; SEQ ID NO: 1542 in this article), SMSIARL (SEQ ID NO: 157 of US9475845; SEQ ID NO: 1543 in this article), HTFEPGV (SEQ ID NO: 158 of US9475845; SEQ ID NO: 1544 in this article), NTSLKRISNKRIRRK (SEQ ID NO: 159 of US9475845; SEQ ID NO: 1545 in this article), LRIKRKRRKRKKTRK (SEQ ID NO: 160 of US9475845; this article (SEQ ID NO: 1546), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH or GTV.

In some embodiments, an AAV serotype may be or may have a sequence as described in U.S. Patent Application Publication No. US 20160369298, which is incorporated herein by reference in its entirety, such as (but not limited to) AAV2 (U.S. SEQ ID NO: 97 of 20160369298; herein SEQ ID NO: 1547) or the site-directed mutation capsid protein of a variant thereof, wherein the specific mutation site is at least one site selected from VP1 or its fragment R447, G453, The sites of S578, N587, N587+1, and S662.

In addition, any of the mutant sequences described in US 20160369298 may be or may have, but is not limited to, any of the following sequences: SDSGASN (SEQ ID NO: 1 and SEQ ID NO: 231 of US20160369298; herein Among them are SEQ ID NO: 1548), SPSGASN (SEQ ID NO: 2 of US20160369298; SEQ ID NO: 1549 in this article), SHSGASN (SEQ ID NO: 3 of US20160369298; SEQ ID NO: 1550 in this article), SRSGASN (SEQ ID NO: 4 of US20160369298; SEQ ID NO: 1551 in this article), SKSGASN (SEQ ID NO: 5 of US20160369298; SEQ ID NO: 1552 in this article), SNSGASN (SEQ ID NO: 6 of US20160369298; SEQ ID NO: 6 in this article) SEQ ID NO: 1553), SGSGASN (SEQ ID NO: 7 of US20160369298; SEQ ID NO: 1554 in this article), SASGASN (SEQ ID NO: 8, 175 and 221 of US20160369298; SEQ ID NO: 221 in this article) 1555), SESGTSN (SEQ ID NO: 9 of US20160369298; SEQ ID NO: 1556 in this article), STTGGSN (SEQ ID NO: 10 of US20160369298; SEQ ID NO: 1557 in this article), SSAGSTN (SEQ ID NO of US20160369298) : 11; in this article, it is SEQ ID NO: 1558), NNDSQA (SEQ ID NO: 12 of US20160369298; in this article, it is SEQ ID NO: 1559), NNRNQA (SEQ ID NO: 13 of US20160369298; in this article, it is SEQ ID NO: 1560), NNNKQA (SEQ ID NO: 14 of US20160369298; SEQ ID NO: 1561 in this article), NAKRQA (SEQ ID NO: 15 of US20160369298; SEQ ID NO: 1562 in this article), NDEHQA (SEQ ID NO of US20160369298) : 16; in this article, it is SEQ ID NO: 1563), NTSQKA (SEQ ID NO: 17 of US20160369298; in this article, it is SEQ ID NO: 1564), YYLSRTNTPSGTDTQSRLVFSQAGA (SEQ ID NO: 18 of US20160369298; in this article, it is SEQ ID NO: 1565), YYLSRTNTDSGTETQSGLDFSQAGA (SEQ ID NO: 19 of US20160369298; SEQ ID NO: 1566 in this article), YYLSRTNTESGTPTQSALEFSQAGA (SEQ ID NO: 20 of US20160369298; SEQ ID NO: 1567 in this article), YYLSRTNTHSGTHTQSPL HFSQAGA (SEQ ID NO of US20160369298 : 21; this article is SEQ ID NO: 1568), YYLSRTNTSSGTITISHLIFSQAGA (US20160369298's SEQ ID NO: 22; this article is SEQ ID NO: 1569), YYLSRTNTRSGIMTKSSLMFSQAGA (US20160369298's SEQ ID NO: 23; this article is SEQ ID NO: 1570), YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 24 of US20160369298; SEQ ID NO: 1571 in this article), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 25 of US20160369298; SEQ ID NO: 1572 in this article), YYLSRTNAASGHATHSDLK FSQPGA (SEQ ID NO of US20160369298 : 26; SEQ ID NO: 1573 in this article), YYLSRTNGQAGSLTMSELGFSQVGA (SEQ ID NO: 27 of US20160369298; SEQ ID NO: 1574 in this article), YYLSRTNSTGGNQTTSQLLFSQLSA (SEQ ID NO: 28 of US20160369298; SEQ ID NO: 28 in this article: 1575), YFLSRTNNNTGLNTNSTLNFSQGRA (SEQ ID NO: 29 of US20160369298; SEQ ID NO: 1576 in this article), SKTGADNNNSEYSWTG (SEQ ID NO: 30 of US20160369298; SEQ ID NO: 1577 in this article), SKTDADNNNSEYSWTG (US201603 SEQ ID NO of 69298 : 31; in this article, SEQ ID NO: 1578), SKTEADNNNSEYSWTG (SEQ ID NO: 32 of US20160369298; in this article, SEQ ID NO: 1579), SKTPADNNNSEYSWTG (SEQ ID NO: 33 of US20160369298; in this article, SEQ ID NO: 1580), SKTHADNNNSEYSWTG (SEQ ID NO: 34 of US20160369298; SEQ ID NO: 1581 in this article), SKTQADNNNSEYSWTG (SEQ ID NO: 35 of US20160369298; SEQ ID NO: 1582 in this article), SKTIADNNNSEYSWTG (SEQ ID NO: 1582 of US20160369298) SEQ ID NO : 36; in this article, it is SEQ ID NO: 1583), SKTMADNNNSEYSWTG (SEQ ID NO: 37 of US20160369298; in this article, it is SEQ ID NO: 1584), SKTRADNNNSEYSWTG (SEQ ID NO: 38 of US20160369298; in this article, it is SEQ ID NO: 1585), SKTNADNNNSEYSWTG (SEQ ID NO: 39 of US20160369298; SEQ ID NO: 1586 in this article), SKTVGRNNNSEYSWTG (SEQ ID NO: 40 of US20160369298; SEQ ID NO: 1587 in this article), SKTADRNNNSEYSWTG (SEQ ID NO: 1587 of US20160369298) EQ ID NO : 41; SEQ ID NO: 1588 in this article), SKKLSQNNNSKYSWQG (SEQ ID NO: 42 of US20160369298; SEQ ID NO: 1589 in this article), SKPTTGNNNSDYSWPG (SEQ ID NO: 43 of US20160369298; SEQ ID NO: 43 in this article) 1590), STQKNENNNSNYSWPG (SEQ ID NO: 44 of US20160369298; SEQ ID NO: 1591 in this article), HKDDEGKF (SEQ ID NO: 45 of US20160369298; SEQ ID NO: 1592 in this article), HKDDNRKF (SEQ ID NO of US20160369298) : 46; in this article, it is SEQ ID NO: 1593), HKDDTNKF (SEQ ID NO: 47 of US20160369298; in this article, it is SEQ ID NO: 1594), HEDSDKNF (SEQ ID NO: 48 of US20160369298; in this article, it is SEQ ID NO: 1595), HRDGADSF (SEQ ID NO: 49 of US20160369298; SEQ ID NO: 1596 in this article), HGDNKSRF (SEQ ID NO: 50 of US20160369298; SEQ ID NO: 1597 in this article), KQGSEKTNVDFEEV (SEQ ID NO of US20160369298) : 51; this article is SEQ ID NO: 1598), KQGSEKTNVDSEEV (US20160369298's SEQ ID NO: 52; this article is SEQ ID NO: 1599), KQGSEKTNVDVEEV (US20160369298's SEQ ID NO: 53; this article is SEQ ID NO: 1600), KQGSDKTNVDDAGV (SEQ ID NO: 54 of US20160369298; SEQ ID NO: 1601 in this article), KQGSSKTNVDPREV (SEQ ID NO: 55 of US20160369298; SEQ ID NO: 1602 in this article), KQGSRKTNVDHKQV (US2016036 SEQ ID NO of 9298 : 56; this article is SEQ ID NO: 1603), KQGSKGGNVDTNRV (US20160369298's SEQ ID NO: 57; this article is SEQ ID NO: 1604), KQGSGEANVDNGDV (US20160369298's SEQ ID NO: 58; this article is SEQ ID NO: 1605), KQDAAADNIDYDHV (SEQ ID NO: 59 of US20160369298; SEQ ID NO: 1606 in this article), KQSGTRSNAAASSV (SEQ ID NO: 60 of US20160369298; SEQ ID NO: 1607 in this article), KENTNTNDTELTNV (SEQ ID NO: 1607 of US20160369298) EQ ID NO : 61; this article is SEQ ID NO: 1608), QRGNNVAATADVNT (US20160369298's SEQ ID NO: 62; this article is SEQ ID NO: 1609), QRGNNEAATADVNT (US20160369298's SEQ ID NO: 63; this article is SEQ ID NO: 1610), QRGNNPAATADVNT (SEQ ID NO: 64 of US20160369298; SEQ ID NO: 1611 in this article), QRGNNHAATADVNT (SEQ ID NO: 65 of US20160369298; SEQ ID NO: 1612 in this article), QEENNIAATPGVNT (SEQ ID NO: 1612 of US20160369298) EQ ID NO : 66; SEQ ID NO: 1613 in this article), QPPNNMAATHEVNT (SEQ ID NO: 67 of US20160369298; SEQ ID NO: 1614 in this article), QHHNNSAATTIVNT (SEQ ID NO: 68 of US20160369298; SEQ ID NO: 68 in this article) 1615), QTTNNRAAFNMVET (SEQ ID NO: 69 of US20160369298; SEQ ID NO: 1616 in this article), QKKNNNAASKKVAT (SEQ ID NO: 70 of US20160369298; SEQ ID NO: 1617 in this article), QGGNNKAADDAVKT (SEQ ID NO: 1617 of US20160369298) SEQ ID NO : 71; this article is SEQ ID NO: 1618), QAAKGGAADDAVKT (US20160369298's SEQ ID NO: 72; this article is SEQ ID NO: 1619), QDDRAAAANESVDT (US20160369298's SEQ ID NO: 73; this article is SEQ ID NO: 1620), QQQHDDAAYQRVHT (SEQ ID NO: 74 of US20160369298; SEQ ID NO: 1621 in this article), QSSSSLAAVSTVQT (SEQ ID NO: 75 of US20160369298; SEQ ID NO: 1622 in this article), QNNQTTAAIRNVTT (US20160369298 SEQ ID NO : 76; in this article, it is SEQ ID NO: 1623), NYNKKSDNVDFT (SEQ ID NO: 77 of US20160369298; in this article, it is SEQ ID NO: 1624), NYNKKSENVDFT (SEQ ID NO: 78 of US20160369298; in this article, it is SEQ ID NO: 1625), NYNKKSLNVDFT (US20160369298 SEQ ID NO: 79; this article is SEQ ID NO: 1626), NYNKKSPNVDFT (US20160369298 SEQ ID NO: 80; this article is SEQ ID NO: 1627), NYSS KKSHCVDFT (US20160369298 SEQ ID NO : 81; this article is SEQ ID NO: 1628), NYRKTIYVDFT (US20160369298's SEQ ID NO: 82; this article is SEQ ID NO: 1629), NYKEKKDVHFT (US20160369298's SEQ ID NO: 83; this article is SEQ ID NO: 1630), NYGHRAIVQFT (SEQ ID NO: 84 of US20160369298; SEQ ID NO: 1631 in this article), NYANHQFVVCT (SEQ ID NO: 85 of US20160369298; SEQ ID NO: 1632 in this article), NYDDDPTGVLLT (SEQ ID NO of US20160369298) : 86; this article is SEQ ID NO: 1633), NYDDPTGVLLT (US20160369298's SEQ ID NO: 87; this article is SEQ ID NO: 1634), NFEQQNSVEWT (US20160369298's SEQ ID NO: 88; this article is SEQ ID NO: 1635), SQSGASN (SEQ ID NO: 89 and SEQ ID NO: 241 of US20160369298; SEQ ID NO: 1636 in this article), NNGSQA (SEQ ID NO: 90 of US20160369298; SEQ ID NO: 1637 in this article), YYLSRTNTPSGTTTWSRLQFSQAGA (SEQ ID NO: 91 of US20160369298; SEQ ID NO: 1638 in this article), SKTSADNNNSEYSWTG (SEQ ID NO: 92 of US20160369298; SEQ ID NO: 1639 in this article), HKDDEEKF (SEQ ID NO: 93, 209 of US20160369298) , 214, 219, 224, 234, 239 and 244; SEQ ID NO: 1640 in this article), KQGSEKTNVDIEEV (SEQ ID NO: 94 of US20160369298; SEQ ID NO: 1641 in this article), QRGNNQAATADVNT (SEQ ID NO of US20160369298) : 95; SEQ ID NO: 1642 in this article), NYNKKSVNVDFT (SEQ ID NO: 96 of US20160369298; SEQ ID NO: 1643 in this article), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSEYSWTGATKYH (SEQ ID NO: 106 of US20160369298; SEQ ID NO: 106 in this article): 1644), SASGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 107 of US20160369298; SEQ ID NO: 1645 in this article), SQSGASNYNTPSGTTTQSRLQFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 108 of US20160369298; SEQ ID in this article) NO: 1646), SASGASNYNTPSGTTTQSRLQFSTSADNNNSEFSWPGATTYH (SEQ ID NO of US20160369298 : 109; SEQ ID NO: 1647 in this article), SQSGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 110 of US20160369298; SEQ ID NO: 1648 in this article), SASGASNYNTPSGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID of US20160369298) NO: 111; in this article, it is SEQ ID NO: 1649), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSDFSWTGATKYH (SEQ ID NO: 112 of US20160369298; SEQ ID NO: 1650 in this article), SGAGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 113 of US20160369298; SEQ ID in this article) NO: 1651), SGAGASN (SEQ ID NO of US20160369298 : 176; SEQ ID NO: 1652 in this article), NSEGGSLTQSSLGFS (SEQ ID NO: 177, 185, 193 and 202 of US20160369298; SEQ ID NO: 1653 in this article), TDGENNNSDFS (SEQ ID NO: 178 of US20160369298; this article Among them are SEQ ID NO: 1654), SEFSWPGATT (SEQ ID NO: 179 of US20160369298; SEQ ID NO: 1655 in this article), TSADNNNSDFSWT (SEQ ID NO: 180 of US20160369298; SEQ ID NO: 1656 in this article), SQSGASNY (SEQ ID NO: 181, 187 and 198 of US20160369298; SEQ ID NO: 1657 in this article), NTPSGTTTQSRLQFS (SEQ ID NO: 182, 188, 191 and 199 of US20160369298; SEQ ID NO: 1658 in this article), TSADNNNSEYSWTGATKYH (SEQ ID NO: 183 of US20160369298; SEQ ID NO: 1659 in this article), SASGASNF (SEQ ID NO: 184 of US20160369298; SEQ ID NO: 1660 in this article), TDGENNNSDFSWTGATKYH (SEQ ID NO: 186, 189 of US20160369298) , 194, 197 and 203; in this article, SEQ ID NO: 1661), SASGASNY (SEQ ID NO: 190 and SEQ ID NO: 195 of US20160369298; in this article, SEQ ID NO: 1662), TSADNNNSEFSWPGATTYH (SEQ ID NO of US20160369298) : 192; in this article, it is SEQ ID NO: 1663), NTPSGSLTQSSLGFS (SEQ ID NO: 196 of US20160369298; in this article, it is SEQ ID NO: 1664), TSADNNNSDFSWTGATKYH (SEQ ID NO: 200 of US20160369298; in this article, it is SEQ ID NO: 1665), SGAGASNF (SEQ ID NO: 201 of US20160369298; SEQ ID NO: 1666 in this article), CTCCAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACACAA (SEQ ID NO: 204 of US20160369298; SEQ ID NO: 1667 in this article), CTCCAGAGAGGCAACA GACAAGCAGCTACCGCAGATGTCAACACACAA (SEQ ID NO of US20160369298 : 205; in this article, it is SEQ ID NO: 1668), SAAGASN (SEQ ID NO: 206 of US20160369298; in this article, it is SEQ ID NO: 1669), YFLSRTNTESGSTTQSTLRFSQAG (SEQ ID NO: 207 of US20160369298; in this article, it is SEQ ID NO: 1670), SKTSADNNNSDFS (SEQ ID NO: 208, 228 and 253 of US20160369298; SEQ ID NO: 1671 in this article), KQGSEKTDVDIDKV (SEQ ID NO: 210 of US20160369298; SEQ ID NO: 1672 in this article), STAGASN (US201603 69298 SEQ ID NO: 211; SEQ ID NO: 1673 in this article), YFLSRTNTTSGIETQSTLRFSQAG (SEQ ID NO: 212 and SEQ ID NO: 247 of US20160369298; SEQ ID NO: 1674 in this article), SKTDGENNNSDFS (SEQ ID NO of US20160369298) : 213 and SEQ ID NO: 248; in this article, SEQ ID NO: 1675), KQGAAADDVEIDGV (SEQ ID NO: 215 and SEQ ID NO: 250 of US20160369298; in this article, SEQ ID NO: 1676), SEAGASN (SEQ of US20160369298) ID NO: 216; SEQ ID NO: 1677 in this article), YYLSRTNTPSGTTTQSRLQFSQAG (SEQ ID NO: 217, 232 and 242 of US20160369298; SEQ ID NO: 1678 in this article), SKTSADNNNSEYS (SEQ ID NO: 218, 233 of US20160369298) , 238 and 243; this article is SEQ ID NO: 1679), KQGSEKTNVDIEKV (US20160369298 SEQ ID NO: 220, 225, and 245; this article is SEQ ID NO: 1680), YFLSRTNDTKSTLFSQAG (U. S20160369298 SEQ ID NO: 222; This article SEQ ID NO: 1681), STTPSENNNSEYS (SEQ ID NO: 223 of US20160369298; SEQ ID NO: 1682 in this article), SAAGATN (SEQ ID NO: 226 and SEQ ID NO: 251 of US20160369298; SEQ ID NO: 251 in this article) NO: 1683), YFLSRTNGEAGSATLSELRFSQAG (SEQ ID NO: 227 of US20160369298; SEQ ID NO: 1684 in this article), HGDDADRF (SEQ ID NO: 229 and SEQ ID NO: 254 of US20160369298; SEQ ID NO: 1685 in this article) , KQGAEKSDVEVDRV (SEQ ID NO: 230 and SEQ ID NO: 255 of US20160369298; SEQ ID NO: 1686 in this article), KQDSGGDNIDIDQV (SEQ ID NO: 235 of US20160369298; SEQ ID NO: 1687 in this article), SDAGASN (US2016036 9298 SEQ ID NO: 236; SEQ ID NO: 1688 in this article), YFLSRTNTEGGHDTQSTLRFSQAG (SEQ ID NO: 237 of US20160369298; SEQ ID NO: 1689 in this article), KEDGGSDVAIDEV (SEQ ID NO: 240 of US20160369298; SEQ ID NO: 240 in this article) SEQ ID NO: 1690), SNAGASN (SEQ ID NO: 246 of US20160369298; SEQ ID NO: 1691 in this article) and YFLSRTNGEAGSATLSELRFSQPG (SEQ ID NO: 252 of US20160369298; SEQ ID NO: 1692 in this article). Non-limiting examples of nucleotide sequences that can encode amino acid mutation sites include the following: AGCVVMDCAGGARSCASCAAC (SEQ ID NO: 97 of US20160369298; herein SEQ ID NO: 1693), AACRACRRSMRSMAGGCA (SEQ ID NO: 98 of US20160369298) ; In this article, it is SEQ ID NO: 1694), CACRRGGACRRCRMSRRSARSTTT (SEQ ID NO: 99 of US20160369298; In this article, it is SEQ ID NO: 1695), TATTTCTTGAGCAGAACAAACRVCVVSRSCGGAMNCVHSACGMHSTCAVVSCTTVDSTTTTTCCAGSBCRGSGCG (SEQ ID NO of US20160369298 : 100; in this article, SEQ ID NO: 1696) , TCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCGTGGMMAGGA (SEQ ID NO: 101 of US20160369298; SEQ ID NO: 1697 in this article), AAGSAARRCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTC (SEQ ID NO: 102 of US20160369298; SEQ ID NO: 16 in this article) 98), CAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACA (SEQ ID NO: 103 of US20160369298 ; SEQ ID NO: 1699 in this article), AACTWCRVSVASMVSVHSDDTGTGSWSTKSACT (SEQ ID NO: 104 of US20160369298; SEQ ID NO: 1700 in this article), TTGTTGAACATCACCACGTGACGCACGTTC (SEQ ID NO: 256 of US20160369298; SEQ ID NO: 1 in this article) 701) , TCCCCGTGGTTCTACTACATAATGTGGCCG (SEQ ID NO: 257 of US20160369298; SEQ ID NO: 1702 in this article), TTCCACACTCCGTTTTGGATAATGTTGAAC (SEQ ID NO: 258 of US20160369298; SEQ ID NO: 1703 in this article), AGGGACATCCCCAGCTCCATGCTGTGGT CG (SEQ ID NO: 259 of US20160369298 ; SEQ ID NO: 1704 in this article), AGGGACAACCCCTCCGACTCGCCCTAATCC (SEQ ID NO: 260 of US20160369298; SEQ ID NO: 1705 in this article), TCCTAGTAGAAGACACCCTCTCACTGCCCG (SEQ ID NO: 261 of US20160369298; SEQ ID NO: 1706 in this article) , AGTACCATGTACACCCACTCTCCCAGTGCC (SEQ ID NO: 262 of US20160369298; SEQ ID NO: 1707 in this article), ATATGGACGTTCATGCTGATCACCATAACCG (SEQ ID NO: 263 of US20160369298; SEQ ID NO: 1708 in this article), AGCAGGAGCTCCTTGGCCTCAGCGTGCGAG (SEQ ID NO: 264 of US20160369298 ; This article is SEQ ID NO: 1709), ACAAGCAGCAGCTCTCTACTACCACCACCTGAC (US20160369298 SEQ ID NO: 265; this article is SEQ ID NO: 1710), Cagcctaggaaactgggaccccctg TTACCGCAGCAGCAGAGAGTCAAMMMAMAVNSRVCSRSRSRSRSRSAAACACATRASTCTCTCTGGGMMAGGGMMAGGGMMAGGGGGGGCACCAATCAGGGGGGACCCCCCCCGGGGGGGGCTAGGCA AGCCACRRRRRRRRMSRSRSARSARSARSARSSARSARSAGCGGGGGGGGGGGGGGGGGGGGAARRSRVARVARVARVRVARVCGMSNHCRVMVRSGAgAgAgAgAgAgAgAC (USA 20160369298 SEQ ID NO: 266; this article is SEQ ID NO: 1711) , TGGGACAATGGCGGTCGTCTCTCAGAGTTKTKKT (SEQ ID NO: 267 of US20160369298; SEQ ID NO: 1712 in this article), AGAGGACCKKTCCTCGATGGTTCATGGTGGAGTTA (SEQ ID NO: 268 of US20160369298; SEQ ID NO: 1713 in this article), CCACTTAGGGCCTGG TCGATACCGTTCGGTG (SEQ ID NO: 269 of US20160369298 ; SEQ ID NO: 1714 in this article) or TCTCGCCCCAAGAGTAGAAACCCTTCSTTYYG (SEQ ID NO: 270 of US20160369298; SEQ ID NO: 1715 in this article).

In some embodiments, AAV serotypes may comprise peptides targeting eye cells as described in International Patent Publication WO2016134375, which is incorporated herein by reference in its entirety, such as (but not limited to) SEQ ID NO of WO2016134375 : 9 or SEQ ID NO: 10. Furthermore, any of the eye cell-targeting peptides or amino acids described in WO2016134375 can be inserted into any parent AAV serotype, such as (but not limited to) AAV2 (SEQ ID NO: 8 of WO2016134375; herein SEQ ID NO: 1716), or AAV9 (SEQ ID NO: 11 of WO2016134375; SEQ ID NO: 1717 in this article). In some embodiments, modifications such as insertions occur at P34-A35, T138-A139, A139-P140, G453-T454, N587-R588 and/or R588-Q589 in the AAV2 protein. In certain embodiments, the insertion occurs at D384, G385, 1560, T561, N562, E563, E564, E565, N704 and/or Y705 of AAV9. The peptide targeting eye cells may be, but is not limited to, any of the following amino acid sequences: GSTPPPM (SEQ ID NO: 1 of WO2016134375; herein SEQ ID NO: 1718), or GETRAPL (SEQ of WO2016134375 ID NO: 4; herein SEQ ID NO: 1719).

In some embodiments, AAV serotypes may be modified as described in U.S. Patent Application Publication No. US 20170145405, which is incorporated by reference in its entirety. AAV serotypes may include modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F, and/or S662V), modified AAV3 (e.g., modifications at Y705F, Y731F, and/or T492V), and modified AAV6 (e.g., modifications at S663V and/or T492V).

In some embodiments, AAV serotypes may be modified as described in International Publication No. WO2017083722, which is incorporated by reference in its entirety. AAV serotypes may include AAV1 (Y705+731F+T492V), AAV2 (Y444+500+730F+T491V), AAV3 (Y705+731F), AAV5, AAV 5 (Y436+693+719F), AAV6 (VP3 variant Y705F /Y731F/T492V), AAV8 (Y733F), AAV9, AAV9 (VP3 variant Y731F) and AAV10 (Y733F).

In some embodiments, as described in International Patent Publication No. WO2017015102, the contents of which are incorporated herein by reference in their entirety, AAV serotypes may comprise engineered epitopes comprising the amino acid SPAKFA (SEQ of WO2017015102 ID NO: 24; herein SEQ ID NO: 1720) or NKDKLN (SEQ ID NO: 2 of WO2017015102; herein SEQ ID NO: 1721). The epitope can be inserted into the region of amino acids 665 to 670 of AAV8 (numbering based on the VP1 capsid) (SEQ ID NO: 3 of WO2017015102) and/or residues 664 to 668 of AAV3B (SEQ ID NO: 3) middle.

In some embodiments, the AAV serotype may be or may have a sequence as described in International Patent Publication No. WO2017058892, which is incorporated herein by reference in its entirety, such as (but not limited to) having a serotype that may include AAV1 One or more of amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500, 528-534, 547-552, 588-597, 709-710 or 716-722 (e.g. 2, 3, 4, 5, 6 or 7) (in any combination) or AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAV variants of the capsid protein with substitutions at equivalent amino acid residues in bovine AAV or avian AAV. The amino acid substitution may be, but is not limited to, any of the amino acid sequences described in WO2017058892. In some embodiments, AAV may comprise residues 256L, 258K, 259Q, 261S, 263A, 264S, 265T, 266G, 272H, 385S, 386Q, S472R, V473D, N500E 547S of AAV1 (SEQ ID NO: 1 of WO2017058892) , 709A, 710N, 716D, 717N, 718N, 720L, A456T, Q457T, N458Q, K459S, T492S, K493A, S586R, S587G, S588N, T589R and/or 722T (in any combination), AAV5 (SEQ of WO2017058892 ID NO: 5) Residues 244N, 246Q, 248R, 249E, 250I, 251K, 252S, 253G, 254S, 255V, 256D, 263Y, 377E, 378N, 453L, 456R, 532Q, 533P, 535N, 536P, 537G, 538T, 539T , 540A, 541T, 542Y, 543L, 546N, 653V, 654P, 656S, 697Q, 698F, 704D, 705S, 706T, 707G, 708E, 709Y and/or 710R (in any combination), AAV5 (SEQ ID NO of WO2017058892: 5) Residues 248R, 316V, 317Q, 318D, 319S, 443N, 530N, 531S, 532Q 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D, 706T, 708E, 709Y and/or 710R (in any combination), residues 264S, 266G, 269N, 272H, 457Q, 588S and/or 589I (in any combination) of AAV6 (SEQ ID NO: 6 of WO2017058892), AAV8 (SEQ ID NO: 6 of WO2017058892 NO: 8) residues 457T, 459N, 496G, 499N, 500N, 589Q, 590N and/or 592A (in any combination), AAV9 (SEQ ID NO: 9 of WO2017058892) residues 451I, 452N, 453G, 454S Amino acid substitutions at , 455G, 456Q, 457N and/or 458Q (in any combination).

In some embodiments, the AAV may include at positions 155, 156, and 157 of VP1 or at positions 17, 18, Amino acid sequences at positions 19 and 20. The amino acid sequence may be, but is not limited to, N-S-S, S-X-S, S-S-Y, N-X-S, N-S-Y, S-X-Y or N-X-Y, where N, X and Y are independently, but not limited to, a non-serine or non-threonine amino group acid, wherein AAV may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In some embodiments, the AAV may include a deletion of at least one amino acid at position 156, 157, or 158 of VP1 or at position 19, 20, or 21 of VP2, wherein the AAV may be (but is not limited to) AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.

In some embodiments, the AAV can be as described by Deverman et al., (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and Serotypes generated by Cre recombination-based targeted evolution of AAV (CREATE) are described in International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of which are incorporated herein by reference in their entirety. In some embodiments, AAV serotypes produced in this manner have improved CNS transduction and/or neuronal and astrocyte tropism compared to AAV serotypes not produced in this manner. As non-limiting examples, AAV serotypes may include targeting peptides such as (but not limited to) PHP.B, PHP.B2, PHP.B3, PHP.A, PHP.S, PHP.N, G2A12, G2A15, G2A3 , G2B4 and G2B5. In some embodiments, these AAV serotypes may be derivatives of AAV9 (SEQ ID NO: 136) or AAV9 K449R (SEQ ID NO: 9), which have an amino acid insertion between amino acids 588 and 589 sequence. Non-limiting examples of such amino acid insertion sequences include TLAVPFK (PHP.B; SEQ ID NO: 1260), SVSKPFL (PHP.B2; SEQ ID NO: 1268), FLTLTTPK (PHP.B3; SEQ ID NO: 1269) ), YTLSQGW (PHP.A; SEQ ID NO: 1275), QAVRTSL (PHP.S; SEQ ID NO: 1319), LAKERLS (G2A3; SEQ ID NO: 1320), MNSTKNV (G2B4; SEQ ID NO: 1321), VSGGHHS (G2B5; SEQ ID NO: 1322) and/or DGTLAVPFKAQ (PHP.N; SEQ ID NO: 1289).

In some embodiments, AAV serotypes can be as described in Jackson et al. (Frontiers in Molecular Neuroscience 9:154 (2016)), the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the AAV serotype is AAV9 (SEQ ID NO: 135 or 136). In some embodiments, the AAV serotype is AAV9 with a peptide insert.

In some embodiments, the AAV serotype is the K449R AAV9 variant (SEQ ID NO: 9). AAV9 K449R has the same functions as wild-type AAV9. In some embodiments, the AAV serotype is AAV9 K449R with a peptide insert.

In some embodiments, the AAV serotype is PHP.B (eg, as described in WO2015038958). In some embodiments, AAV serotypes are paired with a synaptophysin promoter that enhances neuronal transduction compared to using a more general promoter (ie, CBA or CMV).

In some embodiments, the AAV serotype is PHP.N (eg, as described in WO2017100671).

In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.N (PHP.N) peptide or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.B (PHP.B) peptide, or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.A (PHP.A) peptide, or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising the PHP.S peptide or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising the PHP.B2 peptide or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising the PHP.B3 peptide or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising a G2B4 peptide or a variant thereof.

In some embodiments, the AAV serotype is a serotype comprising a G2B5 peptide or a variant thereof.

In some embodiments, the AAV serotype is VOY101 or a variant thereof. In some embodiments, VOY101 comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the capsid sequence comprises the nucleic acid sequence of SEQ ID NO: 1722.

In some embodiments, the AAV serotype is VOY201 or a variant thereof. In some embodiments, VOY201 comprises the amino acid sequence of SEQ ID NO: 1724. In some embodiments, the capsid sequence comprises the nucleic acid sequence of SEQ ID NO: 1723.

In some embodiments, AAV capsids allow penetration of the blood-brain barrier following intravenous administration. Non-limiting examples of such AAV capsids include AAV9, AAV9 K449R, VOY101, VOY201 or AAV capsids that include a peptide insert such as, but not limited to, AAVPHP.N (PHP.N), AAVPHP. B (PHP.B), PHP.S, G2A3, G2B4, G2B5, G2A12, G2A15, PHP.B2, PHP.B3, or AAVPHP.A (PHP.A).

In some embodiments, AAV serotypes may comprise 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% as any of those described above , 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical capsid amino acid sequence. In some embodiments, an AAV serotype comprises a capsid amino acid sequence that is at least 80% identical to SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, an AAV serotype comprises a capsid amino acid sequence that is at least 85% identical to SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, an AAV serotype comprises a capsid amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, an AAV serotype comprises a capsid amino acid sequence that is at least 95% identical to SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, an AAV serotype comprises a capsid amino acid sequence that is at least 99% identical to SEQ ID NO: 1, 2, 3, 9, 136, or 1724. In some embodiments, the AAV serotype comprises the capsid amino acid of SEQ ID NO: 1, 2, 3, 9, 136, or 1724.

In some embodiments, the AAV serotypes may consist of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75% ,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical capsid nucleic acid sequence encoding. In some embodiments, an AAV serotype comprises a capsid nucleic acid sequence that is at least 80% identical to SEQ ID NO: 4, 135, 1722, or 1723. In some embodiments, an AAV serotype comprises a capsid nucleic acid sequence that is at least 85% identical to SEQ ID NO: 4, 135, 1722, or 1723. In some embodiments, an AAV serotype comprises a capsid nucleic acid sequence that is at least 90% identical to SEQ ID NO: 4, 135, 1722, or 1723. In some embodiments, an AAV serotype comprises a capsid nucleic acid sequence that is at least 95% identical to SEQ ID NO: 4, 135, 1722, or 1723. In some embodiments, an AAV serotype comprises a capsid nucleic acid sequence that is at least 99% identical to SEQ ID NO: 4, 135, 1722, or 1723. In some embodiments, the AAV serotype comprises the capsid nucleic acid sequence of SEQ ID NO: 4, 135, 1722, or 1723.

In some embodiments, the initiation codon used to translate the AAV VP1 capsid protein may be CTG, TTG, or GTG as described in U.S. Patent No. 8,163,543, which is incorporated by reference in its entirety.

The present invention relates to structural capsid proteins (including VP1, VP2 and VP3) encoded by the capsid (Cap) gene. These capsid proteins form the protein structural outer shell (ie, capsid) of viral vectors such as AAV. VP capsid proteins synthesized from Cap polynucleotides typically include methionine as the first amino acid (Met1) in the peptide sequence, which corresponds to the start codon (AUG or ATG) in the Cap nucleotide sequence. ) associated. However, it is common for the first methionine (Met1) residue, or generally any first amino acid (AA1), to be cleaved by a protein processing enzyme such as Met-aminopeptidase after or during polypeptide synthesis. This "Met/AA-reduction" process is usually associated with the corresponding acetylation of the second amino acid in the polypeptide sequence (such as alanine, valine, serine, threonine, etc.). Met-reduction is usually performed with the VP1 and VP3 capsid proteins, but can also be performed with the VP2 capsid protein.

In the case of incomplete Met/AA-reduction, mixtures containing one or more (one, two or three) VP capsid proteins of the viral capsid can be produced, some of which may include Met1/AA1 amino acids (Met+/ AA+), and some of them may lack Met1/AA1 amino acids (Met-/AA-) due to Met/AA- reduction. For further discussion of Met/AA-reduction in capsid proteins, see Jin et al. Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods . 2017 Oct 28(5 ): 255-267; Hwang et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science . February 19, 2010. 327(5968): 973-977; the contents of which are each incorporated by reference in full. middle.

According to the present invention, reference to capsid proteins is not limited to abridged (Met-/AA-) or non-abridged (Met+/AA+) sequences, and may in this context refer to individual capsid proteins, viruses comprising mixtures of capsid proteins Capsids and/or polynucleotide sequences (or fragments thereof) encoding, describing, producing or obtaining the capsid proteins of the invention. Direct reference to "capsid protein" or "capsid polypeptide" (such as VP1, VP2 or VP3) may also include VP capsid proteins that include Met1/AA1 amino acids (Met+/AA+), as well as due to Met/AA- reduction The corresponding VP capsid protein lacks Met1/AA1 amino acids (Met-/AA-).

Furthermore, according to the present invention, reference to a specific SEQ ID NO (protein or nucleic acid) each comprising or encoding one or more capsid proteins including one or more Met1/AA1 amino acids (Met+/AA+) should be understood to teach that a lack of Met1/ The AA1 amino acid VP capsid protein, based on a review of the sequences, is obviously any sequence lacking only the first listed amino acid, whether methionine or not.

As a non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and includes the "Met1" amino acid (Met+) encoded by the AUG/ATG initiation codon will also be understood to teach that the length is 735 amino acids. Amino acids and does not include the VP1 polypeptide sequence of the "Met1" amino acid (Met-) in the Met+ sequence of 736 amino acids. As a second non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and includes the "AA1" amino acid (AA1+) encoded by any NNN start codon will also be understood to teach that the length is 735 VP1 polypeptide sequence of amino acids excluding the "AA1" amino acid (AA1-) in the AA1+ sequence of 736 amino acids.

References to viral capsids formed from VP capsid proteins (such as references to specific AAV capsid serotypes) may be accompanied by VP capsid proteins that include Met1/AA1 amino acids (Met+/AA1+), due to Met/AA1- reduction The corresponding VP capsid protein lacking Met1/AA1 amino acids (Met-/AA1-) or its combination (Met+/AA1+ and Met-/AA1-).

As a non-limiting example, AAV capsid serotypes may include VP1 (Met+/AA1+), VP1 (Met-/AA1-), or a combination of VP1 (Met+/AA1+) and VP1 (Met-/AA1-). AAV capsid serotypes may also include VP3 (Met+/AA1+), VP3 (Met-/AA1-), or a combination of VP3 (Met+/AA1+) and VP3 (Met-/AA1-); and may also include VP2 (Met+/ Similar combinations of AA1) and VP2 (Met-/AA1-) exist depending on the situation. Expression vehicle

In some aspects, the AAV particles of the invention serve as expression vehicles encoding FXN. The expression vector is not limited to AAV and may be an adenovirus, retrovirus, lentivirus, plasmid, vector, or any variant thereof.

In some embodiments, an AAV particle expression vector may include an ITR, a promoter, an intron, a nucleic acid sequence encoding FXN, a polyA sequence, and an ITR, stated from 5' to 3' from ITR to ITR. Inverted terminal repeat (ITR)

The AAV particles of the invention comprise a viral genome having at least one ITR region and a payload region encoding FXN. A "viral genome" or "vector genome" as used herein is a polynucleotide comprising at least one inverted terminal ITR and at least one encoded payload. In one embodiment, the viral genome has two ITRs. The two ITRs flank the payload area at the 5' and 3' ends. The ITR serves as the origin of replication and contains the recognition site for replication. ITRs contain complementary and symmetrically configured sequence regions. ITRs incorporated into the viral genome of the invention may comprise naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

The ITR can be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1, or derivatives thereof. The ITR can be of a different serotype than the capsid. In some embodiments, AAV particles have more than one ITR. In some embodiments, the AAV particle has a viral genome containing two ITRs. In some embodiments, the ITRs are of the same serotype as each other. In some embodiments, the ITRs are of different serotypes. Non-limiting examples include zero, one or two ITRs of the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

Independently, each ITR can be from about 100 nucleotides to about 150 nucleotides in length. The ITR can be about 100 to 105 nucleotides in length, 106 to 110 nucleotides in length, 111 to 115 nucleotides in length, 116 to 120 nucleotides in length, 121 to 125 nucleotides in length. Nucleotides, 126 to 130 nucleotides in length, 131 to 135 nucleotides in length, 136 to 140 nucleotides in length, 141 to 145 nucleotides in length or 146 to 150 nucleotides in length Nucleotides. In some embodiments, the ITR is 140 to 142 nucleotides in length. Non-limiting examples of ITR lengths are 102, 105, 119, 130, 140, 141, 142, or 145 nucleotides in length, and lengths of those nucleotides that are at least 95% identical thereto.

In some embodiments, the one or more ITRs are AAV2 ITRs or fragments or variants thereof. In some embodiments, both the 5'ITR and the 3'ITR are AAV2 ITRs or fragments or variants thereof. In some embodiments, the one or more ITRs are 141 nucleotides in length. In some embodiments, both the 5'ITR and the 3'ITR are 141 nucleotides in length. In some embodiments, the 5' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1811. In some embodiments, the 3' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1812. In some embodiments, the 5' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1811, and the 3' ITR comprises a sequence that is at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1812 % consistent sequence. In some embodiments, the viral genome includes a 5' ITR and a 3' ITR as described above and a payload region encoding a cotaxin, such as SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity. . In some embodiments, the viral genome includes a 5' ITR and a 3' ITR as described above and a payload region encoding a cotaxin, such as SEQ ID NO: 1824 or a variant thereof with at least 90% sequence identity. A payload region, such as a variant that retains one or more functional properties of wild-type cotaxin. promoter

Those skilled in the art will recognize that target cells may require specific promoters, including (but not limited to) species-specific, inducible, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997); the contents of which are incorporated herein by reference in their entirety).

In some embodiments, delivery of AAV particles to cells of the central nervous system (eg, parenchyma) comprises a composition, wherein the AAV genome further comprises a cell-specific promoter region. In some embodiments, the delivery comprises a composition, wherein the AAV genome further comprises a ubiquitous promoter region.

In some embodiments, the promoter is effective to drive expression of the payload or transgene. In some embodiments, the promoter is effective to drive the expression of FXN.

In some embodiments, the FXN promoter is used in the viral genome of an AAV particle encoding FXN, or a variant thereof. Certain embodiments provide that the FXN promoter is engineered for optimal FXN performance.

In some embodiments, the promoter is a weaker promoter that provides sustained expression of a payload (eg, FXN) over a period of time in a target tissue such as, but not limited to, nervous system tissue (eg, CNS tissue). Periods of sustainable performance are 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours , 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months , 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Performance lasts 1 to 5 hours, 1 to 12 hours, 1 to 2 days, 1 to 5 days, 1 to 2 weeks, 1 to 3 weeks, 1 to 4 weeks, 1 to 2 months, 1 to 4 months, 1 to 6 months, 2 to 6 months, 3 to 6 months, 3 to 9 months, 4 to 8 months, 6 to 12 months, 1 to 2 years, 1 to 5 years, 2 to 5 years , 3 to 6 years, 3 to 8 years, 4 to 8 years or 5 to 10 years. In some embodiments, the promoter is a weak promoter that consistently expresses the payload in neural tissue.

In some embodiments, the promoter may be less than 1 kb in size. The length of the promoter can be 50, 55, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 332, 340, 350, 360, 361, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 505, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 nucleotides. The length of the promoter can range from 50 to 100, 100 to 150, 150 to 200, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200 to 800, 300 to 400, 300 to 500 , 300 to 600, 300 to 700, 300 to 800, 400 to 500, 400 to 600, 400 to 700, 400 to 800, 500 to 600, 500 to 700, 500 to 800, 600 to 700, 600 to 800 or 700 to 800 nucleotides.

In some embodiments, the promoter may be a combination of two or more components such as, but not limited to, CMV and CBA. The length of each component can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383 ,384,385,386,387,388,389,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570 , 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 Nucleotides. The length of each component can range from 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200 to 800, 300 to 400, 300 to 500, 300 to 600, 300 to 700, 300 to Between 800, 400 to 500, 400 to 600, 400 to 700, 400 to 800, 500 to 600, 500 to 700, 500 to 800, 600 to 700, 600 to 800 or 700 to 800 nucleotides. In some embodiments, the promoter is a combination of a 382 nt CMV enhancer sequence and a 260 nt CBA promoter sequence. In some embodiments, the promoter is a combination of a 380 nucleotide CMV enhancer sequence and a 260 nucleotide CBA promoter sequence.

In some embodiments, the vector genome contains at least one element that enhances target specificity and expression of FXN (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated by reference in their entirety). are incorporated into this article). Non-limiting examples of expression-enhancing elements include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USE), CMV enhancers and introns . In certain embodiments, elements used to enhance target specificity and/or expression of FXN are referred to as "enhancers" or "enhancer" sequences. In some embodiments, a promoter may include enhancer sequences. In some embodiments, the enhancer can be a promoter-independent component of the viral genome. In some embodiments, the enhancer can be 5' to the promoter sequence in the viral genome. In some embodiments, the enhancer can be 3' to the promoter sequence in the viral genome. In some embodiments, the enhancer comprises or consists of SEQ ID NO: 1777.

As used herein, "intron" or "intron sequence" encompasses a full-length intron or fragments thereof. As used herein, "exon" or "exon sequence" encompasses a full-length exon or a fragment thereof. In some embodiments, an enhancer may comprise at least one intron or exon sequence. In some embodiments, an enhancer may comprise at least one intronic sequence. In some embodiments, an enhancer may comprise at least one exonic sequence. In some embodiments, the enhancer includes an intron sequence and an exon sequence. In some embodiments, the enhancer sequence contains two intronic sequences. In some embodiments, the enhancer sequence includes two exon sequences. In some embodiments, the enhancer sequence includes two intron sequences and two exon sequences. In some embodiments, the enhancer comprises SEQ ID NO: 1818. In some embodiments, an enhancer may include two intronic sequences and two exonic sequences. In some embodiments, the enhancer can include an iel exon (eg, exon 1), an iel intron (eg, intron 1), a human beta hemoglobin intron (eg, intron 2), and Human β-hemoglobulin exons (eg, exon 3). In some embodiments, the enhancer from 5' to 3' can include SEQ ID NOs: 1817, 1819, 1820, 1821. In some embodiments, the enhancer may comprise SEQ ID NO: 1816.

Promoters that promote expression in most tissues include, but are not limited to, human elongation factor 1 alpha-subunit (EF1 alpha), immediate early cytomegalovirus (CMV), chicken beta-actin (CBA) and its derivative CAG , β-glucuronidase (GUSB) or ubiquitin C (UBC). Tissue-specific expression elements can be used to limit expression to certain cell types, such as (but not limited to) nervous system promoters that can be used to limit expression to neurons, astrocytes, or oligodendritic glial cells. Non-limiting examples of tissue-specific expression elements for neurons include 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), CaMKII, mGluR2, NFL, NFH, nβ2, PPE, Enk and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for oligodendritic glial cells include the myelin basic protein (MBP) promoter.

In some embodiments, the vector genome contains ubiquitous promoters. Non-limiting examples of ubiquitous promoters include H1, U6, CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp) and UCOE (promoter of HNRPA2B1-CBX3) . Yu et al. (Molecular Pain 2011, 7:63; the content of which is incorporated by reference in its entirety) used lentiviral vectors to evaluate the expression of eGFP under the CAG, EFIα, PGK and UBC promoters in rat DRG cells and primary DRG. Expression in cells, and it was found that UBC showed weaker expression than the other three promoters and only 10-12% glial expression was present in all promoters. Soderblom et al. (E. Neuro 2015; the contents of which are incorporated by reference in their entirety) studied the expression of eGFP in AAV8 via the CMV and UBC promoters and in AAV2 via the CMV promoter after injection into the motor cortex. performance in. Intranasal administration of plasmids containing UBC or EFIα promoters showed sustained respiratory manifestations that were greater than with the CMV promoter (see, e.g., Gill et al., Gene Therapy 2001, Vol. 8, 1539-1546; the contents of which are The full text is incorporated into this article by reference). Husain et al. (Gene Therapy 2009; the contents of which are incorporated by reference in their entirety) evaluated HβH constructs with the hGUSB promoter, HSV-1LAT promoter, and NSE promoter and found that the HβH construct in mouse brain The performance shown is weaker than NSE. Passini and Wolfe (J. Virol. 2001, 12382-12392, the contents of which are incorporated by reference in their entirety) evaluated the long-term effects of an HβH vector after intraventricular injection into neonatal mice and found that there were persistent effects for at least 1 year. . Xu et al. (Gene Therapy 2001, 8, 1323-1332; the contents of which are incorporated by reference in their entirety) found that compared with CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE + wpre, NSE (0.3 kb), NSE (1.8 kb), and NSE (1.8 kb + wpre), showed lower expression in all brain regions when using the NF-L and NF-H promoters. Xu et al. found that the promoter activities in decreasing order are NSE (1.8 kb), EF, NSE (0.3 kb), GFAP, CMV, hENK, PPE, NFL and NFH. NFL is a 650 nucleotide promoter and NFH is a 920 nucleotide promoter. Neither promoter is present in the liver, but NFH is abundant in sensory proprioceptive neurons, brain and spinal cord. of and NFH is present in the heart. Scn8a is a 470-nucleotide promoter that is expressed throughout the DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus, and hypothalamus (see For example, Drews et al. 2007 and Raymond et al. 2004; the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, the vector genome includes a UBC promoter. UBC promoters can be 300 to 350 nucleotides in size. In some embodiments, the UBC promoter is 332 nucleotides in length.

In some embodiments, the vector genome contains the GUSB promoter. The GUSB promoter can be 350 to 400 nucleotides in size. In some embodiments, the GUSB promoter is 378 nucleotides in length. In some embodiments, the construct can be AAV-Promoter-CMV/Intein-FXN-RBG, where the AAV can be self-complementary, and the AAV can be AAV6, AAVrh10, or AAVDJ serotypes.

In some embodiments, the vector genome includes the NFL promoter. NFL promoters can be 600 to 700 nucleotides in size. In some embodiments, the NFL promoter is 650 nucleotides in length.

In some embodiments, the vector genome includes the NFH promoter. NFH promoters can be 900 to 950 nucleotides in size. In some embodiments, the NFH promoter is 920 nucleotides in length.

In some embodiments, the vector genome includes the scn8a promoter. The scn8a promoter can be 450 to 500 nucleotides in size. In some embodiments, the scn8a promoter is 470 nucleotides in length.

In some embodiments, the vector genome includes the FXN promoter.

In some embodiments, the vector genome includes a PGK promoter.

In some embodiments, the vector genome includes a CBA promoter.

In some embodiments, the vector genome includes a CMV promoter.

In some embodiments, the vector genome includes an H1 promoter.

In some embodiments, the vector genome includes a U6 promoter.

In some embodiments, the vector genome contains a liver or skeletal muscle promoter. Non-limiting examples of liver promoters include hAAT and TBG. Non-limiting examples of skeletal muscle promoters include Desmin, MCK, or C5-12.

In some embodiments, AAV vectors include enhancer elements, promoters, and/or 5'UTR introns. The enhancer may be (but is not limited to) a CMV enhancer; the promoter may be (but is not limited to) CMV, CBA, FXN, UBC, GUSB, NSE, synaptophysin, MeCP2 or GFAP promoter; and the 5'UTR/ Introns may be, but are not limited to, SV40 and CBA-MVM. In some embodiments, the enhancers, promoters and/or introns used in combination can be: (1) CMV enhancer, CMV promoter, SV40 5'UTR intron; (2) CMV enhancer, CBA Promoter, SV40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) synaptophysin promoter; (8) MeCP2 promoter; (9) GFAP promoter; (10) H1 promoter; and/or (11) U6 promoter.

In some embodiments, the AAV vector has an engineered promoter.

In some embodiments, the AAV vector comprises a promoter comprising a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1734-1777. In some embodiments, the promoter is or is derived from a CMV promoter and comprises at least 90%, at least 95%, Sequences with at least 99% or 100% sequence identity. In some embodiments, the promoter is or is derived from a CBA promoter and comprises at least 90%, at least 95, %, at least 99% or 100% sequence identity. In some embodiments, the promoter is or is derived from an FXN promoter and comprises at least 90%, at least 95%, at least 99%, or Sequences with 100% sequence identity.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1738. In some embodiments, the promoter is SEQ ID NO: 1738. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1738 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1738 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1738 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1738. In some embodiments, the promoter is SEQ ID NO: 1740. In some embodiments, the AAV vector genome comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1740 and encoding a fascin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1740 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1740 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1742. In some embodiments, the promoter is SEQ ID NO: 1742. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1742 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1742 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1742 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1750. In some embodiments, the promoter is SEQ ID NO: 1750. In some embodiments, the AAV vector genome comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1750 and encoding a cotaxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1750 and encoding a fascin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1750 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the viral genome includes an enhancer, such as an immediate early "ie" enhancer or a CMV/hemoglobulin enhancer. In some embodiments, the enhancer includes ie1 exon 1 and ie1 intron 1 or fragments thereof. In some embodiments, the enhancer includes ie1 exon 1, ie1 intron 1 or a fragment thereof, human beta-hemoglobin intron 2, and human beta-hemoglobulin exon 3. In some embodiments, the enhancer comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1815-1821. In some embodiments, the enhancer comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 1816. In some embodiments, the viral genome includes an enhancer as described above and encodes a cotaxin (e.g., encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity) or includes SEQ ID NO: 1824 A payload region of a nucleic acid sequence or a variant having at least 90% sequence identity thereto. intron

In some embodiments, the vector genome contains at least one intron or fragment or derivative thereof. In some embodiments, the at least one intron can enhance the expression of FXN (see, e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are incorporated herein by reference in their entirety). middle). Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), beta-hemoglobulin SD/immunoglobulin heavy chain splice acceptor (250 bp) , adenovirus splice donor/immunoglobulin splice acceptor (500 bp), SV40 late splice donor/splice acceptor (19S/16S) (180 bp) and hybrid adenovirus splice donor/IgG splice acceptor ( 230 bp).

In some embodiments, the intron can be 100 to 500 nucleotides in length. The length of the intron can be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 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 or 500 nucleotides. The length of the intron can range from 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80- Between 450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500 or 400-500 nucleotides.

In some embodiments, the AAV vector may comprise the SV40 intron or fragments or variants thereof. In some embodiments, the promoter can be CMV. In some embodiments, the promoter can be CBA. In some embodiments, the promoter may be H1.

In some embodiments, an AAV vector may comprise one or more beta-hemoglobulin introns or fragments or variants thereof. In some embodiments, the intron contains one or more human beta-hemoglobulin sequences (eg, including fragments/variants thereof).

In some embodiments, the intron comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the sequence set forth in any of SEQ ID NOs: 1815-1821. In some embodiments, the viral genome includes introns as described above and encodes a cotaxin (e.g., encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity) or includes the nucleic acid sequence SEQ ID NO :1824 or the payload region of a variant having at least 90% sequence identity thereto. In some embodiments, the promoter can be CMV. In some embodiments, the promoter can be CBA. In some embodiments, the promoter may be H1.

In some embodiments, the encoded FXN may be located downstream of an intron in the expression vector such as, but not limited to, the SV40 intron or the beta hemoglobin intron or other introns known in the art. . Additionally, the encoded FXN can also be located upstream of the polyadenylation sequence in the expression vector. In some embodiments, the encoded FXN can be located 1, 2, 3, 4, 5, 6, 7, 8, 9, downstream of the promoter with introns and/or upstream of the polyadenylation sequence in the expression vector. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides. In some embodiments, the encoded FXN can be located 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30 downstream of the intron and/or upstream of the polyadenylation sequence in the expression vector , 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, 15 to 30, 20 to 25, 20 to 30, or 25 to 30 nucleotides. In some embodiments, the encoded FXN can be located in the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, downstream of the intron and/or upstream of the polyadenylation sequence in the expression vector. Within 8%, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides. In some embodiments, the encoded FXN can be located 1-5%, 1-10%, 1-15%, 1-20%, 1 before the sequence downstream of the intron and/or upstream of the polyadenylation sequence in the expression vector -25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20 -25%.

In certain embodiments, the intronic sequence is not an enhancer sequence. In certain embodiments, the intronic sequence is not a secondary component of the promoter sequence. Untranslated region (UTR)

By definition, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. Generally, the 5' UTR begins at the transcription start site and ends at the initiation codon, and the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal.

Features typically found in genes that are well expressed in a specific target organ can be engineered into the UTR to enhance stability and protein production. As a non-limiting example, from mRNA normally expressed in the liver (e.g., albumin, serum amyloid A, lipoprotein A/B/E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) The 5' UTR can be used in the viral genome of the AAV particles of the invention to enhance performance in hepatocyte lines or liver.

While not wishing to be bound by theory, the wild-type 5' untranslated region (UTR) includes features that play a role in translation initiation. Often included in the 5' UTR, Kozak sequences are generally known to be involved in the process by which ribosomes initiate translation of multiple genes. Kozak sequences have the common CCR(A/G)CCAUGG, where R is the purine (adenine or guanine) three bases (ATG) upstream of the start codon, followed by another "G".

In some embodiments, the 5'UTR in the viral genome includes Kozak sequences.

In some embodiments, the 5' UTR in the viral genome does not include Kozak sequences.

While not wishing to be bound by theory, it is known that the wild-type 3' UTR has adenosine and uridine sequence segments embedded in it. Such AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995, the contents of which are incorporated herein by reference in full): Class I AREs, such as (but not limited to) c-Myc and MyoD contain several scattered copies of the AUUUA motif in the U-rich region. Class II AREs, such as (but not limited to) GM-CSF and TNF-a, have two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Class III ARES, such as (but not limited to) c-Jun and Myogenin, are less well defined. These U-rich regions do not contain the AUUUA motif. Most proteins that bind to the ARE are known to destabilize the message, while members of the ELAV family (most notably, HuR) have been documented to increase mRNA stability. HuR binds to all three types of AREs. Engineering a HuR-specific binding site into the 3' UTR of a nucleic acid molecule will cause HuR binding, thus causing message stabilization in vivo.

The introduction, removal, or modification of 3' UTR AU-rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering a specific polynucleotide, such as the payload region of a viral genome, one or more copies of the ARE can be introduced to render the polynucleotide less stable, and therefore reduce translation of the resulting protein and reduce its yield. . Likewise, AREs can be identified and removed or mutated to increase intracellular stability, and thus increase translation and production of the resulting protein.

In some embodiments, the 3' UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

Any UTR from any gene known in the art can be incorporated into the viral genome of the AAV particle. The UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they are selected, or may have their orientation or position changed. In some embodiments, the UTR used in the viral genome of the AAV particle may be inverted, shortened, elongated, or made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term "change" when relating to a UTR means that the UTR has changed in some way relative to the reference sequence. For example, the 3' or 5' UTR may be altered relative to the wild-type or native UTR based on changes in orientation or position as taught above, or may be modified by including additional nucleotides, nucleotide deletions, nucleotide deletions, Changed by transposition or transposition.

In some embodiments, the viral genome of the AAV particle contains at least one artificial UTR that is not a variant of the wild-type UTR.

In some embodiments, the viral genome of the AAV particle includes UTRs that have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property. miRNA target site

In some embodiments, the viral genome may include at least one miRNA binding site. MicroRNAs (or miRNAs or miRs) are non-coding RNAs of 19 to 25 nucleotides that bind to nucleic acid target sites and downregulate gene expression by reducing the stability of nucleic acid molecules or by inhibiting translation. In some embodiments, the 3' UTR of the viral genome can be engineered to include at least one miRNA binding site.

In some embodiments, the viral genome contains at least one sequence encoding a miRNA target site to reduce expression of the transgene in a specific tissue. MiRNAs and the tissues they target are well known in the art. In some embodiments, a miR-122 miRNA target site (miR-122TS) or a tandem copy thereof can be encoded in the viral genome to reduce the expression of the viral genome in the liver where miR-122 is fully expressed.

In some embodiments, the viral genome contains at least one miR122 binding site. In some embodiments, the miR122 binding site comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1827. In some embodiments, the AAV vector genome comprises three copies of a miR122 binding site, such as three copies of SEQ ID NO: 1827 or a variant thereof with at least 90% sequence identity. In some embodiments, the set of miR122 binding sites comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1826. In some embodiments, the viral genome includes one, two, or three miR122 binding sites as described above and encodes a coagulant protein (e.g., encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity ) or a payload region comprising the nucleic acid sequence SEQ ID NO: 1824 or a variant having at least 90% sequence identity thereto. In some embodiments, the viral genome comprises three miR122 binding sites as described above and encodes a syntaxin (e.g., encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity) or comprises the nucleic acid sequence SEQ The payload region of ID NO: 1824 or a variant with at least 90% sequence identity thereto. main chain

In certain embodiments, ipsilateral elements, such as the vector backbone, are incorporated into virions encoding FXN. Backbone sequences regulate transcription during virus production. The backbone sequence can contribute to the stability of FXN performance. The backbone sequence may contribute to the expression of FXN selectively cloned into the pAAVsc or pcDNA3.1 vector backbone. polyadenylation sequence

In some embodiments, the viral genome of the AAV particles of the invention includes at least one polyadenylation sequence. The viral genome of the AAV particle may comprise a polyadenylation sequence located between the 3' end of the payload coding sequence and the 5' end of the 3' UTR.

In some embodiments, the length of the polyadenylation sequence or "polyA sequence" can range from none to about 500 nucleotides. The length of the polyadenylation sequence can be (but is not limited to) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499 or 500 nucleotides.

In some embodiments, the polyadenylation sequence is 50 to 100 nucleotides in length.

In some embodiments, the polyadenylation sequence is 50 to 150 nucleotides in length.

In some embodiments, the polyadenylation sequence is 50 to 160 nucleotides in length.

In some embodiments, the polyadenylation sequence is 50 to 200 nucleotides in length.

In some embodiments, the polyadenylation sequence is 60 to 100 nucleotides in length.

In some embodiments, the polyadenylation sequence is 60 to 150 nucleotides in length.

In some embodiments, the polyadenylation sequence is 60 to 160 nucleotides in length.

In some embodiments, the polyadenylation sequence is 60 to 200 nucleotides in length.

In some embodiments, the polyadenylation sequence is 70 to 100 nucleotides in length.

In some embodiments, the polyadenylation sequence is 70 to 150 nucleotides in length.

In some embodiments, the polyadenylation sequence is 70 to 160 nucleotides in length.

In some embodiments, the polyadenylation sequence is 70 to 200 nucleotides in length.

In some embodiments, the polyadenylation sequence is 80 to 100 nucleotides in length.

In some embodiments, the polyadenylation sequence is 80 to 150 nucleotides in length.

In some embodiments, the polyadenylation sequence is 80 to 160 nucleotides in length.

In some embodiments, the polyadenylation sequence is 80 to 200 nucleotides in length.

In some embodiments, the polyadenylation sequence is 90 to 100 nucleotides in length.

In some embodiments, the polyadenylation sequence is 90 to 150 nucleotides in length.

In some embodiments, the polyadenylation sequence is 90 to 160 nucleotides in length.

In some embodiments, the polyadenylation sequence is 90 to 200 nucleotides in length.

In some embodiments, the encoded FXN can be located upstream of the polyadenylation sequence in the expression vector. Furthermore, the encoded FXN can be located downstream of a promoter with an SV40 intron (such as, but not limited to, CMV, U6, CBA, or the CBA promoter) in an expression vector or fragment thereof (eg, an expression vector disclosed herein). In some embodiments, the encoded FXN can be located downstream of the promoter and/or upstream of the polyadenylation sequence in the expression vector 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or within more than 30 nucleotides. In some embodiments, the encoded FXN can be located 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 1 to 30 downstream of the promoter and/or upstream of the polyadenylation sequence in the expression vector , 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 15 to 20, 15 to 25, 15 to 30, 20 to 25, 20 to 30, or 25 to 30 nucleotides. In some embodiments, the encoded FXN can be located 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% downstream of the promoter and/or upstream of the polyadenylation sequence in the expression vector %, 9%, 10%, 15%, 20%, 25% or more than 25% of the nucleotides. In some embodiments, the encoded FXN can be located 1-5%, 1-10%, 1-15%, 1-20%, 1-1% downstream of the promoter and/or upstream of the polyadenylation sequence in the expression vector. 25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25% or 20- Within 25%.

In some embodiments, the viral genome contains human growth hormone (hGH) polyA sequences. In some embodiments, the viral genome comprises a polyA sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1828. In some embodiments, the viral genome comprises hGH polyA as described above and encodes a cotaxin (e.g., encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity) or comprises the nucleic acid sequence SEQ ID NO: 1824 or the payload region of a variant having at least 90% sequence identity thereto. padding sequence

In some embodiments, the viral genome contains one or more stuffer sequences. The filler sequence can be a wild-type sequence or an engineered sequence. The filler sequence can be a variant of the wild-type sequence. In one embodiment, the stuffer sequence is a derivative of human albumin.

In some embodiments, the viral genome contains one or more stuffer sequences to optimize the length of the viral genome for optimal packaging size. In some embodiments, the viral genome includes at least one stuffer sequence such that the viral genome is approximately 2.3 kb in length. In some embodiments, the viral genome includes at least one stuffer sequence such that the viral genome is approximately 4.6 kb in length.

In some embodiments, the viral genome is a single-stranded (ss) viral genome and includes one or more stuffer sequences that independently or together are about 0.1 kb to 3.8 kb in length, such as (but not Limited to) 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb , 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb or 3.8 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 3.1 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 2.7 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 0.8 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 0.4 kb. In some embodiments, each stuffer sequence in the vector genome is 0.8 kb in length. In some embodiments, each stuffer sequence in the vector genome is 0.4 kb in length.

In some embodiments, the viral genome is a self-complementary (sc) viral genome and includes one or more stuffer sequences that independently or together are about 0.1 kb to 1.5 kb in length, such as (but not Limited to) 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1 kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, or 1.5 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 0.8 kb. In some embodiments, the full-length stuffer sequence in the vector genome is 0.4 kb. In some embodiments, each stuffer sequence in the vector genome is 0.8 kb in length. In some embodiments, each stuffer sequence in the vector genome is 0.4 kb in length.

In some embodiments, the viral genome contains any portion of the stuffer sequence. The viral genome can contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35 of the filler sequences %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

In some embodiments, the viral genome is a single-stranded (ss) viral genome and includes one or more stuffer sequences such that the viral genome is approximately 4.6 kb in length. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 3' end of the 5' ITR sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 5' end of the promoter sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 5' end of the 3' ITR sequence. In some embodiments, the viral genome contains at least one stuffer sequence, and the stuffer sequence is located between two intronic sequences. In some embodiments, the viral genome contains at least one stuffer sequence, and the stuffer sequence is located within an intronic sequence. In some embodiments, the viral genome contains two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. In some embodiments, the viral genome contains two stuffer sequences, and the first stuffer sequence is located at the 5' end of the promoter sequence and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. In some embodiments, the viral genome includes two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 5' end of the 5' ITR sequence.

In some embodiments, the viral genome is a self-complementary (sc) viral genome and includes one or more stuffer sequences such that the viral genome is approximately 2.3 kb in length. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 3' end of the 5' ITR sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 5' end of the promoter sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome includes at least one stuffer sequence, and the stuffer sequence is located at the 5' end of the 3' ITR sequence. In some embodiments, the viral genome contains at least one stuffer sequence, and the stuffer sequence is located between two intronic sequences. As a non-limiting example, the viral genome contains at least one stuffer sequence, and the stuffer sequence is located within an intron sequence. In some embodiments, the viral genome contains two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. In some embodiments, the viral genome contains two stuffer sequences, and the first stuffer sequence is located at the 5' end of the promoter sequence and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. In some embodiments, the viral genome includes two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 5' end of the 5' ITR sequence.

In some embodiments, the viral genome may comprise one or more filler sequences between one of more regions of the viral genome. In some embodiments, stuffing regions may be located such as, but not limited to, payload regions, inverted terminal repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and /or before the region of the exon region. In some embodiments, stuffing regions may be located such as, but not limited to, payload regions, inverted terminal repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and /or after the region of the exon region. In some embodiments, stuffing regions may be located such as, but not limited to, payload regions, inverted terminal repeats (ITRs), promoter regions, intron regions, enhancer regions, polyadenylation signal sequence regions, and /or before and after the region of the exon region.

In some embodiments, the viral genome may comprise one or more stuffer sequences that bifurcate at least one region of the viral genome. The bifurcated region of the viral genome may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% of the region at the 5' end of the stuffer sequence region %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In some embodiments, the stuffer sequence can bifurcate at least one region such that 10% of the region is at the 5' end of the stuffer sequence and 90% of the region is at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 20% of the region is at the 5' end of the stuffer sequence and 80% of the region is at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 30% of the region is located 5' of the stuffer sequence and 70% of the region is located 3' of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 40% of the region is at the 5' end of the stuffer sequence and 60% of the region is at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 50% of the region is at the 5' end of the stuffer sequence and 50% of the region is at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 60% of the region is located at the 5' end of the stuffer sequence and 40% of the region is located at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 70% of the region is at the 5' end of the stuffer sequence and 30% of the region is at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 80% of the region is located at the 5' end of the stuffer sequence and 20% of the region is located at the 3' end of the stuffer sequence. In some embodiments, the stuffer sequence can bifurcate at least one region such that 90% of the region is located at the 5' end of the stuffer sequence and 10% of the region is located at the 3' end of the stuffer sequence.

In some embodiments, the viral genome contains a stuffer sequence following the 5' ITR.

In some embodiments, the viral genome contains stuffer sequences following the promoter region. In some embodiments, the viral genome includes a stuffer sequence following the payload region. In some embodiments, the viral genome contains stuffer sequences following the intronic regions. In some embodiments, the viral genome contains a stuffer sequence following the enhancer region. In some embodiments, the viral genome includes a stuffer sequence following the polyadenylation signal sequence region. In some embodiments, the viral genome contains stuffer sequences following exon regions.

In some embodiments, the viral genome contains stuffer sequences preceding the promoter region. In some embodiments, the viral genome includes a stuffer sequence preceding the payload region. In some embodiments, the viral genome contains stuffer sequences preceding intronic regions. In some embodiments, the viral genome contains a stuffer sequence preceding the enhancer region. In some embodiments, the viral genome contains a stuffer sequence preceding the polyadenylation signal sequence region. In some embodiments, the viral genome contains stuffer sequences preceding exon regions.

In some embodiments, the viral genome contains a stuffer sequence preceding the 3' ITR.

In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a 5' ITR and a promoter region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) between the 5' ITR and the payload region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a 5' ITR and an intronic region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a 5' ITR and an enhancer region. In some embodiments, a stuffer sequence can be located between two regions, such as (but not limited to) between a 5' ITR and a polyadenylation signal sequence region.

In some embodiments, the stuffer sequence may be located between two regions, such as (but not limited to) between the 5' ITR and the exonic region.

In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and a payload region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and an intron region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and an enhancer region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and a polyadenylation signal sequence region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and an exon region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a promoter region and a 3' ITR.

In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a payload region and an intron region. In some embodiments, a filler sequence may be located between two regions, such as (but not limited to) a payload region and a booster region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a payload region and a polyadenylation signal sequence region. In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) a payload region and an exon region.

In some embodiments, a stuffer sequence may be located between two regions, such as (but not limited to) between the payload region and the 3' ITR. Self-complementary and single-stranded carriers

In some embodiments, AAV vectors used in the present invention are single-stranded vectors (ssAAV).

In some embodiments, the AAV vector may be a self-complementary AAV vector (scAAV). See, for example, U.S. Patent No. 7,465,583. scAAV vectors contain two DNA strands that are bonded together to form double-stranded DNA. By skipping second-strand synthesis, scAAV achieves rapid expression in cells.

In some embodiments, the AAV vector used in the invention is scAAV.

Methods for generating and/or modifying AAV vectors are disclosed in the art, such as pseudotyped AAV vectors (International Patent Publications Nos. WO200028004, WO200123001, WO2004112727, WO 2005005610 and WO 2005072364, The contents of each of these are incorporated herein by reference in their entirety). genome size

In some embodiments, the viral genome of the AAV particles of the invention can be single-stranded or double-stranded. The size of the vector genome can be small, medium, large or maximum size.

In some embodiments, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a small single-stranded vector genome. The small single-stranded vector genome may be about 2.7 kb to about 3.5 kb in size, such as about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, or about 3.5 kb in size. In some embodiments, the small single-stranded vector genome may be 3.2 kb in size.

In some embodiments, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a small double-stranded vector genome. The small double-stranded vector genome may be about 1.3 to about 1.7 kb in size, such as about 1.3, about 1.4, about 1.5, about 1.6, or about 1.7 kb in size. In some embodiments, the small double-stranded vector genome may be 1.6 kb in size.

In some embodiments, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a medium single-stranded vector genome. The size of a medium single-stranded vector genome can range from about 3.6 to about 4.3 kb, such as a size of about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, or about 4.3 kb. In some embodiments, a medium single-stranded vector genome may be 4.0 kb in size.

In some embodiments, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a medium double-stranded vector genome. The size of the medium double-stranded vector genome can range from about 1.8 to about 2.1 kb, such as a size of about 1.8, about 1.9, about 2.0, or about 2.1 kb. In some embodiments, the medium double-stranded vector genome may be 2.0 kb in size. Additionally, the vector genome may include a promoter and polyA tail.

In one embodiment, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a large single-stranded vector genome. Large single-stranded vector genomes can range in size from 4.4 to 6.0 kb, such as sizes of approximately 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0 kb. As a non-limiting example, a large single-stranded vector genome may be 4.7 kb in size. As another non-limiting example, a large single-stranded vector genome may be 4.8 kb in size. As yet another non-limiting example, a large single-stranded vector genome may be 6.0 kb in size.

In one embodiment, a vector genome comprising a nucleic acid sequence encoding FXN described herein can be a large double-stranded vector genome. Large double-stranded vector genomes can be 2.2 to 3.0 kb in size, such as about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 kb in size. As a non-limiting example, the large double-stranded vector genome may be 2.4 kb in size. payload

In some embodiments, the invention herein provides constructs that achieve improved expression of FXN delivered by gene therapy vectors.

In some aspects, the present invention relates to compositions containing or comprising a nucleic acid sequence encoding FXN or functional fragments thereof and administering the same in vitro or in vivo in humans and/or animal models of disease. and other composition methods.

The AAV particles of the invention may comprise a nucleic acid sequence encoding at least one "payload". As used herein, "payload" or "payload region" refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome, or such polynucleotides or polynucleotides Expression products of the nucleotide region, such as transgenic genes, polynucleotides encoding polypeptides or polypeptides (such as FXN or its variants). The payload may comprise methods known in the art that are suitable for expression of FXN in target cells transduced with or in contact with AAV particles carrying the payload (either by supplementing the protein product or by gene replacement using regulatory nucleic acids). of any nucleic acid.

The payload construct may contain a combination of coding and non-coding nucleic acid sequences.

Any segment, fragment, or entire viral genome and the payload constructs therein can be codon-optimized.

In some embodiments, the nucleic acid sequence of the AAV particle can be a payload construct comprising at least a portion encoding FXN.

In some embodiments, a payload construct encodes more than one payload. As a non-limiting example, payload constructs encoding more than one payload can be replicated and encapsulated into viral particles. Target cells transduced with viral particles containing more than one payload can express each of the payloads in a single cell.

In some embodiments, the payload construct may encode coding or non-coding RNA. In certain embodiments, the adeno-associated virus vector particles further comprise at least one ipsilateral element selected from the group consisting of Kozak sequences, backbone sequences, and intron sequences.

In some embodiments, the payload is a polypeptide, which can be a peptide or a protein. Proteins encoded by the payload construct may include secreted proteins, intracellular proteins, extracellular proteins, and/or membrane proteins. Encoded proteins may be structural or functional. Proteins encoded by payload constructs include, but are not limited to, mammalian proteins. In certain embodiments, the AAV particle contains a viral genome encoding FXN or a variant thereof. AAV particles encoding payloads may be suitable for use in human diseases, veterinary applications, and various in vivo and in vitro environments.

In some embodiments, payloads may include polypeptides that serve as marker proteins to assess cell transformation and performance, fusion proteins, polypeptides with desired biological activities, gene products that complement genetic defects, RNA molecules, transcription factors, and regulatory proteins. and/or other gene products whose performance is of concern. In some embodiments, the payload may comprise a nucleotide sequence (e.g., transposon, transcription factor) that provides a desired action or regulatory function.

The encoded payload may comprise a gene therapy product. Gene therapy products may include, but are not limited to, polypeptides, RNA molecules, or other gene products that provide the desired therapeutic effect when expressed in target cells. In some embodiments, gene therapy products may include non-functional genes or replacements of genes that are absent, expressed in insufficient amounts, or mutated. In some embodiments, gene therapy products may include non-functional proteins or polypeptides or substitutions of proteins or polypeptides that are absent, expressed in insufficient amounts, fold abnormally, degrade too rapidly, or are mutated. For example, a gene therapy product may include an FXN polypeptide or polynucleotide encoding an FXN polypeptide to treat FXN deficiency or FA.

In some embodiments, the payload encodes messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of translation to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. Certain embodiments provide mRNA encoding FXN or a variant thereof.

Components of mRNA include, but are not limited to, coding region, 5'-UTR (untranslated region), 3'-UTR, 5'-cap, and poly-A tail. In some embodiments, the encoded mRNA or any portion of the AAV genome may be codon optimized.

In some embodiments, the protein or polypeptide encoded by the payload construct encoding FXN or a variant thereof is between about 50 and about 4500 amino acid residues in length (hereinafter in this context, "length is "X amino acids" refers to X amino acid residues). In some embodiments, the encoded protein or polypeptide is between 50 and 2000 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 1000 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 1500 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 1000 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 800 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 600 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 400 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 200 amino acids in length. In some embodiments, the encoded protein or polypeptide is between 50 and 100 amino acids in length.

The payload construct encoding the payload may contain or encode a selectable marker. A selectable marker may comprise a gene sequence expressed in a host cell or a protein or polypeptide encoded by a gene sequence, which allows the identification, selection and/or purification of host cells from a population of cells that may or may not express the selectable marker. In some embodiments, the selectable marker provides resistance to a selection process (such as treatment with antibiotics) that would otherwise kill the host cell. In some embodiments, antibiotic selectable markers can include one or more antibiotic resistance factors, including, but not limited to, neomycin resistance (e.g., neo), hygromycin resistance, conmycin resistance, and /or puromycin resistance.

In some embodiments, any nucleic acid sequence encoding a protein or polypeptide can be used to comprise a selectable marker recognized by a specific antibody.

In some embodiments, a payload construct encoding a payload may include a selectable marker including, but not limited to, beta-lactamase, luciferase, beta-galactosidase, or any other reporter gene , as that term is understood in the art, includes cell surface markers such as CD4 or truncated nerve growth factor (NGFR) (for GFP, see WO 96/23810; Heim et al., Current Biology 2:178-182 (1996); Heim et al., Proc. Natl. Acad. Sci. US A (1995); or Heim et al., Science 373:663-664 (1995); for beta-lactamase, see WO 96/30540 ); the contents of each of these documents are incorporated herein by reference in their entirety.

In some embodiments, a payload construct encoding a selectable marker may comprise a fluorescent protein. Fluorescent proteins as described herein may comprise any fluorescent label, including but not limited to green, yellow and/or red fluorescent proteins (GFP, YFP and/or RFP). In some embodiments, a payload construct encoding a selectable marker may comprise a human influenza hemagglutinin (HA) tag.

In certain embodiments, the nucleic acid used to express the payload in the target cell will be incorporated into the viral genome and located between two ITR sequences. Payload: Fataxin

In some embodiments, the payload is syntaxin. As used herein, the term "Fataxin" or "FXN protein" is used interchangeably with "Fataxin polypeptide" or "FXN polypeptide" and encompasses wild-type FXN as well as functional variants thereof. Functional variants are variants that retain some or all of the activity of their wild-type counterpart in order to achieve the desired therapeutic effect. For example, in some embodiments, functional variants may be useful in gene therapy to treat a disorder or condition, such as FXN deficiency or FA. Unless otherwise indicated, variants of FXN as described herein (eg, in the context of constructs, vectors, genomes, methods, kits, compositions, etc. of the invention) are functional variants.

Friedrich's ataxia (FA) is an autosomal recessive disorder that occurs when the ataxin (FXN) gene contains an expanded intronic GAA repeat (an example of a trinucleotide repeat expansion). Genetic diseases. See Parkinson et al., Journal of Neurochemistry , 2013, 126 (Suppl. 1), 103-117, the contents of which are incorporated by reference in their entirety. The expansion of the GAA repeat sequence within the gene causes a decrease in FXN protein content. FXN is an iron-binding protein responsible for the formation of iron-sulfur clusters. One result of FXN protein deficiency is mitochondrial iron overload that causes damage to many proteins. See Nageshwaran and Festenstein, Frontiers in Neurology , Volume 6, Art. 262 (2015), the contents of which are incorporated by reference in their entirety. The FXN gene is located on chromosome 9. See Sandi et al., Frontiers in Genetics , Volume 5, Art. 165 (June 2014), the contents of which are incorporated by reference in their entirety.

The mutated genes contain expanded GAA trinucleotide repeats in the first intron, and in a few cases, point mutations have been detected. Because the defect is located in an intron that is removed from the mRNA transcript between transcription and translation, this mutation does not result in the production of abnormal FXN protein. See Nageshwaran and Festenstein, Frontiers in Neurology , Volume 6, Art. 262 (2015). Alternatively, the mutation causes gene silencing (ie, the mutation reduces transcription of the gene) by inducing heterochromatin structure in a manner similar to position-effect variegation. In addition to reducing FXN protein expression, long GAA repeats induced chromosome breakage in in vivo yeast studies.

Low levels of FXN protein cause insufficient biosynthesis of iron-sulfur clusters required for mitochondrial electron transport and assembly of functional aconitase, as well as dysregulation of iron metabolism throughout the cell. See Nageshwaran and Festenstein, Frontiers in Neurology , Volume 6, Art. 262 (2015). In normal individuals, the FXN gene encodes the mitochondrial matrix FXN protein. This globular protein consists of two α-helices and seven β-strands and is highly conserved and is found in all eukaryotes and some prokaryotes. The FXN protein has various known functions; most notably, it contributes to iron-sulfur protein synthesis in the electron transport chain to ultimately produce adenosine triphosphate (ATP), the energy currency necessary for metabolic functions in cells. FXN protein also regulates iron transfer in mitochondria to provide appropriate amounts of reactive oxygen species (ROS) to maintain normal processes. In the absence of FXN protein, energy in mitochondria declines, and excess iron leads to the production of additional ROS, causing further cell damage.

Other disorders of the central nervous system may eventually be found to be related to abnormal expressions or lack of quantity or function of the FXN protein. Such disorders may include, but are not limited to, neurological or neuromuscular disorders such as Alzheimer's disease, Huntington's disease, autism, Parkinson's disease, and spinal cord disease. muscular dystrophy, or other neurological or neuromuscular diseases, disorders or conditions described herein.

As used herein, "associated with reduced levels of fastaxin" or "associated with reduced manifestations" means that one or more symptoms of a disease are caused by lower than normal levels of fastaxin in a target tissue or biological fluid such as blood. . Diseases or conditions associated with reduced levels or expression of fataxin may be disorders of the central nervous system. The disease or condition may be a neuromuscular or neurological disorder or condition. For example, the disease associated with decreased fataxin content may be FA, or may be another neurological or neuromuscular disorder described herein.

The present invention addresses the need for new technologies by providing FXN-related treatments that can be delivered through AAV-based compositions and complexes for the treatment of FA.

Although delivery is exemplified in the context of AAV, other viral vectors, non-viral vectors, nanoparticles, or liposomes can be similarly used to deliver treatment for FXN and include, but are not limited to, AAV serotypes or other viral delivery vehicles or slow delivery vehicles. Vector genome of any virus, etc. The observations and teachings extend to any macromolecular structure, including modified cells, introduced into the CNS in a manner as described herein.

Sequence identifiers for representative polynucleotide and polypeptide sequences of the cotaxin that may be used in the viral genomes disclosed herein and that may constitute the cotaxin payload are given in Table 2. Functional variants may also be used, such as those that retain at least about 90% or at least 95% sequence identity to the sequences shown in Table 2. Other variants that are codon optimized and encode the same or substantially the same FXN amino acid sequence (eg, those having at least about 90% amino acid sequence identity) may also be used. Table 2. Representative syntaxin sequences SEQ ID NO: Type Species describe 1725 PRT Homo sapiens NP_000135.2 1726 PRT Homo sapiens NP_852090.1 1727 PRT Homo sapiens NP_001155178.1 1728 DNA Homo sapiens NM_000144.4 encoding NP_000135.2 1729 DNA Homo sapiens NM_181425.2 encoding NP_852090.1 1730 DNA Homo sapiens NM_001161706.1 encoding NP_001155178.1 1731 PRT long tail macaque A0A2K5VX49 (UniProt) 1732 PRT long tail macaque NP_001271967.1 1733 PRT rhesus macaque NP_001247670.1

In some embodiments, the viral genome includes a payload region encoding a syntaxin. The encoded syntaxin may originate from any species, such as, but not limited to, humans, non-human primates, or rodents.

In some embodiments, the viral genome contains a payload region encoding human (Homo sapiens) faxin or a variant thereof.

Various embodiments of the invention provide herein an adeno-associated virus (AAV) particle comprising a viral genome comprising at least one inverted terminal repeat region and encoding SEQ ID NO: 1725, 1726 and/or 1727 The nucleic acid sequence of a polypeptide or a variant thereof that has at least 90% sequence identity to the sequence of human fascin (hFXN).

In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 1725. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 1725. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 98% sequence identity to SEQ ID NO: 1725. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to SEQ ID NO: 1725. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and the nucleic acid sequence encoding SEQ ID NO: 1725.

In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 1728 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 1728 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO: 1728 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO: 1728 or a fragment thereof. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and the nucleic acid sequence of SEQ ID NO: 1728 or a fragment thereof. In some embodiments, the fragment of SEQ ID NO: 1728 comprises nucleotides 221-853 of SEQ ID NO: 1728.

In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and the nucleic acid sequence of SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the nucleic acid sequence further includes a stop codon.

In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 1824 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO: 1824 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO: 1824 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO: 1824 or a fragment thereof. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and the nucleic acid sequence of SEQ ID NO: 1824 or a fragment thereof.

In some embodiments, the FXN polypeptide is derived from the FXN sequence (cynoFXN) of a non-human primate, such as the cynomolgus macaque (long-tailed macaque). Certain embodiments provide FXN polypeptides that are humanized versions of the long-tailed macaque (HcynoFXN) sequence. In some embodiments, the FXN polypeptide sequence has at least about 90% sequence identity to the art-recognized typical human FXN amino acid sequence of SEQ ID NO: 1725 encoded by the nucleic acid sequence of SEQ ID NO: 1728. In some embodiments, the FXN polypeptide sequence has at least about 90% sequence identity to the art-recognized typical human FXN amino acid sequence of SEQ ID NO: 1726 encoded by the nucleic acid sequence of SEQ ID NO: 1729. In some embodiments, the FXN polypeptide sequence has at least about 90% sequence identity to the art-recognized typical human FXN amino acid sequence of SEQ ID NO: 1727 encoded by the nucleic acid sequence of SEQ ID NO: 1730.

In some embodiments, the viral genome includes a payload region encoding cynomolgus macaque (long-tailed macaque/ Macaca fascicularis ) fascicularin or a variant thereof.

In some embodiments, the viral genome contains a payload region encoding macaque macaque (rhesus macaque) fataxin or a variant thereof.

In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 1822 or a fragment thereof. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and a nucleic acid sequence that has at least 95% sequence identity to SEQ ID NO: 1822 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO: 1822 or a fragment thereof. In some embodiments, the AAV viral genome comprises at least one inverted terminal repeat region and a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO: 1822 or a fragment thereof. In some embodiments, the AAV viral genome includes at least one inverted terminal repeat region and the nucleic acid sequence of SEQ ID NO: 1822 or a fragment thereof.

In some embodiments, a Fataxin polypeptide may comprise 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% identical to any of the sequences described above. ,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%,72%,73%,74 %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, An amino acid sequence that is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

In some embodiments, a fataxin polypeptide may consist of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% ,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity of the nucleic acid sequence encoding. Viral genome: promoter

In some embodiments, the payload region of the viral genome contains elements that enhance or modulate payload performance, such as (but not limited to) promoters. The promoter can be a wild-type or engineered promoter or a combination thereof. In some embodiments, the viral genome contains at least one promoter. In some embodiments, the viral genome contains more than one promoter.

In some embodiments, the promoter is a wild-type cotaxin promoter or a derivative (eg, a truncated or variant) thereof. Suitable wild-type cotaxin promoter derivatives are those that are functional, eg, those that are effective in expressing the payload in at least a minimal detectable amount.

In some embodiments, the promoter is an engineered syntaxin promoter. Shorter variants of the cotaxin promoter are included herein. The length of the syntaxin promoter can be 200-1400 nt, or any length in between. In some embodiments, the cotaxin promoter variant can be 223, 363, 534, 747, 906, 1060, 1226, or 1353 nucleotides in length. Fataxin promoter variants may be less specific than wild type due to deletions in any region of the promoter sequence, such as, but not limited to, the 5' end of the promoter sequence, the 3' end of the promoter sequence, or within the promoter sequence. The cotaxin promoter sequence is short.

In some embodiments, the promoter is a combination of any one or more of the promoters described herein. In some embodiments, a promoter with an enhancer sequence is used. In some embodiments, the enhancer sequence may be derived from the cytomegalovirus immediate early gene (CMVie). In some embodiments, the enhancer can be located upstream (5') of the promoter. In some embodiments, the enhancer comprises SEQ ID NO: 1777.

In some embodiments, the promoter is a CBA promoter or a derivative (eg, a truncated or variant) thereof. It will be understood that suitable CBA promoter derivatives are functional, eg, can efficiently express a payload.

In some embodiments, when recited from 5' to 3', the CBA promoter includes the CMVie enhancer, backbone sequence, and CB promoter sequence. Each of the three components (CMVie enhancer, backbone and CB sequence) can have a different length between variants.

In some embodiments, when recited from 5' to 3', the CBA promoter includes the backbone sequence and the CB promoter sequence.

In some embodiments, the CBA promoter comprises a CB promoter sequence.

In some embodiments, the CBA promoter can be 100 to 700 nt in length or any length therebetween. In some embodiments, CBA promoter variants can be 100, 180, 260, 270, 332, 412, 492, or 572 nucleotides in length. CBA promoter variants may be attributed to deletions in the enhancer, backbone, or any region of the promoter sequence, such as, but not limited to, the 5' end of the promoter sequence, the 3' end of the promoter sequence, or within the promoter sequence. And shorter than the wild-type CBA promoter sequence.

In some embodiments, the promoter is a CMV promoter or a derivative (eg, a truncated or variant) thereof. It will be appreciated that suitable CMV promoter derivatives are functional, eg, can efficiently express a payload. A CMV promoter may comprise a CMV enhancer and a CMV promoter sequence, or only a CMV promoter sequence. CMV enhancer and CMV promoter sequences can have different lengths between promoter variants.

In some embodiments, the CMV promoter can be 50 to 700 nt in length or any length therebetween. In some embodiments, CMV promoter variants can be 55, 109, 163, 217, 289, 361, 433, or 505 nucleotides in length. CMV promoter variants may be less specific than wild type due to deletions in the enhancer or any region of the promoter sequence, such as, but not limited to, the 5' end of the promoter sequence, the 3' end of the promoter sequence, or within the promoter sequence. The CMV promoter sequence is short.

In some embodiments, the promoter is a deletion variant of the parental promoter sequence, in which one or more nucleotides have been removed from the parental sequence.

In some embodiments, the promoter is an insertional variant of the parent promoter sequence, in which one or more nucleotides are added to the parent sequence.

In some embodiments, the promoter contains one or more mutations compared to the parental promoter sequence.

In some embodiments, the promoter is modified in one or more ways (eg, deletions, mutations, and/or insertions) to create promoter variants.

In some embodiments, the promoter may comprise a sequence, fragment, or variant thereof of any of the sequences in Table 3. For example, a promoter may comprise a sequence that is at least 90%, at least 95%, at least 99%, or 100% sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 1734-1777, such as a sequence selected from the group consisting of SEQ ID NOs: 1734-1777. Sequences forming the group NO: 1734-1777 have a specified percent identity and provide some or all of the same functions. In some embodiments, the promoter is or is derived from a CMV promoter and comprises at least 90%, at least 95%, at least Sequences with 99% or 100% sequence identity. In some embodiments, the promoter is or is derived from a CBA promoter and comprises at least 90%, at least 95% similarity to a sequence selected from the group consisting of SEQ ID NOs: 1734-1742, 1760-1766, 1768, and 1775-1776 , a sequence with at least 99% or 100% sequence identity. In some embodiments, the promoter is or is derived from an FXN promoter and comprises at least 90%, at least 95%, at least 99%, or 100% similarity to a sequence selected from the group consisting of SEQ ID NOs: 1752-1759 and 1769-1770 % sequence identity of the sequence.

In some embodiments, a promoter may comprise any combination of more than one of those sequences listed in Table 3. In some embodiments, the promoter sequence may further comprise at least one of the intron/exon sequences set forth in Table 6.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1738. In some embodiments, the promoter is SEQ ID NO: 1738. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1738 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1738 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1738 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload area). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1740. In some embodiments, the promoter is SEQ ID NO: 1740. In some embodiments, the AAV vector genome comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1740 and encoding a fascin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1740 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1740 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload area). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1742. In some embodiments, the promoter is SEQ ID NO: 1742. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1742 and encodes a syntaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1742 and encoding a fascin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1742 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload area). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1750. In some embodiments, the promoter is SEQ ID NO: 1750. In some embodiments, the AAV vector genome comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1750 and encoding a cotaxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1750 and encoding a fascin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1750 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload area). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, promoters used in viral genomes disclosed herein comprise any of the promoter sequences in Table 3. In Table 3, CMV stands for "cellular giant virus"; CBA stands for "chicken beta-actin", which can have CMV IE ("immediate early") enhancer region and promoter region; CAG stands for CMV enhancer, CBA promoter and rabbit beta-hemoglobin splice acceptor site; FXN stands for "cotaxin"; and mCBA stands for a variant of the CBA promoter generated using PCR. Table 3. Representative promoters promoter name initial promoter promoter length Promoter SEQ ID NO CBA CBA 652 1734 CBA-D1 CBA 572 1735 CBA-D2 CBA 492 1736 CBA-D3 CBA 412 1737 CBA-D4 CBA 332 1738 CBA-D5 CBA 270 1739 CBA-D6 CBA 260 1740 CBA-D7 CBA 180 1741 CBA-D8 CBA 100 1742 CMV CMV 588 1743 CMV-D1 CMV 505 1744 CMV-D2 CMV 433 1745 CMV-D3 CMV 361 1746 CMV-D4 CMV 289 1747 CMV-D5 CMV 217 1748 CMV-D6 CMV 163 1749 CMV-D7 CMV 109 1750 CMV-D8 CMV 55 1751 FXNpro223 FXN 223 1752 FXNpro363 FXN 363 1753 FXNpro534 FXN 534 1754 FXNpro907 FXN 907 1755 FXNpro1060 FXN 1060 1756 FXNpro1226 FXN 1226 1757 FXNpro1353 FXN 1353 1758 FXNproN1336 FXN 1336 1759 mCBA mCBA 610 1760 mCBA-D1 mCBA 526 1761 mCBA-D2 mCBA 441 1762 mCBA-D3 mCBA 366 1763 mCBA-D4 mCBA 286 1764 mCBA-D5 mCBA 224 1765 mCBA-D6 mCBA 214 1766 CMV-80 CMV 80 1767 CBA-90 CBA 90 1768 FXN-150 FXN 150 1769 FXN-200 FXN 198 1770 CAG CAG 1715 1771 CMV-205 CMV 205 1772 CMV-299 CMV 299 1773 CMV-380 CMV 380 1774 CBAmin CBA 283 1775 CBA-654 CBA 654 1776 CMV enhancer CMV 383 1777

In some embodiments, the promoter is used to regulate syntaxin expression in cells of interest. In certain embodiments, a promoter can be used to increase fataxin expression in a target cell to a level above normal endogenous fataxin expression. In certain embodiments, a promoter can be used to induce fataxin expression in a cell of interest to a level that is close to or equivalent to normal endogenous fataxin expression.

In some embodiments, the adapter sequences can be used in combination with promoters described herein, such as, but not limited to, those listed in Table 3. In certain embodiments, the junction sequence can be located 5' to the promoter in the viral genome. In certain embodiments, the junction sequence can be located 3' to the promoter in the viral genome. In certain embodiments, the viral genome may include more than one conjugative sequence. As a non-limiting example, the viral genome may contain junction sequences on the 5' end of the promoter and on the 3' end of the promoter. The joining sequence can be the same sequence, two different sequences, or a sequence split on either side of the promoter sequence. In certain embodiments, the joining sequence includes SEQ ID NO: 1813. In certain embodiments, the joining sequence includes SEQ ID NO: 1814.

In some embodiments, the promoter is used to enhance fataxin expression in target cells (eg, nervous system or cardiac tissue). Fataxin performance can be increased by 0.01 to 100 (0.01-100×) times compared to endogenous Fataxin performance on target cells. In some embodiments, the promoter is used to maintain 0.5 to 3× ( For example, 0.5 to 1×, 1 to 1.5×, 1.5 to 2×, 2 to 2.5×, 2.5 to 3×).

In some embodiments, a promoter (eg, the promoter in Table 3) is used in an AAV vector genome further comprising a sequence encoding a Fataxin polypeptide sequence (eg, a human Fataxin polypeptide sequence). In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1742. In some embodiments, the promoter is SEQ ID NO: 1742. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1742 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1742 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1742 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1750. In some embodiments, the promoter is SEQ ID NO: 1750. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1750 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1750 and encoding a cotaxin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1750 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1738. In some embodiments, the promoter is SEQ ID NO: 1738. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1738 and encodes a syntaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome comprises a promoter sequence having at least 95% sequence identity to SEQ ID NO: 1738 and encoding a fascin polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1738 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In some embodiments, the promoter comprises a sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1740. In some embodiments, the promoter is SEQ ID NO: 1740. In some embodiments, the AAV vector genome includes a promoter sequence that has at least 90% sequence identity to SEQ ID NO: 1740 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 90% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes a promoter sequence that has at least 95% sequence identity to SEQ ID NO: 1740 and encodes a cotaxin polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1725 A payload region (e.g., a payload region comprising a nucleic acid sequence at least 95% identical to SEQ ID NO: 1824). In some embodiments, the AAV vector genome includes the promoter sequence of SEQ ID NO: 1740 and a payload region encoding a cotaxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., including the sequence of SEQ ID NO: 1824 payload region), and/or further comprising one or more sequences as provided in Tables 5 to 11 or 95% identical variants thereof. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1728 or a fragment thereof (optionally nucleotides 221-853 of SEQ ID NO: 1728). In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

In various embodiments, any of the promoters disclosed herein (e.g., a promoter from Table 3 or a promoter having 90% or greater homology thereto) may be used alone or with additional sequences (e.g., stuffer sequences) The combination is paired in the AAV viral vector genome with one or more of the components disclosed in Tables 5 to 11 or components with 90% or greater homology thereto. In some embodiments, an AAV vector genome may comprise multiple copies (eg, two, three, or more copies) of one or more viral genome components described herein. In some embodiments, the viral genome contains two miR binding sites (eg, two miR122 binding sites). In some embodiments, the viral genome contains three miR binding sites (eg, three miR122 binding sites). In some embodiments, the viral genome includes any of the promoters disclosed herein in the 5' to 3' order shown in any of Tables 4, 12, 13, 14, 15, 16, or 17 (eg, a promoter from Table 3 or a promoter having 90% or greater homology thereto) and one or more components provided in any of Tables 5 to 11 or otherwise described herein. In some embodiments, the viral genome includes the promoter provided in Table 3 in the 5' to 3' order shown in any of Tables 4, 12, 13, 14, 15, 16, or 17, and Table 3 One or more components provided in any of 5 to Table 11 or otherwise described herein. In some embodiments, the viral genome includes all components and the 5' to 3' sequence shown in any of Tables 4, 12, 13, 14, 15, 16, or 17.

For example, a promoter comprising SEQ ID NO: 1742 or having 90% or higher homology thereto may be any component of Table 5 to Table 11 in the AAV vector genome (or having 90% or higher homology thereto). component) pairing, e.g. the promoter is located between the 5' ITR sequence and ie1 exon 1 (e.g. directly in contact with two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome contains three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a syntaxin.

In another example, a promoter comprising SEQ ID NO: 1750 or having 90% or greater homology thereto may be any component of Tables 5 to 11 in the AAV vector genome (or having 90% or greater homology thereto). (e.g., directly in contact with two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome contains three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a syntaxin.

For example, a promoter comprising SEQ ID NO: 1738 or having 90% or higher homology thereto may be any component of Table 5 to Table 11 in the AAV vector genome (or having 90% or higher homology thereto). component) pairing, e.g. the promoter is located between the 5' ITR sequence and ie1 exon 1 (e.g. directly in contact with two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome contains three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a syntaxin.

In another example, a promoter comprising SEQ ID NO: 1740 or having 90% or greater homology thereto may be any component of Tables 5 to 11 in the AAV vector genome (or having 90% or greater homology thereto). (e.g., directly in contact with two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome contains three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a syntaxin.

In some embodiments, the promoter is or is derived from a CBA promoter. The CBA promoter can drive the expression of the payload in various tissues of the individual. As a non-limiting example, the performance of FXN using the CBA promoter (the promoter provided as SEQ ID NO: 1776, and the ITR to ITR provided as SEQ ID NO: 1778) is shown in the contents, which are incorporated herein by reference in their entirety. Example 4 (including Table 16 to Table 28) of the jointly owned international patent application No. PCT/US2019/032387. The expression of FXN in mice after IV injection is shown in Table 16 of co-owned international patent application No. PCT/US2019/032387, in which expression was seen in the cortex, lumbar spinal cord, and lumbar region with the help of VOY101 particles with CBA promoter. In dorsal root ganglia, trigeminal ganglia, heart and liver. The expression of FXN in NHP after IV injection is shown in Table 18 of co-owned International Patent Application No. PCT/US2019/032387, where expression was seen in the brainstem, cervical spinal cord, In the thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar/sacral DRG, ventricle, atrium, liver, soleus muscle and jejunum. The performance of FXN in NHP after IV injection at different doses (6.3×10 ¹¹ VG/kg, 2×10 ¹² VG/kg or 2×10 ¹³ VG/kg) is demonstrated in co-owned international patent application No. PCT/US2019 In Table 19 of No./032387, with the help of VOY201 particles with CBA promoter, the manifestations are found in the brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar/sacral DRG, ventricle, In the atria, liver, kidneys, lungs, soleus muscle and/or spleen. The performance of FXN in NHP after IV injection at different doses (6.7×10 ¹² VG/kg or 4.89×10 ¹³ VG/kg) is shown in Table 20 of co-owned international patent application No. PCT/US2019/032387, Among them, with the help of VOY101 particles with CBA promoter, the manifestations are found in the brainstem, cerebellum, cortex, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar/sacral DRG, ventricle, atrium, liver, kidney, In the soleus muscle, sympathetic thoracic chain ganglia, and/or adrenal gland. The distribution of the vector genome in mice after IV injection is shown in Table 17 of the jointly owned international patent application No. PCT/US2019/032387, in which the distribution was found in the cortex and waist with the help of VOY101 and AAV9 particles with CBA promoter. In the spinal cord, thoracic dorsal root ganglia, trigeminal ganglia, heart and liver. The distribution of the vector genome in NHPs after IV injection is shown in Table 18 of co-owned international patent application No. PCT/US2019/032387, where the distribution was found in the frontal cortex, striatum with the help of VOY101 particles with CBA promoter Body, brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar/sacral dorsal root ganglion, ventricle, atrium, liver, kidney, lung, soleus muscle, jejunum and In the spleen. The distribution of vector genomes in NHPs after IV injection at different doses (6.3×10 ¹¹ VG/kg, 2×10 ¹² VG/kg or 2×10 ¹³ VG/kg) is shown in co-owned international patent application No. PCT/ In Table 19 of US2019/032387, with the help of VOY201 particles with CBA promoter, the distribution is found in the frontal cortex, striatum, brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, and chest In DRG, lumbar/sacral DRG, ventricles, atria, liver, kidneys, lungs, soleus and/or spleen. The distribution of vector genomes in NHPs after IV injection at different doses (6.7×10 ¹² VG/kg or 4.89×10 ¹³ VG/kg) is shown in Table 20 of co-owned international patent application No. PCT/US2019/032387 , with the help of VOY101 particles with CBA promoter, the distribution is found in the motor cortex, sensorimotor cortex, striatum, brainstem, cerebellar cortex, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, thoracic spinal cord, cervical DRG, and thoracic DRG , lumbar/sacral DRG, ventricle, atrium, liver, kidney, soleus muscle, jejunum, spleen, sympathetic thoracic chain ganglion, and/or adrenal gland.

In some embodiments, the promoter is or is derived from a promoter including the CMVie enhancer, CBA, CMV, cotaxin promoter, truncated CBA, and/or truncated CMV promoter. The promoter drives the expression of the payload in various tissues of the individual. As a non-limiting example, a mouse model of Friedrich's ataxia used to evaluate the in vivo distribution, performance, and efficacy of IV administration of VOY101 particles with FXN can be used, as the content is incorporated by reference in its entirety. This is shown in Example 5 of commonly owned International Patent Application No. PCT/US2019/032387, which is incorporated herein by reference. In certain embodiments, the performance of driven FXN in mice can be assessed such as (but not limited to) those in Table 3 as outlined in Example 5 of co-owned International Patent Application No. PCT/US2019/032387. Promoters of other promoters. As another non-limiting example, an NHP model of Friedrich's ataxia used to evaluate the in vivo distribution and performance of IV administration of VOY101 particles with FXN is shown in the content, which is incorporated herein by reference in its entirety. Example 5 of the jointly owned international patent application No. PCT/US2019/032387. In certain embodiments, performance evaluations such as (but not limited to) those in Table 3 for driving FXN in NHP can be performed as outlined in Example 5 of co-owned International Patent Application No. PCT/US2019/032387. promoter of promoter.

In some embodiments, the promoter is or is derived from a CBA promoter that drives expression of the payload in various tissues of the individual. As a non-limiting example, the performance of FXN using the CBA promoter (the promoter provided as SEQ ID NO: 1776, and the ITR to ITR provided as SEQ ID NO: 1778) is shown in the contents, which are incorporated herein by reference in their entirety. In Example 14 (including Table 33 to Table 34) of the jointly owned international patent application No. PCT/US2019/032387. The performance of FXN in mice after IV injection is shown in Table 33 of co-owned International Patent Application No. PCT/US2019/032387, with the help of VOY101, VOY801 and/or VOY1101 particles with CBA promoter. The performance is shown in In the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, thoracic DRG, heart and/or liver. The distribution of the vector genome in mice after IV injection is shown in Table 34 of co-owned International Patent Application No. PCT/US2019/032387, where the distribution was achieved with the help of VOY101, VOY801 and/or VOY1101 particles with CBA promoter. Found in the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, heart and liver. As another non-limiting example, performance of FXN using the CBA promoter (promoter provided as SEQ ID NO: 1776, and ITR to ITR provided as SEQ ID NO: 1778) in VOY701 and VOY101 capsids is shown in the text at Example 14 (including Tables 35 through 36) of co-owned Provisional Patent Application No. 62/839,889 is incorporated herein by reference in its entirety. The performance of FXN in mice after IV injection is shown in Table 35 of co-owned Provisional Patent Application No. 62/839,889, in which expression was seen in cortex, striatum, with the help of VOY701 and/or VOY101 particles with CBA promoter. body, hippocampus, brainstem, thoracic spinal cord, and/or liver. The distribution of the vector genome in mice after IV injection is shown in Table 36 of co-owned Provisional Patent Application No. 62/839,889, where the distribution was seen in the cortex, striae, with the help of VOY701 and/or VOY101 particles with CBA promoter. In the corpus, hippocampus, brainstem, thoracic spinal cord, and/or liver.

In some embodiments, AAV particles described herein comprise a viral genome having a payload region encoding a syntaxin. Viral genomes can be engineered to optimize syntaxin expression in target cells. Viral genome : ITR to ITR sequence including cotaxin payload

Any of the components described herein can be used to design and optimize the ITR-to-ITR sequences of the viral genome for desired syntaxin expression. Viral genomes may contain any number of components, such as (but not limited to) ITRs, enhancers, promoters, introns, UTRs, payload regions, tags or selectable markers, miR binding or target sites, backbones One or more of the region, polyA sequence and/or filler sequence. Each of these components may be present at zero, one, two, or more times in a given viral genome.

Each of the ITR, promoter, enhancer, intron, exon, payload, tag, miR binding site, polyA and/or stuffer component may be selected independently or in any combination from Table 3 and Sequences provided in Tables 5 to 11.

In some embodiments, the AAV viral genome includes a 5' ITR, an enhancer, an intron, a payload region, an optional tag, up to three miR binding sites, a polyA sequence, an optional stuffer sequence, and 3 'ITR. In some embodiments, the 5' ITR is an AAV2 ITR. In some embodiments, the 5' ITR comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1811. In some embodiments, the enhancer includes ie1 exon 1 and ie1 intron 1 or fragments thereof. In some embodiments, the enhancer comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1817 and/or 1819. In some embodiments, the enhancer comprises one or more human beta-hemoglobin sequences, e.g., at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1816, 1820, and/or 1821 sequence. In some embodiments, enhancers include SEQ ID NOs: 1817, 1819, 1820, and 1821. In some embodiments, the enhancer comprises SEQ ID NO: 1816.

In some embodiments, the payload region comprises a coding sequence that has at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 1725, 1726, 1727, 1731, 1732 or 1733 (e.g., to SEQ ID NO: 1725, 1726, 1727, 1731, 1732 or 1733 NO: 1725 Nucleic acid sequence of a polypeptide having at least 90%, at least 95%, at least 99% or 100% sequence identity). In some embodiments, the payload region comprises a nucleic acid sequence that has at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1728, 1729, 1730, or a fragment thereof. In some embodiments, the fragment of SEQ ID NO: 1728 comprises nucleotides 221-853 of SEQ ID NO: 1728. In some embodiments, a Fataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824. In some embodiments, no tags are present. In some embodiments, the tag is present and is a human influenza hemagglutinin HA tag. In some embodiments, the HA tag includes SEQ ID NO: 1825. In some embodiments, no miR binding site is present. In some embodiments, at least one miR binding site is present and includes a miR122 binding site. In some embodiments, the miR122 binding site comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1827. In some embodiments, the AAV vector genome comprises three copies of a miR122 binding site, such as three copies of SEQ ID NO: 1827 or a variant thereof with at least 90% sequence identity. In some embodiments, a series of miR binding sites comprising three copies of the miR122 binding site comprises SEQ ID NO: 1826. In some embodiments, the viral genome contains human growth hormone polyA sequences. In some embodiments, the viral genome comprises a polyA sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1828. In some embodiments, the AAV viral genome further includes stuffer sequences, such as albumin stuffer sequences. In some embodiments, the filler sequence includes any of those set forth in SEQ ID NOs: 1829-1842. In some embodiments, the 3' ITR is an AAV2 ITR. In some embodiments, the 3' ITR comprises a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1812.

In certain embodiments, AAV particles include at least one ipsilateral element, including but not limited to Kozak sequences, backbone sequences, and/or intron sequences. Certain embodiments provide that the AAV particle further comprises a promoter region. For example, promoters may include promoters from any of the CBA, CMV, FXN and/or SV40 genes, or variants thereof. Non-limiting examples of ITR to ITR sequences for AAV particles containing viral genomes with payload regions encoding cotaxin are described in Table 4.

In Table 4, cFXN indicates cynomolgus macaque (long-tailed macaque) fataxin, hFXN indicates human (Homo sapiens) fataxin, hβglobin indicates human β-hemoglobin, HA indicates human influenza hemagglutinin HA tag, and hGH indicates Human Growth Hormone. Alb indicates albumin. The number after alb indicates the length of the albumin filling. miR-122 BS is the binding site of miR-122. The "-" symbol indicates that the construct does not have that component or sequence. The "+" symbol indicates that the construct has that component or sequence. Table 4. Representative ITR to ITR sequences Structure name 5'ITR promoter intron payload label miR-122 BS (3x) Poly(A) filler 3'ITR SEQ ID NO cFXN1 + CBA hβglobin ikB HA - hGH - + 1778 cFXN2 + CBA hβglobin ikB HA + hGH - + 1779 cFXN3 + CBA-D4 hβglobin ikB HA + hGH - + 1780 cFXN4 + CBA-D6 hβglobin ikB HA + hGH - + 1781 cFXN5 + CBA-D6 hβglobin ikB HA + hGH Alb450 + 1782 cFXN6 + CBA-D8 hβglobin ikB HA + hGH - + 1783 cFXN7 + CBA-D8 hβglobin ikB HA + hGH Alb450 + 1784 cFXN8 + mCBA hβglobin ikB HA + hGH - + 1785 cFXN9 + mCBA-D1 hβglobin ikB HA + hGH - + 1786 cFXN10 + mCBA-D2 hβglobin ikB HA + hGH - + 1787 cFXN11 + CMV hβglobin ikB HA - hGH - + 1788 cFXN12 + CMV hβglobin ikB HA + hGH - + 1789 cFXN13 + CMV-D1 hβglobin ikB HA + hGH - + 1790 cFXN14 + CMV-D3 hβglobin ikB HA + hGH - + 1791 cFXN15 + CMV-D7 hβglobin ikB HA + hGH - + 1792 cFXN16 + CMV-D7 hβglobin ikB HA + hGH Alb450 + 1793 cFXN17 + FXNpro534 hβglobin ikB HA + hGH - + 1794 cFXN18 + FXNpro1060 hβglobin ikB HA + hGH - + 1795 hFXN1 + CBA hβglobin htK HA + hGH + 1796 hFXN2 + CBA-D8 hβglobin htK - + hGH Alb2266 + 1797 hFXN3 + CBA-D8 hβglobin htK - - hGH Alb2335 + 1798 AHr + CMV hβglobin htK - + hGH Alb1785 + 1799 ikB + CMV hβglobin htK - - hGH Alb1856 + 1800 hFXN6 + CMV-D7 hβglobin htK - + hGH Alb2264 + 1801 htK + CMV-D7 hβglobin htK - - hGH Alb2335 + 1802 AHr + FXNpro1060 hβglobin htK - + hGH Alb1313 + 1803 AHr + FXNpro1060 hβglobin htK - - hGH Alb1384 + 1804 hFXN10 + CAG intron htK - + hGH Alb570 + 1805 hFXN11 + CMV-D1 hβglobin htK - + hGH Alb1870 + 1806 hFXN12 + CMV-D3 hβglobin htK - + hGH Alb2014 + 1807 hFXN13 + CBA-D4 hβglobin htK - + hGH Alb2034 + 1808 hFXN14 + CBA-D6 hβglobin htK - + hGH Alb2106 + 1809 hFXN15 + CMV/CBA hβglobin htK - + hGH Alb1790 + 1810

In some embodiments, the AAV particle comprises a viral genome comprising a sequence that has a certain percent identity to any of SEQ ID NOs: 1778-1810. The viral genome may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% Or 100% consistency. The viral genome may be 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90% identical to any one of SEQ ID NO: 1778-1810 %, 50-99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99 %, 70-100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99% or 90-100% consistency. In some embodiments, the viral genome comprises a sequence that is at least 80% identical to any of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 85% identical to any of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 90% identical to any of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 95% identical to any of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 99% identical to any of SEQ ID NOs: 1778-1810.

In some embodiments, the viral genome comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1797. In some embodiments, the viral genome comprises SEQ ID NO: 1797. In some embodiments, the viral genome comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1801. In some embodiments, the viral genome comprises SEQ ID NO: 1801. In some embodiments, the viral genome comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1808. In some embodiments, the viral genome comprises SEQ ID NO: 1808. In some embodiments, the viral genome comprises a sequence that has at least 95% sequence identity to SEQ ID NO: 1809. In some embodiments, the viral genome comprises SEQ ID NO: 1809. In some embodiments, the viral genome of the AAV particles of the invention may comprise any combination of the sequence regions described in Tables 2 to 11 or otherwise described herein, encapsulated in a coat listed in Table 1 or described herein. In any one of the shells.

In some embodiments, the AAV particle viral genome may comprise at least one sequence region as described in Tables 2 to 11. These regions may precede or follow any of the other sequence regions described herein. The viral genome may further comprise more than one copy of one or more sequence regions as described in Tables 2 to 11. Viral genome: inverted terminal repeats (ITR)

In some embodiments, the AAV particle viral genome may comprise at least one inverted terminal repeat (ITR) region. The ITR regions may independently have lengths such as (but not limited to) 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 ,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117 ,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142 ,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167 , 168, 169, 170, 171, 172, 173, 174 and 175 nucleotides. The length of the ITR region of the viral genome can be 75-80, 75-85, 75-100, 80-85, 80-90, 80-105, 85-90, 85-95, 85-110, 90-95, 90 -100, 90-115, 95-100, 95-105, 95-120, 100-105, 100-110, 100-125, 105-110, 105-115, 105-130, 110-115, 110-120 ,110-135,115-120,115-125,115-140,120-125,120-130,120-145,125-130,125-135,125-150,130-135,130-140,130 -155, 135-140, 135-145, 135-160, 140-145, 140-150, 140-165, 145-150, 145-155, 145-170, 150-155, 150-160, 150-175 , 155-160, 155-165, 160-165, 160-170, 165-170, 165-175 and 170-175 nucleotides. As a non-limiting example, the viral genome contains a 5' ITR that is approximately 141 nucleotides in length. As a non-limiting example, the viral genome contains a 5' ITR that is approximately 130 nucleotides in length. As a non-limiting example, the viral genome contains a 5' ITR that is approximately 119 nucleotides in length. As a non-limiting example, the viral genome contains a 3' ITR of approximately 141 nucleotides in length. As a non-limiting example, the viral genome contains a 3' ITR of approximately 130 nucleotides in length. As a non-limiting example, the viral genome contains a 3' ITR that is approximately 119 nucleotides in length. As a non-limiting example, the 5' ITR and the 3' ITR may include the same length and/or the same sequence. In another non-limiting example, the 5' ITR and 3' ITR are different in length and/or sequence.

In some embodiments, the AAV particle viral genome contains at least one inverted terminal repeat region. Non-limiting examples of ITR sequence regions are described in Table 5. Table 5. Representative inverted terminal repeat (ITR) sequence regions Sequence area name sequence length SEQ ID NO ITR1 141 1811 ITR2 141 1812

In some embodiments, the AAV particle viral genome may have an ITR that includes ITR1. In some embodiments, the AAV particle viral genome can have an ITR that includes ITR2. In some embodiments, the AAV particle viral genome may have two ITRs. As a non-limiting example, the two ITRs may be ITR1 and ITR2. Viral genome: intron and exon sequences of payload region

In some embodiments, the AAV particle virus genome includes at least one intron and/or exon sequence region. Non-limiting examples of intronic and exonic sequence regions are described in Table 6. Table 6. Representative intron and exon sequence regions Sequence area name sequence length SEQ ID NO intron 1016 1815 hBglobin intron/exon 566 1816 ie1 exon 1 134 1817 CMV/hemoglobulin intron 379 1818 ie1 intron 1 (part) 32 1819 hBglobin intron 2 347 1820 hBglobin exon 3 53 1821

In some embodiments, the AAV particle viral genome may comprise at least one intronic sequence region. Intron sequence regions may independently have lengths such as (but not limited to) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350 and More than 350 nucleotides. The length of the intron sequence region of the viral genome can be 25-35, 25-50, 35-45, 45-55, 50-75, 55-65, 65-75, 75-85, 75-100, 85- 95, 95-105, 100-125, 105-115, 115-125, 125-135, 125-150, 135-145, 145-155, 150-175, 155-165, 165-175, 175-185, 175-200, 185-195, 195-205, 200-225, 205-215, 215-225, 225-235, 225-250, 235-245, 245-255, 250-275, 255-265, 265- 275, 275-285, 275-300, 285-295, 295-305, 300-325, 305-315, 315-325, 325-335, 325-350 and 335-345 nucleotides. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 32 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 53 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 134 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 347 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 379 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 566 nucleotides in length. As a non-limiting example, the viral genome contains an intronic sequence region that is approximately 1016 nucleotides in length. As a non-limiting example, viral genomes include intronic sequence regions that are greater than about 1016 nucleotides in length.

In some embodiments, the AAV particle viral genome contains two intronic sequence regions. In some embodiments, the AAV particle virus genome contains three intronic sequence regions. In some embodiments, the AAV particle viral genome contains more than three intronic sequence regions.

In some embodiments, the AAV particle viral genome may comprise at least one exonic sequence region. The exon sequence regions may independently have the following lengths, such as (but not limited to) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348,349,350 and More than 350 nucleotides. The length of the exon sequence region of the viral genome can be 25-35, 25-50, 35-45, 45-55, 50-75, 55-65, 65-75, 75-85, 75-100, 85- 95, 95-105, 100-125, 105-115, 115-125, 125-135, 125-150, 135-145, 145-155, 150-175, 155-165, 165-175, 175-185, 175-200, 185-195, 195-205, 200-225, 205-215, 215-225, 225-235, 225-250, 235-245, 245-255, 250-275, 255-265, 265- 275, 275-285, 275-300, 285-295, 295-305, 300-325, 305-315, 315-325, 325-335, 325-350 and 335-345 nucleotides. As a non-limiting example, the viral genome contains an exon region that is approximately 32 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region that is approximately 53 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region approximately 134 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region that is approximately 347 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region that is approximately 379 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region that is approximately 566 nucleotides in length. As a non-limiting example, the viral genome contains an exon sequence region of approximately 1016 nucleotides in length. As a non-limiting example, viral genomes include exon sequence regions that are greater than about 1016 nucleotides in length.

In some embodiments, the AAV particle virus genome contains two exon sequence regions. In some embodiments, the AAV particle virus genome contains three exon sequence regions. In some embodiments, the AAV particle virus genome contains more than three exon sequence regions.

In some embodiments, the AAV particle virus genome includes a hybrid intron/exon sequence region including at least one intron and at least one exon. In some embodiments, a hybrid intron/exon sequence region includes an intron and an exon. In some embodiments, a hybrid intron/exon sequence region includes two introns and two exons. In some embodiments, an intron or exon sequence may comprise a full-length intron or exon. In some embodiments, the intron or exon sequence may comprise fragments or variants of the intron or exon sequence.

Hybrid intron/exon sequence regions may independently have lengths such as (but not limited to) 15-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600- 700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200 and more than 1200 nucleotides. As a non-limiting example, the viral genome contains a hybrid intron/exon sequence region of approximately 379 nucleotides in length. As a non-limiting example, the viral genome contains a hybrid intron/exon sequence region of approximately 566 nucleotides in length. As a non-limiting example, the viral genome contains a hybrid intron/exon region that is approximately 379 nucleotides in length.

In some embodiments, the intron/exon sequence regions are enhancer sequences. In some embodiments, the intron/exon sequence region is not an enhancer sequence.

In some embodiments, the intron/exon sequence region is a component of the promoter sequence. In some embodiments, the intron/exon sequence region is not a component of the promoter sequence. Viral genomes: cotaxin payload

In some embodiments, the payload may include any of the sequences listed in Table 7. Table 7. Representative cotaxin payload sequences Sequence area name sequence length SEQ ID NO ikB 630 1822 htK 630 1823 hFXN+termination 633 1824

In some embodiments, the payload sequence encodes a cotaxin derived from Cynomolgus macaques (long-tailed macaques) or a variant thereof. In some embodiments, the payload sequence encodes a syntaxin derived from the cynomolgus macaque (long-tailed macaque) but differs by at least one amino acid. In some embodiments, the payload sequence encodes a syntaxin derived from cynomolgus macaques (long-tailed macaques) but differs from wild type in at least one amino acid. In some embodiments, the payload sequence encodes a syntaxin derived from cynomolgus macaques (long-tailed macaques) but differs from wild type by at least two amino acids.

In some embodiments, the payload sequence encodes a syntaxin of human (Homo sapiens) origin or a variant thereof. In some embodiments, the payload sequence includes a stop codon. Viral genomes: tag sequences

In some embodiments, the AAV particle viral genome may comprise at least one tag sequence region. As used herein, the term "tag" refers to a polynucleotide sequence attached to a payload that, upon expression, can be used to identify the expressed payload. Alternatively, the term "tag" may refer to a polynucleotide sequence attached to a payload that signals retention of the expressed payload in a specific region of the cell, such as the endoplasmic reticulum. Tag sequence regions may independently have lengths such as (but not limited to) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides. The length of the tag sequence region in the viral genome can be 10-15, 15-20, 20-25, 25-30 or more than 30 nucleotides. As a non-limiting example, the viral genome contains a tag sequence region that is approximately 27 nucleotides in length.

In some embodiments, the AAV particle viral genome contains at least one tag sequence region. Non-limiting examples of tag sequence regions are shown in Table 8. Table 8. Representative tag sequence regions Sequence area name sequence length SEQ ID NO HA 27 1825

In some embodiments, the AAV particle viral genome contains a tag sequence region. In some embodiments, the tag sequence region is a human influenza hemagglutinin (HA) tag.

In some embodiments, the AAV particle viral genome contains more than one tag sequence region. In one embodiment, the AAV particle viral genome contains two tag sequence regions. In one embodiment, the AAV particle virus genome contains three tag sequence regions. In one embodiment, the AAV particle viral genome contains more than three tag sequence regions. Viral genomes : microRNA ( i.e., miR ) binding sites

In some embodiments, the AAV particle viral genome may comprise at least one miR binding site. Non-limiting examples of miR binding site sequence regions are shown in Table 9. Table 9. Representative miR binding site sequence regions Sequence area name sequence length SEQ ID NO miR binding site series 71 1826 Single miR binding site twenty three 1827

In some embodiments, the AAV particle viral genome contains a single miR binding site sequence. As a non-limiting example, the miR binding site sequence may be a miR-122 binding site.

In some embodiments, the viral genome may contain more than one miR binding site sequence. As non-limiting examples, viral genomes may contain two, three, four or five miR binding site sequences.

In some embodiments, the viral genome may comprise a series of miR binding sites (SEQ ID NO: 1826), including three single miR binding site sequences (SEQ ID NO: 1827).

The miR binding site sequence region may have the following length, such as (but not limited to) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 ,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51 ,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76 ,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 cores glycosides. Viral genome: polyA signal

In some embodiments, the AAV particle viral genome may comprise at least one polyadenylation (polyA) sequence region. Polyadenylation sequence regions may independently have lengths such as, but not limited to, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44 ,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69 ,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94 ,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119 ,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144 ,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169 ,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194 ,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219 ,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244 ,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269 ,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294 ,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319 ,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344 ,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369 ,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392,393,394 ,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419 ,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442,443,444 ,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467,468,469 ,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492,493,494 ,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517,518,519 ,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542,543,544 ,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567,568,569 ,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592,593,594 , 595, 596, 597, 598, 599 and 600 nucleotides. The length of the polyadenylation sequence region of the viral genome can be 4-10, 10-20, 10-50, 20-30, 30-40, 40-50, 50-60, 50-100, 60-70, 70 -80, 80-90, 90-100, 100-110, 100-150, 110-120, 120-130, 130-140, 140-150, 150-160, 150-200, 160-170, 170-180 ,180-190,190-200,200-210,200-250,210-220,220-230,230-240,240-250,250-260,250-300,260-270,270-280,280 -290, 290-300, 300-310, 300-350, 310-320, 320-330, 330-340, 340-350, 350-360, 350-400, 360-370, 370-380, 380-390 ,390-400,400-410,400-450,410-420,420-430,430-440,440-450,450-460,450-500,460-470,470-480,480-490,490 -500, 500-510, 500-550, 510-520, 520-530, 530-540, 540-550, 550-560, 550-600, 560-570, 570-580, 580-590 and 590-600 nucleotides. In some embodiments, the viral genome includes a polyadenylation sequence region that is approximately 477 nucleotides in length.

In some embodiments, the AAV particle viral genome contains at least one polyA sequence region. Non-limiting examples of polyA sequence regions are described in Table 10. Table 10. Representative PolyA sequence regions Sequence area name sequence length SEQ ID NO AHr 477 1828

In some embodiments, the AAV particle viral genome contains a polyA sequence region. As a non-limiting example, the polyA sequence includes the human growth hormone polyadenylation sequence.

In one embodiment, the AAV particle viral genome contains more than one polyA sequence region. Viral Genomes : Stuffing ( or Stuffing ) Sequences

In one embodiment, the AAV particle virus genome may comprise at least one or more stuffer sequence regions. The padding regions may independently have lengths such as (but not limited to) 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 ,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92 ,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117 ,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142 ,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167 ,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192 ,193,194,195,196,197,198,199,200,201,202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217 ,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242 ,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267 ,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292 ,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317 ,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342 ,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367 ,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,391,392 ,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,410,411,412,413,414,415,416,417 ,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434,435,436,437,438,439,440,441,442 ,443,444,445,446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,464,465,466,467 ,468,469,470,471,472,473,474,475,476,477,478,479,480,481,482,483,484,485,486,487,488,489,490,491,492 ,493,494,495,496,497,498,499,500,501,502,503,504,505,506,507,508,509,510,511,512,513,514,515,516,517 ,518,519,520,521,522,523,524,525,526,527,528,529,530,531,532,533,534,535,536,537,538,539,540,541,542 ,543,544,545,546,547,548,549,550,551,552,553,554,555,556,557,558,559,560,561,562,563,564,565,566,567 ,568,569,570,571,572,573,574,575,576,577,578,579,580,581,582,583,584,585,586,587,588,589,590,591,592 ,593,594,595,596,597,598,599,600,601,602,603,604,605,606,607,608,609,610,611,612,613,614,615,616,617 ,618,619,620,621,622,623,624,625,626,627,628,629,630,631,632,633,634,635,636,637,638,639,640,641,642 ,643,644,645,646,647,648,649,650,651,652,653,654,655,656,657,658,659,660,661,662,663,664,665,666,667 ,668,669,670,671,672,673,674,675,676,677,678,679,680,681,682,683,684,685,686,687,688,689,690,691,692 ,693,694,695,696,697,698,699,700,701,702,703,704,705,706,707,708,709,710,711,712,713,714,715,716,717 ,718,719,720,721,722,723,724,725,726,727,728,729,730,731,732,733,734,735,736,737,738,739,740,741,742 ,743,744,745,746,747,748,749,750,751,752,753,754,755,756,757,758,759,760,761,762,763,764,765,766,767 ,768,769,770,771,772,773,774,775,776,777,778,779,780,781,782,783,784,785,786,787,788,789,790,791,792 ,793,794,795,796,797,798,799,800,801,802,803,804,805,806,807,808,809,810,811,812,813,814,815,816,817 ,818,819,820,821,822,823,824,825,826,827,828,829,830,831,832,833,834,835,836,837,838,839,840,841,842 ,843,844,845,846,847,848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867 ,868,869,870,871,872,873,874,875,876,877,878,879,880,881,882,883,884,885,886,887,888,889,890,891,892 ,893,894,895,896,897,898,899,900,901,902,903,904,905,906,907,908,909,910,911,912,913,914,915,916,917 ,918,919,920,921,922,923,924,925,926,927,928,929,930,931,932,933,934,935,936,937,938,939,940,941,942 ,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967 ,968,969,970,971,972,973,974,975,976,977,978,979,980,981,982,983,984,985,986,987,988,989,990,991,992 ,993,994,995,996,997,998,999,1000,1001,1002,1003,1004,1005,1006,1007,1008,1009,1010,1011,1012,1013,1014,1015,1016,1017 ,1018,1019,1020,1021,1022,1023,1024,1025,1026,1027,1028,1029,1030,1031,1032,1033,1034,1035,1036,1037,1038,1039,1040,1041, 1042 ,1043,1044,1045,1046,1047,1048,1049,1050,1051,1052,1053,1054,1055,1056,1057,1058,1059,1060,1061,1062,1063,1064,1065,1066, 1067 ,1068,1069,1070,1071,1072,1073,1074,1075,1076,1077,1078,1079,1080,1081,1082,1083,1084,1085,1086,1087,1088,1089,1090,1091, 1092 ,1093,1094,1095,1096,1097,1098,1099,1100,1101,1102,1103,1104,1105,1106,1107,1108,1109,1110,1111,1112,1113,1114,1115,1116, 1117 ,1118,1119,1120,1121,1122,1123,1124,1125,1126,1127,1128,1129,1130,1131,1132,1133,1134,1135,1136,1137,1138,1139,1140,1141, 1142 ,1143,1144,1145,1146,1147,1148,1149,1150,1151,1152,1153,1154,1155,1156,1157,1158,1159,1160,1161,1162,1163,1164,1165,1166, 1167 ,1168,1169,1170,1171,1172,1173,1174,1175,1176,1177,1178,1179,1180,1181,1182,1183,1184,1185,1186,1187,1188,1189,1190,1191, 1192 ,1193,1194,1195,1196,1197,1198,1199,1200,1201,1202,1203,1204,1205,1206,1207,1208,1209,1210,1211,1212,1213,1214,1215,1216, 1217 ,1218,1219,1220,1221,1222,1223,1224,1225,1226,1227,1228,1229,1230,1231,1232,1233,1234,1235,1236,1237,1238,1239,1240,1241, 1242 ,1243,1244,1245,1246,1247,1248,1249,1250,1251,1252,1253,1254,1255,1256,1257,1258,1259,1260,1261,1262,1263,1264,1265,1266, 1267 ,1268,1269,1270,1271,1272,1273,1274,1275,1276,1277,1278,1279,1280,1281,1282,1283,1284,1285,1286,1287,1288,1289,1290,1291, 1292 ,1293,1294,1295,1296,1297,1298,1299,1300,1301,1302,1303,1304,1305,1306,1307,1308,1309,1310,1311,1312,1313,1314,1315,1316, 1317 ,1318,1319,1320,1321,1322,1323,1324,1325,1326,1327,1328,1329,1330,1331,1332,1333,1334,1335,1336,1337,1338,1339,1340,1341, 1342 ,1343,1344,1345,1346,1347,1348,1349,1350,1351,1352,1353,1354,1355,1356,1357,1358,1359,1360,1361,1362,1363,1364,1365,1366, 1367 ,1368,1369,1370,1371,1372,1373,1374,1375,1376,1377,1378,1379,1380,1381,1382,1383,1384,1385,1386,1387,1388,1389,1390,1391, 1392 ,1393,1394,1395,1396,1397,1398,1399,1400,1401,1402,1403,1404,1405,1406,1407,1408,1409,1410,1411,1412,1413,1414,1415,1416, 1417 ,1418,1419,1420,1421,1422,1423,1424,1425,1426,1427,1428,1429,1430,1431,1432,1433,1434,1435,1436,1437,1438,1439,1440,1441, 1442 ,1443,1444,1445,1446,1447,1448,1449,1450,1451,1452,1453,1454,1455,1456,1457,1458,1459,1460,1461,1462,1463,1464,1465,1466, 1467 ,1468,1469,1470,1471,1472,1473,1474,1475,1476,1477,1478,1479,1480,1481,1482,1483,1484,1485,1486,1487,1488,1489,1490,1491, 1492 ,1493,1494,1495,1496,1497,1498,1499,1500,1501,1502,1503,1504,1505,1506,1507,1508,1509,1510,1511,1512,1513,1514,1515,1516, 1517 ,1518,1519,1520,1521,1522,1523,1524,1525,1526,1527,1528,1529,1530,1531,1532,1533,1534,1535,1536,1537,1538,1539,1540,1541, 1542 ,1543,1544,1545,1546,1547,1548,1549,1550,1551,1552,1553,1554,1555,1556,1557,1558,1559,1560,1561,1562,1563,1564,1565,1566, 1567 ,1568,1569,1570,1571,1572,1573,1574,1575,1576,1577,1578,1579,1580,1581,1582,1583,1584,1585,1586,1587,1588,1589,1590,1591, 1592 ,1593,1594,1595,1596,1597,1598,1599,1600,1601,1602,1603,1604,1605,1606,1607,1608,1609,1610,1611,1612,1613,1614,1615,1616, 1617 ,1618,1619,1620,1621,1622,1623,1624,1625,1626,1627,1628,1629,1630,1631,1632,1633,1634,1635,1636,1637,1638,1639,1640,1641, 1642 ,1643,1644,1645,1646,1647,1648,1649,1650,1651,1652,1653,1654,1655,1656,1657,1658,1659,1660,1661,1662,1663,1664,1665,1666, 1667 ,1668,1669,1670,1671,1672,1673,1674,1675,1676,1677,1678,1679,1680,1681,1682,1683,1684,1685,1686,1687,1688,1689,1690,1691, 1692 ,1693,1694,1695,1696,1697,1698,1699,1700,1701,1702,1703,1704,1705,1706,1707,1708,1709,1710,1711,1712,1713,1714,1715,1716, 1717 ,1718,1719,1720,1721,1722,1723,1724,1725,1726,1727,1728,1729,1730,1731,1732,1733,1734,1735,1736,1737,1738,1739,1740,1741, 1742 ,1743,1744,1745,1746,1747,1748,1749,1750,1751,1752,1753,1754,1755,1756,1757,1758,1759,1760,1761,1762,1763,1764,1765,1766, 1767 ,1768,1769,1770,1771,1772,1773,1774,1775,1776,1777,1778,1779,1780,1781,1782,1783,1784,1785,1786,1787,1788,1789,1790,1791, 1792 ,1793,1794,1795,1796,1797,1798,1799,1800,1801,1802,1803,1804,1805,1806,1807,1808,1809,1810,1811,1812,1813,1814,1815,1816, 1817 ,1818,1819,1820,1821,1822,1823,1824,1825,1826,1827,1828,1829,1830,1831,1832,1833,1834,1835,1836,1837,1838,1839,1840,1841, 1842 ,1843,1844,1845,1846,1847,1848,1849,1850,1851,1852,1853,1854,1855,1856,1857,1858,1859,1860,1861,1862,1863,1864,1865,1866, 1867 ,1868,1869,1870,1871,1872,1873,1874,1875,1876,1877,1878,1879,1880,1881,1882,1883,1884,1885,1886,1887,1888,1889,1890,1891, 1892 ,1893,1894,1895,1896,1897,1898,1899,1900,1901,1902,1903,1904,1905,1906,1907,1908,1909,1910,1911,1912,1913,1914,1915,1916, 1917 ,1918,1919,1920,1921,1922,1923,1924,1925,1926,1927,1928,1929,1930,1931,1932,1933,1934,1935,1936,1937,1938,1939,1940,1941, 1942 , 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967 , 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992 , 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017 , 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042 ,2043,2044,2045,2046,2047,2048,2049,2050,2051,2052,2053,2054,2055,2056,2057,2058,2059,2060,2061,2062,2063,2064,2065,2066, 2067 ,2068,2069,2070,2071,2072,2073,2074,2075,2076,2077,2078,2079,2080,2081,2082,2083,2084,2085,2086,2087,2088,2089,2090,2091, 2092 ,2093,2094,2095,2096,2097,2098,2099,2100,2101,2102,2103,2104,2105,2106,2107,2108,2109,2110,2111,2112,2113,2114,2115,2116, 2117 ,2118,2119,2120,2121,2122,2123,2124,2125,2126,2127,2128,2129,2130,2131,2132,2133,2134,2135,2136,2137,2138,2139,2140,2141, 2142 ,2143,2144,2145,2146,2147,2148,2149,2150,2151,2152,2153,2154,2155,2156,2157,2158,2159,2160,2161,2162,2163,2164,2165,2166, 2167 ,2168,2169,2170,2171,2172,2173,2174,2175,2176,2177,2178,2179,2180,2181,2182,2183,2184,2185,2186,2187,2188,2189,2190,2191, 2192 ,2193,2194,2195,2196,2197,2198,2199,2200,2201,2202,2203,2204,2205,2206,2207,2208,2209,2210,2211,2212,2213,2214,2215,2216, 2217 ,2218,2219,2220,2221,2222,2223,2224,2225,2226,2227,2228,2229,2230,2231,2232,2233,2234,2235,2236,2237,2238,2239,2240,2241, 2242 ,2243,2244,2245,2246,2247,2248,2249,2250,2251,2252,2253,2254,2255,2256,2257,2258,2259,2260,2261,2262,2263,2264,2265,2266, 2267 ,2268,2269,2270,2271,2272,2273,2274,2275,2276,2277,2278,2279,2280,2281,2282,2283,2284,2285,2286,2287,2288,2289,2290,2291, 2292 ,2293,2294,2295,2296,2297,2298,2299,2300,2301,2302,2303,2304,2305,2306,2307,2308,2309,2310,2311,2312,2313,2314,2315,2316, 2317 ,2318,2319,2320,2321,2322,2323,2324,2325,2326,2327,2328,2329,2330,2331,2332,2333,2334,2335,2336,2337,2338,2339,2340,2341, 2342 ,2343,2344,2345,2346,2347,2348,2349,2350,2351,2352,2353,2354,2355,2356,2357,2358,2359,2360,2361,2362,2363,2364,2365,2366, 2367 ,2368,2369,2370,2371,2372,2373,2374,2375,2376,2377,2378,2379,2380,2381,2382,2383,2384,2385,2386,2387,2388,2389,2390,2391, 2392 ,2393,2394,2395,2396,2397,2398,2399,2400,2401,2402,2403,2404,2405,2406,2407,2408,2409,2410,2411,2412,2413,2414,2415,2416, 2417 ,2418,2419,2420,2421,2422,2423,2424,2425,2426,2427,2428,2429,2430,2431,2432,2433,2434,2435,2436,2437,2438,2439,2440,2441, 2442 ,2443,2444,2445,2446,2447,2448,2449,2450,2451,2452,2453,2454,2455,2456,2457,2458,2459,2460,2461,2462,2463,2464,2465,2466, 2467 ,2468,2469,2470,2471,2472,2473,2474,2475,2476,2477,2478,2479,2480,2481,2482,2483,2484,2485,2486,2487,2488,2489,2490,2491, 2492 ,2493,2494,2495,2496,2497,2498,2499,2500,2501,2502,2503,2504,2505,2506,2507,2508,2509,2510,2511,2512,2513,2514,2515,2516, 2517 ,2518,2519,2520,2521,2522,2523,2524,2525,2526,2527,2528,2529,2530,2531,2532,2533,2534,2535,2536,2537,2538,2539,2540,2541, 2542 ,2543,2544,2545,2546,2547,2548,2549,2550,2551,2552,2553,2554,2555,2556,2557,2558,2559,2560,2561,2562,2563,2564,2565,2566, 2567 ,2568,2569,2570,2571,2572,2573,2574,2575,2576,2577,2578,2579,2580,2581,2582,2583,2584,2585,2586,2587,2588,2589,2590,2591, 2592 ,2593,2594,2595,2596,2597,2598,2599,2600,2601,2602,2603,2604,2605,2606,2607,2608,2609,2610,2611,2612,2613,2614,2615,2616, 2617 ,2618,2619,2620,2621,2622,2623,2624,2625,2626,2627,2628,2629,2630,2631,2632,2633,2634,2635,2636,2637,2638,2639,2640,2641, 2642 ,2643,2644,2645,2646,2647,2648,2649,2650,2651,2652,2653,2654,2655,2656,2657,2658,2659,2660,2661,2662,2663,2664,2665,2666, 2667 ,2668,2669,2670,2671,2672,2673,2674,2675,2676,2677,2678,2679,2680,2681,2682,2683,2684,2685,2686,2687,2688,2689,2690,2691, 2692 ,2693,2694,2695,2696,2697,2698,2699,2700,2701,2702,2703,2704,2705,2706,2707,2708,2709,2710,2711,2712,2713,2714,2715,2716, 2717 ,2718,2719,2720,2721,2722,2723,2724,2725,2726,2727,2728,2729,2730,2731,2732,2733,2734,2735,2736,2737,2738,2739,2740,2741, 2742 ,2743,2744,2745,2746,2747,2748,2749,2750,2751,2752,2753,2754,2755,2756,2757,2758,2759,2760,2761,2762,2763,2764,2765,2766, 2767 ,2768,2769,2770,2771,2772,2773,2774,2775,2776,2777,2778,2779,2780,2781,2782,2783,2784,2785,2786,2787,2788,2789,2790,2791, 2792 ,2793,2794,2795,2796,2797,2798,2799,2800,2801,2802,2803,2804,2805,2806,2807,2808,2809,2810,2811,2812,2813,2814,2815,2816, 2817 ,2818,2819,2820,2821,2822,2823,2824,2825,2826,2827,2828,2829,2830,2831,2832,2833,2834,2835,2836,2837,2838,2839,2840,2841, 2842 ,2843,2844,2845,2846,2847,2848,2849,2850,2851,2852,2853,2854,2855,2856,2857,2858,2859,2860,2861,2862,2863,2864,2865,2866, 2867 ,2868,2869,2870,2871,2872,2873,2874,2875,2876,2877,2878,2879,2880,2881,2882,2883,2884,2885,2886,2887,2888,2889,2890,2891, 2892 ,2893,2894,2895,2896,2897,2898,2899,2900,2901,2902,2903,2904,2905,2906,2907,2908,2909,2910,2911,2912,2913,2914,2915,2916, 2917 ,2918,2919,2920,2921,2922,2923,2924,2925,2926,2927,2928,2929,2930,2931,2932,2933,2934,2935,2936,2937,2938,2939,2940,2941, 2942 ,2943,2944,2945,2946,2947,2948,2949,2950,2951,2952,2953,2954,2955,2956,2957,2958,2959,2960,2961,2962,2963,2964,2965,2966, 2967 ,2968,2969,2970,2971,2972,2973,2974,2975,2976,2977,2978,2979,2980,2981,2982,2983,2984,2985,2986,2987,2988,2989,2990,2991, 2992 ,2993,2994,2995,2996,2997,2998,2999,3000,3001,3002,3003,3004,3005,3006,3007,3008,3009,3010,3011,3012,3013,3014,3015,3016, 3017 ,3018,3019,3020,3021,3022,3023,3024,3025,3026,3027,3028,3029,3030,3031,3032,3033,3034,3035,3036,3037,3038,3039,3040,3041, 3042 ,3043,3044,3045,3046,3047,3048,3049,3050,3051,3052,3053,3054,3055,3056,3057,3058,3059,3060,3061,3062,3063,3064,3065,3066, 3067 ,3068,3069,3070,3071,3072,3073,3074,3075,3076,3077,3078,3079,3080,3081,3082,3083,3084,3085,3086,3087,3088,3089,3090,3091, 3092 ,3093,3094,3095,3096,3097,3098,3099,3100,3101,3102,3103,3104,3105,3106,3107,3108,3109,3110,3111,3112,3113,3114,3115,3116, 3117 ,3118,3119,3120,3121,3122,3123,3124,3125,3126,3127,3128,3129,3130,3131,3132,3133,3134,3135,3136,3137,3138,3139,3140,3141, 3142 ,3143,3144,3145,3146,3147,3148,3149,3150,3151,3152,3153,3154,3155,3156,3157,3158,3159,3160,3161,3162,3163,3164,3165,3166, 3167 ,3168,3169,3170,3171,3172,3173,3174,3175,3176,3177,3178,3179,3180,3181,3182,3183,3184,3185,3186,3187,3188,3189,3190,3191, 3192 ,3193,3194,3195,3196,3197,3198,3199,3200,3201,3202,3203,3204,3205,3206,3207,3208,3209,3210,3211,3212,3213,3214,3215,3216, 3217 ,3218,3219,3220,3221,3222,3223,3224,3225,3226,3227,3228,3229,3230,3231,3232,3233,3234,3235,3236,3237,3238,3239,3240,3241, 3242 , 3243, 3244, 3245, 3246, 3247, 3248, 3249 and 3250 nucleotides. The length of any stuffing region of the viral genome can be 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150, 1150- 1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750, 1750-1 800, 2 400- 2450, 2450-2500, 2500-2550, 2550-2600, 2600-2650, 2650-2700, 2700-2750, 2750-2800, 2800-2850, 2850-2900, 2900-2950, 2950-3000, 3000-3 050. 3050-3100, 3100-3150, 3150-3200 and 3200-3250 nucleotides. As a non-limiting example, the viral genome contains a stuffer region that is approximately 450 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 570 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1313 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1384 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1785 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1790 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1856 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1868 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 1870 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2012 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2014 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2034 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2106 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2264 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2266 nucleotides in length. As a non-limiting example, the viral genome contains a stuffer region that is approximately 2335 nucleotides in length.

In some embodiments, the AAV particle viral genome contains at least one stuffer sequence region. Non-limiting examples of padding sequence regions are described in Table 11. Table 11. Representative filler sequence regions Sequence area name sequence length SEQ ID NO Alb450 450 1829 Alb570 570 1830 Alb1313 1313 1831 Alb1384 1384 1832 Alb1785 1785 1833 Alb1790 1790 1834 Alb1856 1856 1835 Alb1870 1870 1836 Alb2014 2014 1837 Alb2034 2034 1838 Alb2106 2106 1839 Alb2264 2264 1840 Alb2266 2266 1841 Alb2335 2335 1842

In some embodiments, the AAV particle viral genome includes a stuffer sequence region that includes human albumin sequences. In some embodiments, the AAV particle virus genome contains the stuffer sequence region Alb450. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb570. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb1313. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb1384. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb1785. In some embodiments, the AAV particulate viral genome includes the stuffer sequence region Alb1790. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb1856. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb1870. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb2014. In some embodiments, the AAV particle virus genome contains the stuffer sequence region Alb2034. In some embodiments, the AAV particle virus genome contains the stuffer sequence region Alb2106. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb2264. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb2266. In some embodiments, the AAV particle virus genome includes the stuffer sequence region Alb2335. Viral genomes: conjugation sequences

In some embodiments, the joining sequences can be used in combination with any of the viral genome components described herein, such as, but not limited to, those listed in Tables 5-11. In certain embodiments, the junction sequence can be located at the 5' end of a viral genomic component (eg, promoter, enhancer, intron/exon, miR binding site, tag, polyA) within the viral genome. In certain embodiments, the junction sequence can be located at the 3' end of a viral genomic component (eg, promoter, enhancer, intron/exon, miR binding site, tag, polyA) within the viral genome. In certain embodiments, the viral genome may include more than one conjugative sequence. As a non-limiting example, the viral genome can include junction sequences on the 5' end of the viral genome component and on the 3' end of the viral genome component. The joining sequence may be the same sequence, two different sequences, or a sequence split on either side of the viral genome component. In certain embodiments, the joining sequence includes SEQ ID NO: 1813. In certain embodiments, the joining sequence includes SEQ ID NO: 1814. Viral Genomes : ITR to ITR Modularity

In some embodiments, the ITR to ITR sequences of the viral genome may comprise any of the sequences set forth in Tables 12 to 17. Table 12. Representative sequence regions from ITR to ITR sequences cFXN1 cFXN2 cFXN3 cFXN4 cFXN5 cFXN6 ITR to ITR 1778 1779 1780 1781 1782 1783 5'ITR 1811 1811 1811 1811 1811 1811 promoter 1776 1776 1738 1740 1740 1742 join 1813 1813 1813 1813 intron / exon 1816 1816 1816 1816 1816 1816 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1822 1822 1822 1822 1822 1822 label 1825 1825 1825 1825 1825 1825 miR122 BS (3x) 1826 1826 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 1828 filler 1829 3'ITR 1812 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1778 (cFXN1), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA promoter region, including the ie1 exon. Region 1, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, shared by crab-eating macaques Ecoprotein payload sequence, HA tag sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1779 (cFXN2), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA promoter region, including the ie1 exon. Region 1, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, shared by crab-eating macaques The economic protein payload sequence, the HA tag sequence, the miR binding site series containing three repeats of a single miR122 binding site sequence, and the human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1780 (cFXN3), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D4 promoter region, and a junction sequence, Human β-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1781 (cFXN4), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D6 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1782 (cFXN5), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D6 promoter region, and a junction sequence, Human beta-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1783 (cFXN6), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D8 promoter region, and a junction sequence, Human β-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence. Table 13. Representative sequence regions from ITR to ITR sequences cFXN7 cFXN8 cFXN9 cFXN10 cFXN11 cFXN12 ITR to ITR 1784 1785 1786 1787 1788 1789 5'ITR 1811 1811 1811 1811 1811 1811 enhancer 1777 1777 promoter 1742 1760 1761 1762 1772 1772 join 1813 1814 1813 1813 intron / exon 1816 1816 1816 1816 1816 1816 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1822 1822 1822 1822 1822 1822 label 1825 1825 1825 1825 1825 1825 miR122 BS (3x) 1826 1826 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 1828 filler 1829 3'ITR 1812 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1784 (cFXN7), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D8 promoter region, and a junction sequence, Human beta-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1785 (cFXN8), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a mCBA promoter region, and a junction sequence, including ie1 Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, food Cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1786 (cFXN9), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a mCBA-D1 promoter region, and a junction sequence, Human β-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1787 (cFXN10), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a mCBA-D2 promoter region, and a junction sequence, Human beta-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region , cynomolgus macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1788 (cFXN11), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV enhancer and promoter region, including ie1 Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, food Crab macaque syntaxin payload sequence, HA tag sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1789 (cFXN12), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV enhancer and promoter region, including ie1 Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, food Crab macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence. Table 14. Representative sequence regions from ITR to ITR sequences cFXN13 cFXN14 cFXN15 cFXN16 cFXN17 cFXN18 ITR to ITR 1790 1791 1792 1793 1794 1795 5'ITR 1811 1811 1811 1811 1811 1811 promoter 1744 1746 1750 1750 1754 1756 intron / exon 1816 1816 1816 1816 1816 1816 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1822 1822 1822 1822 1822 1822 label 1825 1825 1825 1825 1825 1825 miR122 BS (3x) 1826 1826 1826 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 1828 filler 1829 3'ITR 1812 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1790 (cFXN13), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D1 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1791 (cFXN14), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D3 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1792 (cFXN15), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D7 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1793 (cFXN16), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D7 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1794 (cFXN17), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a cotaxin promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1795 (cFXN18), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a cotaxin promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, eating crab Macaque syntaxin payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and human growth hormone polyadenylation sequence. Table 15. Representative sequence regions from ITR to ITR sequences hFXN1 hFXN2 hFXN3 AHr ikB ITR to ITR 1796 1797 1798 1799 1800 5'ITR 1811 1811 1811 1811 1811 enhancer 1777 1777 promoter 1776 1742 1742 1772 1772 join 1813 1813 intron / exon 1816 1816 1816 1816 1816 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1823 1824 1824 1824 1824 label 1825 miR122 BS (3x) 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 filler 1841 1842 1833 1835 3'ITR 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1796 (hFXN1), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA promoter region, including the ie1 exon. Region 1, ie1 intron region 1, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human fataxin The payload sequence, HA tag sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, and a human growth hormone polyadenylation sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1797 (hFXN2), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D8 promoter region, and a junction sequence, Human beta-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region , human syntaxin payload sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1798 (hFXN3), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D8 promoter region, and a junction sequence, Human β-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region , human syntaxin payload sequence, human growth hormone polyadenylation sequence, and albumin filler sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1799 (hFXN4), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV enhancer and promoter region, including ie1 Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, human The syntaxin payload sequence consists of a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1800 (hFXN5), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV enhancer and promoter region, including ie1 Exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region, human β-hemoglobulin intron/exon region, human Fataxin payload sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence. Table 16. Representative sequence regions from ITR to ITR sequences hFXN6 htK AHr AHr hFXN10 ITR to ITR 1801 1802 1803 1804 1805 5'ITR 1811 1811 1811 1811 1811 promoter 1750 1750 1756 1756 1771 promoter components 1773, 1775 intron / exon 1816 1816 1816 1816 1815 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1824 1824 1824 1824 1824 miR122 BS (3x) 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 filler 1840 1842 1831 1832 1830 3'ITR 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1801 (hFXN6), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D7 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common The economic protein payload sequence includes a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1802 (hFXN7), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D7 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common Ecorin payload sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1803 (hFXN8), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a cotaxin promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common The economic protein payload sequence includes a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1804 (hFXN9), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a cotaxin promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common Ecorin payload sequence, human growth hormone polyadenylation sequence, and albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1805 (hFXN10), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, including a CMV promoter region and a CBA promoter region The CAG promoter region, introns, human syntaxin payload sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, human growth hormone polyadenylation sequence, and albumin filler sequence. Table 17. Representative sequence regions from ITR to ITR sequences hFXN11 hFXN12 hFXN13 hFXN14 hFXN15 ITR to ITR 1806 1807 1808 1809 1810 5'ITR 1811 1811 1811 1811 1811 promoter 1744 1746 1738 1740 1774 promoter components 1740 join 1813 intron / exon 1816 1816 1816 1816 1816 Intron / exon components 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 1817, 1819, 1820, 1821 FXN payload 1824 1824 1824 1824 1824 miR122 BS (3x) 1826 1826 1826 1826 1826 miR122BS 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) 1827 (3x) Poly(A) 1828 1828 1828 1828 1828 filler 1836 1837 1838 1839 1834 3'ITR 1812 1812 1812 1812 1812

In some embodiments, the AAV particle genome includes SEQ ID NO: 1806 (hFXN11), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D1 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common The economic protein payload sequence includes a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1807 (hFXN12), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CMV-D3 promoter region, including an ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common The economic protein payload sequence includes a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1808 (hFXN13), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D4 promoter region, and a junction sequence, Human β-hemoglobulin intron/exon region including ie1 exon 1 region, ie1 intron 1 region, human β-hemoglobulin intron region, human β-hemoglobulin exon region , human syntaxin payload sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1809 (hFXN14), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, a CBA-D6 promoter region, including the ie1 outer Exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region, human beta-hemoglobulin intron/exon region, human common The economic protein payload sequence includes a series of three repeats of a single miR122 binding site, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence.

In some embodiments, the AAV particle genome includes SEQ ID NO: 1810 (hFXN15), which includes a 5' inverted terminal repeat (ITR) sequence region and a 3' ITR sequence region, including a promoter region of the CMV region and the CBA region , including the ie1 exon 1 region, ie1 intron 1 region, human beta-hemoglobulin intron region, human beta-hemoglobulin exon region and human beta-hemoglobulin intron/exon Region, human syntaxin payload sequence, a series of miR binding sites containing three repeats of a single miR122 binding site sequence, a human growth hormone polyadenylation sequence, and an albumin stuffer sequence. Certain embodiments provide the viral genome encapsulated in a capsid having a serotype selected from Table 1. For example, the capsid serotype may be selected from the group consisting of: VOY101, VOY102, AAVPHP.B, AAVPHP.N, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV9 K449R, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVDJ and AAVDJ8 or any variant thereof. In some embodiments, the capsid serotype is AAVPHP.B, AAV9, AAV6, AAVrh10, and/or AAVDJ.

In some embodiments, a viral genome as provided in any of Tables 4, 12, 13, 14, 15, 16, or 17 is encapsulated in an AAV capsid to produce AAV particles. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a VOY101 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a VOY201 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and an AAV9 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and an AAV9 K449R capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and an AAVPHP.B capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and an AAVPHP.N capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 1. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 1724. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 136. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 3. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 2. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 9.

In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a VOY101 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a VOY201 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and an AAV9 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and an AAV9 K449R capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and an AAVPHP.B capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and an AAVPHP.N capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 1. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 1724. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 136. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 3. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 2. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 9.

In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a VOY101 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a VOY201 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and an AAV9 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and an AAV9 K449R capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and an AAVPHP.B capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and an AAVPHP.N capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 1. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 1724. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 136. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 3. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 2. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 9.

In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a VOY101 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a VOY201 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and an AAV9 capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and an AAV9 K449R capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and an AAVPHP.B capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and an AAVPHP.N capsid. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 1. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 1724. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 136. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 3. In some embodiments, an AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 2. In some embodiments, the AAV particle comprises a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 9.

In some embodiments, AAV particles comprising a viral genome and capsid as set forth in any of Tables 4, 12, 13, 14, 15, 16, or 17 are formulated in a solution suitable for administration, For example, formulations containing one or more salts and one or more surfactants. In some embodiments, the formulation includes one or more of sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and pluronic F-68, with a pH of about 7-8. In some embodiments, the formulation includes 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate), and 0.001% Pluronic F- 68 (v/v), pH 7.4 (Formulation 1 of the invention). In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a VOY101 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a VOY201 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and an AAV9 capsid are formulated to comprise 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and an AAV9 K449R capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and an AAVPHP.B capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and an AAVPHP.N capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 1 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 1724 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 136 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 3 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 2 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1797 and a capsid comprising SEQ ID NO: 9 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4.

In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a VOY101 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a VOY201 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and an AAV9 capsid are formulated to comprise 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and an AAV9 K449R capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and an AAVPHP.B capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and an AAVPHP.N capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 1 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 1724 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 136 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 3 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 2 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1801 and a capsid comprising SEQ ID NO: 9 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4.

In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a VOY101 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a VOY201 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and an AAV9 capsid are formulated to comprise 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and an AAV9 K449R capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and an AAVPHP.B capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and an AAVPHP.N capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 1 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 1724 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 136 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 3 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 2 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1808 and a capsid comprising SEQ ID NO: 9 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4.

In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a VOY101 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a VOY201 capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and an AAV9 capsid are formulated to comprise 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride , 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Plotonic F-68 (v/v), in a solution with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and an AAV9 K449R capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and an AAVPHP.B capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and an AAVPHP.N capsid are formulated to contain 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM chloride Potassium phosphate, 2 mM potassium dihydrogen phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 1 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1722 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 1724 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 1723 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 136 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 135 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 3 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid encoded by a nucleic acid sequence comprising SEQ ID NO: 4 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate ( Disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), pH 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 2 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4. In some embodiments, AAV particles comprising a viral genome comprising SEQ ID NO: 1809 and a capsid comprising SEQ ID NO: 9 are formulated in a solution containing 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate) , 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate) and 0.001% Pluronic F-68 (v/v), with a pH of 7.4.

In some embodiments, the viral genome is single-stranded. In some embodiments, the viral genome is self-complementary. Certain embodiments of the AAV particles of the invention comprise two inverted terminal repeat (ITR) regions. In some embodiments, the ITR is an AAV2 ITR. In some embodiments, one ITR includes SEQ ID NO: 1811 and the other ITR includes SEQ ID NO: 1812. Certain embodiments provide a viral genome comprising a first ITR region located 5' relative to the polynucleotide sequence and a second inverted terminal repeat region located 3' relative to the polynucleotide sequence. II. Virus production General virus production process

In some embodiments, cells used to produce AAV (eg, rAAV) particles may include mammalian cells (such as HEK293 cells) and/or insect cells (such as Sf9 cells).

In various embodiments, AAV production includes processes and methods for generating AAV particles and vectors that can contact target cells to deliver a payload (eg, a recombinant viral construct) that includes nucleotides encoding the payload molecule. In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector, such as a recombinant adeno-associated virus (rAAV) vector. In certain embodiments, the AAV particles are adeno-associated virus (AAV) particles, such as recombinant adeno-associated virus (rAAV) particles.

In various embodiments, provided herein are methods of producing AAV particles or vectors by: (a) contacting a virus-producing cell with one or more viral expression constructs encoding at least one AAV capsid protein and one or more encoding payload molecules contact with a payload construct, the payload molecule may be selected from: transgenic genes, protein-encoding polynucleotides and regulatory nucleic acids; (b) culturing virus-producing cells under conditions that produce at least one AAV particle or vector, and (c) spontaneous flow separation of AAV particles or vectors.

In such methods, the viral expression construct may encode at least one structural protein and/or at least one non-structural protein. Structural proteins may include any of the native or wild-type capsid proteins VP1, VP2 and/or VP3 or chimeric proteins thereof. Non-structural proteins may include any of the native or wild-type Rep78, Rep68, Rep52 and/or Rep40 proteins or chimeric proteins thereof.

In certain embodiments, contacting occurs via transient transfection, viral transduction, and/or electroporation.

In certain embodiments, the virus-producing cell line is selected from mammalian cells and insect cells. In certain embodiments, the insect cells include Spodoptera frugiperda insect cells. In certain embodiments, the insect cells include Sf9 insect cells. In certain embodiments, the insect cells include Sf21 insect cells.

In various embodiments, the payload construct vectors of the present invention may include at least one inverted terminal repeat (ITR) and may include mammalian DNA.

AAV particles and viral vectors produced according to the methods described herein are also provided.

In various embodiments, the AAV particles of the invention may be formulated into pharmaceutical compositions with one or more acceptable excipients.

In certain embodiments, AAV particles or viral vectors can be produced by the methods described herein.

In certain embodiments, AAV particles can be produced by contacting virus-producing cells (eg, insect cells or mammalian cells) with at least one viral expression construct encoding at least one capsid protein and at least one payload construct vector. Virus-producing cells can be contacted by transient transfection, viral transduction and/or electroporation. Payload Construct Vectors may include payload constructs encoding payload molecules such as, but not limited to, transgenes, protein-encoding polynucleotides, and regulatory nucleic acids. The virus-producing cells may be in a state that allows the production, isolation (e.g., using temperature-induced lysis, mechanical lysis, and/or chemical lysis), and/or purification (e.g., using filtration, chromatography, and/or immunoaffinity purification) of at least one AAV particle or vector. cultured under conditions. As a non-limiting example, the payload construct vector may include mammalian DNA.

In certain embodiments, AAV particles are produced in insect cells, such as Fall Armyworm (Sf9) cells, using the methods described herein. As a non-limiting example, insect cells are contacted using viral transduction, which may include baculovirus transduction.

In certain embodiments, AAV particles are produced in mammalian cells (eg, HEK293 cells) using the methods described herein. As a non-limiting example, mammalian cells are contacted using viral transduction, which may include multiplastid transient transfection, such as tripleplastid transient transfection.

In certain embodiments, the AAV particle production methods described herein produce greater than 10 ¹ , greater than 10 ² , greater than 10 ³ , greater than 10 ⁴ or greater than 10 ⁵ AAV particles in a virus-producing cell.

In certain embodiments, processes of the present invention include producing virus particles in virus-producing cells using a virus production system that includes at least one viral expression construct and at least one payload construct. At least one viral expression construct and at least one payload construct can be co-transfected (eg, double transfection, triple transfection) into virus-producing cells. Transfection is accomplished using standard molecular biology techniques known and routinely performed by those skilled in the art. Virus-producing cells provide the cellular machinery required for protein expression and other biological materials required to produce AAV particles, including the Rep protein that replicates the payload construct and the Cap protein that assembles the capsid that forms the payload construct that encloses replication. The resulting AAV particles are extracted from virus-producing cells and processed into pharmaceutical preparations for administration.

In various embodiments, once administered, without being bound by theory, the AAV particles disclosed herein can contact a target cell and enter the cell, such as endosomes. AAV particles, such as those released from endosomes, can then contact the nucleus of the target cell to deliver the payload construct. For example, upon delivery of the payload construct of a recombinant viral construct to the nucleus of a target cell, the payload molecule encoded by the payload construct can be expressed in the target cell.

In certain embodiments, processes for producing viral particles utilize seed cultures of virus-producing cells that include one or more baculoviruses (e.g., baculoviruses that have been transfected with viral expression constructs and payload construct vectors). expression vector (BEV) or baculovirus-infected insect cells (BIIC)). In certain embodiments, the seed culture is harvested, divided into aliquots and frozen, and can be used at a later time point to initiate infection of the primary producing cell population.

In some embodiments, large-scale production of AAV particles utilizes bioreactors. Without being bound by theory, the use of bioreactors may allow precise measurement and/or control of variables that support the growth and activity of virus-producing cells, such as mass, temperature, mixing conditions (impeller RPM or wave oscillation), CO ₂ concentration, _O2 concentration, gas injection rate and volume, gas coverage rate and volume, pH, viable cell density (VCD), cell viability, cell diameter and/or optical density (OD). In certain embodiments, the bioreactor is used for batch production, where the entire culture is harvested and the AAV particles purified at experimentally determined time points. In some embodiments, the bioreactor is used for continuous production, where a portion of the culture is harvested for AAV particle purification at experimentally determined time points, and the remaining culture in the bioreactor is regenerated with additional growth medium components. new.

In various embodiments, AAV viral particles can be extracted from virus-producing cells in a process that includes cell lysis, clarification, sterilization, and purification. Cytolysis includes any process that destroys the structure of virus-producing cells, thereby releasing AAV particles. In certain embodiments, cell lysis may include thermal shock, chemical or mechanical lysis methods. Clarification may include overall purification of a mixture of lysed cells, media components, and AAV particles. In certain embodiments, clarification includes centrifugation and/or filtration, including (but not limited to) deep end, tangential flow, and/or hollow fiber filtration.

In various embodiments, the end result of virus production is a collection of purified AAV particles comprising the following two components: (1) a payload construct (eg, a recombinant AAV vector genome construct) and (2) a viral capsid.

In certain embodiments, the virus production system or process of the present invention includes the step of producing baculovirus-infected insect cells (BIIC) using virus-producing cells (VPC) and plastid constructs. Virus-producing cells (VPC) from the cell bank (CB) are thawed and amplified to obtain the target working volume and VPC concentration. The obtained VPC aggregate is divided into a Rep/Cap VPC aggregate and a payload VPC aggregate. One or more Rep/Cap plastid constructs (viral expression constructs) are processed into Rep/Cap shuttle vector polynucleotides and transfected into Rep/Cap VPC aggregates. One or more payload plasmid constructs (payload constructs) are processed into payload shuttle polynucleotides and transfected into the payload VPC pool. Two VPC pools were grown to generate the P1 Rep/Cap baculovirus expression vector (BEV) and the P1 payload BEV. The two BEV pools are amplified into a plaque pool, of which a single plaque is selected for pure line plaque (CP) purification (also known as single plaque amplification). The process may include a single CP purification step or may include multiple CP purification steps either sequentially or separated by other processing steps. One or more CP purification steps provide a CP Rep/Cap BEV aggregate and a CP payload BEV aggregate. These two BEV pools can then be stored and used in future generation steps, or they can then be transfected into VPCs to generate Rep/Cap BIIC pools and payload BIIC pools.

In certain embodiments, the virus production system or process of the present invention includes the step of producing AAV particles using virus-producing cells (VPC) and baculovirus-infected insect cells (BIIC). Virus-producing cells (VPC) from the cell bank (CB) are thawed and amplified to obtain the target working volume and VPC concentration. A working volume of virus-producing cells is inoculated into a production bioreactor and can be further expanded to a working volume of 200 to 2000 L with the target VPC concentration for BIIC infection. The working volume of VPC in the production bioreactor was then co-infected with Rep/Cap BIIC and payload BIIC at the target VPC:BIIC ratio and the target BIIC:BIIC ratio. VCD infection can also exploit BEV. Co-infected VPCs are cultured and expanded in production bioreactors to generate bulk harvests of AAV particles and VPCs. viral expression construct

In various embodiments, the virus production systems of the invention include one or more virus expression constructs that can be transfected/transduced into virus-producing cells. In certain embodiments, the viral expression construct or payload construct of the invention can be a shuttle vector, also known as a baculovirus plasmid or a recombinant baculovirus genome. In certain embodiments, viral expression includes a protein-encoding nucleotide sequence and at least one expression control sequence for expression in a virus-producing cell. In certain embodiments, viral expression includes a protein-encoding nucleotide sequence operably linked to at least one expression control sequence for expression in a virus-producing cell. In certain embodiments, viral expression constructs contain parvovirus genes under the control of one or more promoters. Parvovirus genes may include nucleotide sequences encoding non-structural AAV replication proteins, such as Rep genes encoding Rep52, Rep40, Rep68 or Rep78 proteins. Parvovirus genes may include nucleotide sequences encoding structural AAV proteins, such as the Cap gene encoding VP1, VP2, and VP3 proteins.

Viral expression constructs of the invention may include any biological or chemical compound or formulation that facilitates transformation, transfection, or transduction of cells with nucleic acids. Exemplary biological viral expression constructs include plasmids, linear nucleic acid molecules, and recombinant viruses, including baculoviruses. Exemplary chemical carriers include lipoplexes. Viral expression constructs are used to incorporate nucleic acid sequences into virus-replicating cells according to the invention. (O'Reilly, David R., Lois K. Miller and Verne A. Luckow. Baculovirus expression vectors: a laboratory manual. Oxford University Press, 1994.); Maniatis et al., ed. Molecular Cloning. CSH Laboratory, NY, N.Y. (1982) ); and Philipport and Scluber, eds. Liposomes as tools in Basic Research and Industry. CRC Press, Ann Arbor, Mich. (1995), each of which is incorporated by reference in its entirety for their respective content on viral expression constructs and their uses.

In certain embodiments, the viral expression construct is an AAV expression construct that includes one or more nucleotide sequences encoding a non-structural AAV replication protein, a structural AAV capsid protein, or a combination thereof.

In certain embodiments, the viral expression constructs of the invention can be plastid vectors. In certain embodiments, the viral expression constructs of the invention may be baculovirus constructs.

The invention is not limited by the number of viral expression constructs used to generate AAV particles or viral vectors. In certain embodiments, one, two, three, four, five, six or more viral expression constructs can be used to produce AAV particles in virus-producing cells according to the invention. In certain embodiments of the invention, viral expression constructs can be used to produce AAV particles in insect cells. In certain embodiments, wild-type AAV sequences of the capsid and/or rep genes can be modified, for example, to improve properties of the viral particles, such as to increase infectivity or specificity, or to increase yield.

In certain embodiments, a viral expression construct may contain a nucleotide sequence that includes an initiation codon region, such as a sequence encoding an AAV capsid protein that includes one or more initiation codon regions. In certain embodiments, the initiation codon region may be within a expression control sequence. The start codon can be an ATG or a non-ATG codon (ie, a suboptimal start codon, where the start codon of the AAV VP1 capsid protein is non-ATG).

In certain embodiments, viral expression constructs for AAV production may contain nucleotide sequences encoding AAV capsid proteins, wherein the initiation codon of the AAV VP1 capsid protein is non-ATG, i.e., suboptimal initiation. Codons that allow expression of modified ratios of viral capsid proteins in production systems to provide improved host cell infectivity. In a non-limiting example, the viral construct vector may contain a nucleic acid construct comprising nucleotide sequences encoding AAV VP1, VP2, and VP3 capsid proteins, wherein the initiation codon for translation of the AAV VP1 capsid protein be CTG, TTG or GTG, as described in US Pat. No. 8,163,543, which is incorporated herein by reference in its entirety with respect to AAV capsid proteins and their production.

In certain embodiments, the viral expression construct of the invention can be a plasmid vector or a baculovirus construct encoding the parvovirus Rep protein for expression in insect cells. In certain embodiments, a single coding sequence is used for Rep78 and Rep52 proteins, wherein the initiation codon used to translate the Rep78 protein is a suboptimal initiation codon selected from the group consisting of ACG, TTG, CTG, and GTG, wherein Achieving partial exon skipping after expression in insect cells, as described in U.S. Patent No. 8,512,981, the contents of which are incorporated herein by reference in its entirety, e.g., to promote expression of Rep78 versus promoting high vector yields Rep52 is less adequate.

In certain embodiments, the VP coding region encodes one or more AAV capsid proteins of a particular AAV serotype. The AAV serotypes in the VP coding region may be the same or different. In certain embodiments, the VP coding region may be codon optimized. In certain embodiments, the VP coding region or nucleotide sequence may be codon-optimized for mammalian cells. In certain embodiments, the VP coding region or nucleotide sequence may be codon-optimized for insect cells. In certain embodiments, the VP coding region or nucleotide sequence may be codon-optimized for S. frugiperda cells. In certain embodiments, the VP coding region or nucleotide sequence can be codon-optimized for Sf9 or Sf21 cell lines.

In certain embodiments, a nucleotide sequence encoding one or more VP capsid proteins can be codon-optimized to have less than 100% nucleotide homology to a reference nucleotide sequence. In certain embodiments, the nucleotide homology between the codon-optimized VP nucleotide sequence and the reference VP nucleotide sequence is less than 100%, less than 99%, less than 98%, less than 97% %, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, less than 85%, Less than 84%, less than 83%, less than 82%, less than 81%, less than 80%, less than 78%, less than 76%, less than 74%, less than 72%, less than 70%, less than 68%, less than 66%, less than 64 %, less than 62%, less than 60%, less than 55%, less than 50% and less than 40%.

In certain embodiments, the viral expression construct or payload construct of the invention can be a shuttle vector, also known as a baculovirus plasmid or a recombinant baculovirus genome. In certain embodiments, viral expression constructs or payload constructs (e.g., shuttle vectors) of the invention may include polynucleotides prepared by standard molecular biology techniques known and performed by those skilled in the art. Homologous recombination (transposon donor/acceptor system) is incorporated into the shuttle vector.

In certain embodiments, polynucleotides incorporated into shuttle vectors (ie, polynucleotide inserts) may include expression control sequences operably linked to protein-encoding nucleotide sequences. In certain embodiments, polynucleotides incorporated into the shuttle vector can include expression control sequences that include a promoter, such as p10 or polh, and that are operably linked to the encoding structural AAV capsid protein (e.g., VP1 , VP2 VP3 or combination thereof) nucleotide sequence. In certain embodiments, polynucleotides incorporated into the shuttle vector may include expression control sequences that include a promoter, such as p10 or polh, and that are operably linked to encoding a nonstructural AAV capsid protein (e.g., Rep78, Rep52 or combination thereof) nucleotide sequence.

The method of the present invention is not limited to the use of specific presentation control sequences. However, when a certain stoichiometry of VP products is reached (close to 1:1:10 for VP1, VP2, and VP3, respectively), and when the amount of Rep52 or Rep40 (also called p19 Rep) is significantly higher than that of Rep78 or Rep68 ( Also known as p5 Rep), improved AAV production in producing cells, such as insect cells, can be achieved. In certain embodiments, the p5/p19 ratio is below 0.6, below 0.4, or below 0.3, but is always at least 0.03. These ratios can be measured in terms of protein content or can be related to the relative amounts of specific mRNAs.

In certain embodiments, AAV particles are produced in virus-producing cells, such as mammalian or insect cells, in which all three VP proteins are expressed in a stoichiometry close to, about, or at: 1:1:10 (VP1 :VP2:VP3), 2:2:10 (VP1:VP2:VP3), 2:0:10 (VP1:VP2:VP3), 1-2:0-2:10 (VP1:VP2:VP3), 1 -2:1-2:10 (VP1:VP2:VP3), 2-3:0-3:10 (VP1:VP2:VP3), 2-3:2-3:10 (VP1:VP2:VP3), 3:3:10 (VP1:VP2:VP3), 3-5:0-5:10 (VP1:VP2:VP3) or 3-5:3-5:10 (VP1:VP2:VP3).

In certain embodiments, the performance control region is engineered to produce a VP1:VP2:VP3 ratio selected from the group consisting of: about or exactly 1:0:10, about or exactly 1:1:10, about or exactly 2:1:10, about or exactly 2:1:10, about or exactly 2:2:10, about or exactly 3:0:10, about or exactly 3:1:10, about or exactly 3:2:10 , about or exactly 3:3:10, about or exactly 4:0:10, about or exactly 4:1:10, about or exactly 4:2:10, about or exactly 4:3:10, about or exactly 4 :4:10, about or exactly 5:5:10, about or exactly 1-2:0-2:10, about or exactly 1-2:1-2:10, about or exactly 1-3:0-3 :10, about or exactly 1-3:1-3:10, about or exactly 1-4:0-4:10, about or exactly 1-4:1-4:10, about or exactly 1-5:1 -5:10, about or exactly 2-3:0-3:10, about or exactly 2-3:2-3:10, about or exactly 2-4:2-4:10, about or exactly 2-5 :2-5:10, about or exactly 3-4:3-4:10, about or exactly 3-5:3-5:10 and about or exactly 4-5:4-5:10.

In certain embodiments of the invention, Rep52 or Rep78 is transcribed from a baculovirus-derived polyhedral promoter (polh). Rep52 or Rep78 can also be transcribed from a weaker promoter. For example, the transcription activity of the Δie-1 promoter, a deletion mutant of the ie-1 promoter, is about 20% of that of the ie-1 promoter. A promoter that is substantially homologous to the Δie-1 promoter can be used. For promoters, a homology of at least 50%, 60%, 70%, 80%, 90% or greater is considered to be a substantially homologous promoter. mammalian cells

The virus production of the invention disclosed herein describes processes and methods for producing AAV particles or viral vectors that contact target cells to deliver a payload construct, such as a recombinant AAV particle or viral construct that includes an encoded payload Molecules of nucleotides. Virus-producing cells can be selected from any organism, including prokaryotic (eg, bacterial) cells, and eukaryotic cells, including insect cells, yeast cells, and mammalian cells.

In certain embodiments, AAV particles of the invention can be produced in virus-producing cells, including mammalian cells. Virus-producing cells may include mammalian cells such as A549, WEH1, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, HEK293T (293T ), Saos, C2C12, L cells, HT1080, Huh7, HepG2, C127, 3T3, CHO, HeLa cells, KB cells, BHK and fibroblasts, hepatocytes and myoblasts derived from mammals. Virus-producing cells may include cells derived from any mammalian species, including, but not limited to, human, monkey, mouse, rat, rabbit, and hamster, or any cell type, including, but not limited to, fibroblasts, liver cells, tumor cells, cell lines transformed cells, etc.

AAV virus-producing cells commonly used to produce recombinant AAV particles include, but are not limited to, other mammalian cell lines as described in: U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 6,428,988 and No. 5,688,676; U.S. Patent Application No. 2002/0081721; and International Patent Publications Nos. WO 00/47757, WO 00/24916, and WO 96/17947, the contents of each of which are at To the extent that they do not conflict with the present invention, they are incorporated herein by reference in their entirety. In certain embodiments, the AAV virus-producing cells are trans-complementing encapsulating cell lines that provide the function of replication-deficient helper virus deletion, such as HEK293 cells or other Ea trans-complementing cells.

In certain embodiments, the encapsulating cell line 293-10-3 (ATCC registration number PTA-2361) can be used to produce AAV particles, as described in U.S. Patent No. 6,281,010 for the 293-10-3 encapsulating cell The contents of the strains and their uses are incorporated herein by reference in their entirety.

In certain embodiments of the present invention, cell lines for trans-complementing E1-deleted adenoviral vectors, such as HeLA cell lines, can be used for AAV particle production. These adenoviral vectors have a phosphoglycerate kinase (PGK) promoter. Encoding adenovirus E1a and adenovirus E1b under control, as described in U.S. Patent No. 6,365,394, which is incorporated herein by reference in its entirety regarding HeLA cell lines and their uses.

In certain embodiments, AAV particles are produced in mammalian cells using multiplastid transient transfection, such as tripleplastid transient transfection. In certain embodiments, the polyplastid transient transfection method includes transfecting three different constructs: (i) a payload construct, (ii) a Rep/Cap construct (parvovirus Rep and parvovirus Cap), and (iii) Auxiliary structures. In certain embodiments, a triple transfection method of three components of AAV particle production can be used to generate small batches of virus for analysis including transduction efficiency, target tissue (tropism) assessment, and stability. In certain embodiments, a triple transfection method of three components of AAV particle production can be used to generate large batches of material for clinical or commercial applications.

AAV particles to be formulated can be produced by triple transfection or baculovirus-mediated virus production or any other method known in the art. Any suitable permissive or encapsulating cell known in the art may be used to generate the vector. In certain embodiments, a trans-complementing encapsulating cell line that provides functionality deleted from a replication-deficient helper virus is used, such as 293 cells or other E1a trans-complementing cells.

Gene cassettes may contain some or all of the cap and rep genes of parvovirus (eg, AAV). In certain embodiments, some or all of the cap and rep functions are provided in trans by introducing encapsulation vectors encoding capsid and/or Rep proteins into the cell. In certain embodiments, the gene cassette does not encode capsid or Rep protein. Alternatively, encapsulation cell lines stably transformed to express cap and/or rep genes are used.

In certain embodiments, recombinant AAV virions are produced and purified from culture supernatants according to procedures as described in US2016/0032254, which is incorporated by reference in its entirety with respect to the production and processing of recombinant AAV virions. in this article. Production may also involve methods known in the art, including methods using 293T cells, triple transfection, or any suitable production method.

In certain embodiments, mammalian virus-producing cells (eg, 293T cells) can be in an adherent/adhesive state (eg, with calcium phosphate) or in a suspended state (eg, with polyethylenediimide (PEI)). Mammalian virus-producing cells are transfected with the plasmids required for AAV production (ie, AAV rep/cap construct, adenoviral helper construct, and/or ITR-flanked vehicle construct). In some embodiments, the transfection process may include optional media replacement (e.g., media replacement for cells in adherent form, no media replacement for cells in suspension form, media replacement when necessary for cells in suspension form) ). In certain embodiments, the transfection process may include transfection media such as DMEM or F17. In some embodiments, the transfection medium may include serum or may be serum-free (eg, cells in a state of adhesion to calcium phosphate and containing serum, cells in a suspension state with PEI and no serum).

Cells can then be collected by scraping (adherent form) and/or pelleting (suspended form and scraped adherent form) and transferred to a container. The collection step may be repeated as necessary to completely collect the resulting cells. Next, the cell culture can be lysed by continuous freeze-thaw cycles (-80°C to 37°C), chemical lysis (such as adding the detergent triton), mechanical lysis, or by allowing the cell culture to reach approximately 0% viability. Degradation to achieve cell lysis. Cell debris is removed by centrifugation and/or depth filtration. Samples were quantified by DNA qPCR with anti-DNase genomic titration against AAV particles.

AAV particle titers were measured based on genome copy number (number of genome particles per milliliter). Genomic particle concentration was based on DNA qPCR of vector DNA as previously reported (Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278, Their respective contents regarding particle concentration measurement are incorporated by reference in their entirety). insect cells

Viral production of the present invention includes processes and methods for producing AAV particles or viral vectors that contact target cells to deliver a payload construct, such as a recombinant viral construct that includes a core encoding a payload molecule. glycosides. In certain embodiments, AAV particles or viral vectors of the invention can be produced in virus-producing cells, including insect cells.

Growth conditions of insect cells in culture and the production of heterologous products in insect cells in culture are well known in the art, see U.S. Patent No. 6,204,059 on the growth and use of insect cells in virus production. It is incorporated herein by reference in its entirety.

Any insect cell that allows the replication of parvovirus and can be maintained in culture can be used according to the present invention. AAV virus-producing cells commonly used to produce recombinant AAV particles include (but are not limited to) F. frugiperda, including (but not limited to) Sf9 or Sf21 cell lines; Drosophila cell lines; or mosquito cell lines, such as Aedes albopictus ( Aedes albopictus) derived cell lines. The use of insect cells for expressing heterologous proteins is well documented, as are methods of introducing nucleic acids (such as vectors, e.g., insect cell compatible vectors) into such cells and methods of maintaining such cells in culture. It is well documented. See, for example, Methods in Molecular Biology, Richard, ed., Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63: 3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kimbauer et al. Human, Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); and Samulski et al., U.S. Patent No. 6,204,059, each regarding the use of insect cells in virus production. The content is incorporated by reference in its entirety.

In some embodiments, AAV particles are prepared using methods described in WO2015/191508, the contents of which are incorporated herein by reference in their entirety to the extent that they do not conflict with the present invention.

In certain embodiments, a combination of insect host cell systems and baculovirus systems may be used (eg, as described in Luckow et al., Bio/Technology 6: 47 (1988)). In certain embodiments, the expression system used to prepare chimeric peptides is the Trichoplusia ni , Tn 5B1-4 insect cell/baculovirus system, which can be used for high protein content, such as U.S. Patent No. 6660521 No. 1, the content of this patent regarding the production of viral particles is incorporated herein by reference in its entirety.

Expansion, culture, transfection, infection and storage of insect cells can be performed in any cell culture medium, cell transfection medium or storage medium known in the art, including Hyclone ^TM SFX-Insect ^TM Cell Culture Medium, Expression System ESF AF ^TM Insect cell culture medium, ThermoFisher Sf-900II ^TM medium, ThermoFisher Sf-900III ^TM medium or ThermoFisher Grace's insect medium. Insect cell mixtures of the present invention may also include any of the formulation additives or elements described in the present invention, including but not limited to salts, acids, bases, buffers, surfactants (such as poloxamer 188/Plonik F-68) and other known media elements. Formulation additives may be incorporated gradually or as "spikes" (large volumes incorporated in a short period of time). Baculovirus production system

In certain embodiments, methods of the invention may include producing AAV particles or viral vectors in a baculovirus system using viral expression constructs and payload construct vectors. In certain embodiments, the baculovirus system includes a baculovirus expression vector (BEV) and/or a baculovirus-infected insect cell (BIIC). In certain embodiments, the viral expression construct or payload construct of the invention can be a shuttle vector, also known as a baculovirus plasmid or a recombinant baculovirus genome. In certain embodiments, viral expression constructs or payload constructs of the invention may be polynucleotides that are produced by homologous recombination (transformation) by standard molecular biology techniques known and performed by those skilled in the art. transposon donor/acceptor system) is incorporated into the shuttle vector. Transfection of independent viral replicating cell populations produces two or more groups (e.g., two, three) of baculoviruses (BEVs), one or more of which may include a viral expression construct (expression of BEV), and one of which One or more groups may include a payload construct (payload BEV). Baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors.

In certain embodiments, the process includes transfecting a single population of virus-replicating cells to generate a single baculovirus (BEV) panel that includes both a viral expression construct and a payload construct. These baculoviruses can be used to infect virus-producing cells to produce AAV particles or viral vectors.

In certain embodiments, BEVs are generated using shuttle vector transfection reagents such as Promega FuGENE® HD, WFI water, or ThermoFisher Cellfectin® II reagent. In certain embodiments, BEVs are produced and expanded in virus-producing cells, such as insect cells.

In certain embodiments, methods utilize a seed culture of virus-producing cells including one or more BEVs, including baculovirus-infected insect cells (BIIC). The seed BIIC has been transfected/transduced/infected with an expression BEV including a viral expression construct and a payload BEV including a payload construct. In certain embodiments, the seed culture is harvested, divided into aliquots and frozen, and can be used later to initiate transfection/transduction/infection of the primary production cell population. In certain embodiments, a set of inoculum BIIC is stored at -80°C or in LN2 vapor.

Baculoviruses are made of several essential proteins that are necessary for baculovirus function and replication, such as replication, envelope, and capsid proteins. The baculovirus genome therefore includes several essential gene nucleotide sequences encoding essential proteins. As a non-limiting example, the genome may include essential gene regions that include essential gene nucleotide sequences encoding essential proteins for use in baculovirus constructs. Essential proteins may include: GP64 baculovirus envelope protein, VP39 baculovirus capsid protein, or other similar essential proteins for use in baculovirus constructs.

Baculovirus expression vectors (BEVs) used to produce AAV particles in insect cells including, but not limited to, fall armyworm (Sf9) cells provide high titer viral vector products. Recombinant baculoviruses encoding viral expression constructs and payload constructs initiate productive infection of viral vector-replicating cells. Infectious baculovirus particles released from the primary infection secondarily infect additional cells in the culture, exponentially infecting the entire cell culture population over the number of infection cycles as a function of the initial infection rate, see Urabe, M. et al. J Virol. 2006 Feb;80(4):1874-85, which is incorporated by reference in its entirety for its content regarding the generation and use of BEVs and viral particles.

Generating AAV particles with baculovirus in insect cell systems resolves the known genetic and physical instabilities of baculoviruses.

In certain embodiments, the production systems of the present invention address baculovirus instability over multiple passages by utilizing a null-infected cell collection and scaling up the system. Small-scale seed cultures of virus-producing cells are transfected with viral expression constructs encoding structural and/or non-structural components of AAV particles. Baculovirus-infected virus-producing cells are collected into aliquots that can be cryopreserved in liquid nitrogen; the aliquots retain viability and infectivity for use in infecting large-scale virus-producing cell cultures. Wasilko DJ et al. Protein Expr Purif. 2009 Jun;65(2):122-32 is incorporated by reference in its entirety regarding the generation and use of BEVs and viral particles.

Genetically stable baculoviruses can be used to generate a source of one or more of the components used to produce AAV particles in invertebrate cells. In certain embodiments, defective baculovirus expression vectors can be maintained episomally in insect cells. In such embodiments, the corresponding shuttle vector is engineered with replication control elements, including, but not limited to, promoters, enhancers, and/or cell cycle regulated replication elements.

In certain embodiments, stable virus-producing cells that permit baculovirus infection are engineered with at least one stable integrated copy of any of the elements required for AAV replication and vector production, the at least one copy including but not Limited to) complete AAV genome, Rep and Cap genes, Rep gene, Cap gene, each Rep protein in the form of a separate transcription cassette, each VP protein in the form of a separate transcription cassette, AAP (Assembly Activation Protein) or at least one native Or baculovirus helper genes with non-native promoters.

In some embodiments, AAV particles of the invention can be produced in insect cells (eg, Sf9 cells).

In some embodiments, AAV particles of the invention can be produced using triple transfection.

In some embodiments, AAV particles of the invention can be produced in mammalian cells.

In some embodiments, AAV particles of the invention can be produced in mammalian cells by triple transfection.

In some embodiments, AAV particles of the invention can be produced in HEK293 cells by triple transfection.

The AAV viral genomes encoding syntaxin described herein are applicable to the fields of human disease, veterinary applications, and various in vivo and in vitro environments. The AAV particles of the present invention can be used in the field of medicines for treating, preventing, alleviating or improving neurological or neuromuscular diseases and/or disorders. In some embodiments, the AAV particles of the invention are used to prevent and/or treat Friedrich's ataxia.

Various embodiments of the present invention provide herein a pharmaceutical composition comprising an AAV particle as described herein and a pharmaceutically acceptable excipient.

Various embodiments of the invention herein provide a method of treating an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition described herein.

Certain embodiments of the methods provide for treating a subject by a route of administration of a pharmaceutical composition selected from the group consisting of intravenous, intracerebroventricular, intraparenchymal, intrathecal, subpial, and intramuscular, or combinations thereof. Certain embodiments of the methods provide for treating a subject for Friedrich's ataxia and/or other neurological disorders resulting from deficiencies in the amount or function of fataxin. In one aspect of the method, alleviating pathological features of Friedrich's ataxia or another neurological condition, and/or stopping, slowing, ameliorating or reversing Friedrich's ataxia or another neurological condition progress.

Various embodiments of the present invention describe herein a method of increasing fataxin content in the central nervous system of an individual in need thereof, comprising administering to the individual via infusion an effective amount of a pharmaceutical composition described herein.

Also described herein are compositions, methods, processes, kits, and devices for designing, preparing, manufacturing, and/or formulating AAV particles. In some embodiments, a payload such as, but not limited to, FXN may be encoded by a payload construct or contained within a plasmid or vector or recombinant adeno-associated virus (AAV).

The invention also provides methods of administration and/or delivery of vectors and viral particles (eg, AAV particles) for treating or ameliorating Friedrich's ataxia. Such methods may involve gene replacement or gene activation. Such results are achieved by utilizing the methods and compositions taught herein. III. Pharmaceutical compositions

The invention further provides a method for treating FA and disorders associated with lack of function or expression of fataxin in a mammalian subject, including a human subject, comprising administering to the subject an AAV polynucleotide or AAV described herein Either one of the genomes (i.e., a "vector genome", a "viral genome" or a "VG") or a particle comprising the AAV polynucleotide or AAV genome is administered to an individual, or the described combination is administered to an individual Any of the substances (including pharmaceutical compositions).

As used herein, the term "composition" includes an AAV polynucleotide or AAV genome or AAV particle and at least one excipient.

As used herein, the term "pharmaceutical composition" includes an AAV polynucleotide or AAV genome or AAV particle and one or more pharmaceutically acceptable excipients.

Although the descriptions of pharmaceutical compositions (eg, AAVs containing a payload encoding a FXN construct to be delivered) provided herein are generally directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that , such compositions are generally suitable for administration to any other animal (eg, a non-human animal, such as a non-human mammal). It is fully understood that modifications of pharmaceutical compositions suitable for administration to humans may be made to render the compositions suitable for administration to various animals, and that the ordinarily skilled veterinary pharmacologist may design and/or use only ordinary experimentation, if any or make such modifications. Subjects contemplated by the administration of pharmaceutical compositions include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese and/or turkeys.

In some embodiments, the composition is administered to a human, human patient or individual.

In some embodiments, AAV particle formulations described herein can contain nucleic acid encoding at least one payload. In some embodiments, a formulation may contain nucleic acids encoding 1, 2, 3, 4, or 5 payloads. In some embodiments, the formulation may contain a nucleic acid encoding a payload construct encoding a protein selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, Antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic proteins, cytoskeletal proteins, intracellular membrane-bound proteins, nuclear proteins, proteins related to human diseases and/or related to non-human diseases of protein. In some embodiments, the formulation contains at least three payload constructs encoding proteins. Certain embodiments provide that at least one of the payloads is FXN or a variant thereof.

Pharmaceutical compositions according to the present invention may be prepared, packaged and/or sold in bulk, in single unit dosage form and/or in a plurality of single unit dosage forms. As used herein, "unit dose" refers to a discrete quantity of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient will generally equal the dose of active ingredient to be administered to the subject and/or an appropriate fraction of such dose, such as one-half or one-third of such dose. IV. Formulation

Formulations of the AAV pharmaceutical compositions described herein may be prepared by any method known in the pharmacological art or hereafter developed. Generally, such preparation methods include the steps of bringing into association the active ingredient with the excipients and/or one or more other accessory ingredients, and subsequently, if necessary and/or desired, dividing, shaping and/or encapsulating the product into single or multiple dose units as desired.

The relative amounts of the active ingredients, pharmaceutically acceptable excipients and/or any additional ingredients in the pharmaceutical compositions according to the invention will depend on the identity, size and/or condition of the individual being treated and further on the composition to which the composition will be administered. changes along the way.

For example, the composition may contain between 0.1% and 99% (w/w) active ingredient. For example, the composition may comprise between 0.1% and 100%, such as between 5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) of the active ingredient .

The AAV particles of the present invention can be formulated using one or more excipients to (1) increase stability; (2) increase cell transfection or transduction; (3) allow sustained or delayed release; (4) alter biodistribution (e.g., targeting viral particles to specific tissues or cell types); (5) increasing translation of the encoded protein in vivo; (6) altering the release profile of the encoded protein in vivo; and/or (7) enabling the payload to be Regulate performance.

Formulations of the invention may include, but are not limited to, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, transfected with viral vectors cells (e.g., for transplantation into an individual), nanoparticle mimics, and combinations thereof. In addition, the viral vector of the present invention can be formulated using self-assembling nucleic acid nanoparticles.

In some embodiments, viral vectors encoding FXN can be formulated to optimize baricity and/or osmolality. In some embodiments, the specific gravity and/or osmolality of the formulation may be optimized to ensure optimal drug distribution in the central nervous system or a region or component of the central nervous system.

In some embodiments, the AAV particles of the present invention can be formulated in PBS (pH about 7.0) with 0.001% plonic acid (F-68).

In some embodiments, the AAV particles of the present invention can be formulated in combination with ethylene oxide/propylene oxide copolymer (also known as plonik or poloxamer) in PBS.

In some embodiments, AAV particles of the present invention can be formulated in PBS (pH about 7.0) with 0.001% plonic acid (F-68) (poloxamer 188).

In some embodiments, AAV particles of the present invention can be formulated in PBS (pH about 7.3) with 0.001% plonic acid (F-68) (poloxamer 188).

In some embodiments, AAV particles of the invention can be formulated in PBS (pH about 7.4) with 0.001% plonic acid (F-68) (poloxamer 188).

In some embodiments, the AAV particles of the present invention can be formulated in a solution containing sodium chloride, sodium phosphate, and ethylene oxide/propylene oxide copolymer.

In some embodiments, the AAV particles of the present invention can be formulated in a solution containing sodium chloride, disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate and poloxamer 188/plonic acid (F-68) middle.

In some embodiments, the AAV particles of the present invention can be formulated to include 192 mM sodium chloride, 10 mM sodium phosphate (disodium hydrogen phosphate), 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate), and 0.001 % Pluronic F-68 (v/v) solution (pH 7.4). This formulation is referred to herein as Formulation 1.

In some embodiments, the AAV particles of the invention can be formulated in a solution (pH of about 7.3) containing about 192 mM sodium chloride, about 10 mM disodium hydrogen phosphate, and about 0.001% poloxamer 188. The concentration of sodium chloride in the final solution can range from 150 mM to 200 mM. As non-limiting examples, the concentration of sodium chloride in the final solution may be 150 mM, 160 mM, 170 mM, 180 mM, 190 mM or 200 mM. The concentration of disodium hydrogen phosphate in the final solution can range from 1 mM to 50 mM. As a non-limiting example, the concentration of disodium hydrogen phosphate in the final solution may be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20mM, 25mM, 30mM, 40mM or 50mM. The concentration of Poloxamer 188 (Plonic acid (F-68)) can be 0.0001% to 1%. As a non-limiting example, the concentration of poloxamer 188 (Plonic acid (F-68)) may be 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The pH of the final solution can be 6.8 to 7.7. Non-limiting examples of pH of the final solution include a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7.

In one embodiment, the AAV particles of the present invention may contain about 1.05% sodium chloride, about 0.212% sodium hydrogen phosphate disodium, heptahydrate, about 0.025% sodium hydrogen phosphate monohydrate, and 0.001% poloxa Prepare a solution of M188 (pH of approximately 7.4). As a non-limiting example, the concentration of AAV particles in the formulated solution can be about 0.001%. The concentration of sodium chloride in the final solution can be 0.1-2.0%, with non-limiting examples being 0.1%, 0.25%, 0.5%, 0.75%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1.00% , 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.10%, 1.25%, 1.5%, 1.75% or 2%. The concentration of disodium hydrogen phosphate in the final solution can be 0.100-0.300%, where non-limiting examples include 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.210%, 0.211%, 0.212%, 0.213%, 0.214 %, 0.215%, 0.225%, 0.250%, 0.275%, 0.300%. The concentration of sodium dihydrogen phosphate in the final solution can be 0.010-0.050%, with non-limiting examples being 0.010%, 0.015%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027 %, 0.028%, 0.029%, 0.030%, 0.035%, 0.040%, 0.045% or 0.050%. The concentration of Poloxamer 188 (Plonic acid (F-68)) can be 0.0001% to 1%. As a non-limiting example, the concentration of poloxamer 188 (Plonic acid (F-68)) may be 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, or 1%. The pH of the final solution can be 6.8 to 7.7. Non-limiting examples of pH of the final solution include a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. Excipients

The formulations of the present invention may include one or more excipients, each of which together increase the stability of the AAV particles, increase cell transfection or transduction by the viral particles, increase the expression of proteins encoded by the viral particles, and/or alter the AAV The quantity of the release profile of the protein encoded by the particle. In some embodiments, the purity of a pharmaceutically acceptable excipient can be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipients are approved for human use and for veterinary use. In some embodiments, the excipients may be approved by the United States Food and Drug Administration. In some embodiments, the excipients may be of pharmaceutical grade. In some embodiments, the excipients may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Excipients as used herein include, but are not limited to, any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, suitable for the particular desired dosage form, Thickening or emulsifiers, preservatives and the like. Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed., AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the contents of which are incorporated herein by reference in their entirety). The use of any customary excipient medium is prohibited unless any customary excipient medium may be incompatible with the substance or its derivatives, such as to produce any undesirable biological effects or otherwise interact in a deleterious manner with any other component of the pharmaceutical composition. can be included within the scope of the present invention. inactive ingredients

In some embodiments, AAV formulations can include at least one excipient as an inactive ingredient. As used herein, the term "inactive ingredient" refers to one or more agents that do not contribute to the activity of the pharmaceutical composition included in the formulation. In some embodiments, all, none, or some of the inactive ingredients useful in the formulations of the present invention may be approved by the United States Food and Drug Administration (FDA).

The formulations of AAV particles disclosed herein may include cations or anions. In one embodiment, the formulation includes metal cations such as, but not limited to, Zn ²⁺ , Ca ²⁺ , Cu ²⁺ , Mg ⁺ , or combinations thereof. In some embodiments, formulations may include polymers or polynucleotides complexed with metal cations (see, for example, U.S. Patent Nos. 6,265,389 and 6,555,525, the contents of each of which are incorporated by reference in their entirety). are incorporated into this article). V.Use and Application

The compositions of the invention herein may be administered to an individual or used in the manufacture of a medicament for administration to an individual who is deficient in the amount or function of FXN or who suffers from a disease or condition associated with reduced expression of FXN. In some embodiments, the disease is FA. In certain embodiments, AAV particles including FXN can be administered to an individual to treat FA. In some embodiments, administration of AAV particles comprising a viral genome encoding FXN protects central pathways from degradation.

In some embodiments, the payload delivered by the AAV particle is a polynucleotide encoding an FXN polypeptide that has at least 90% sequence identity to the human FXN sequence of SEQ ID NOs: 1725-1727. In some embodiments, the fataxin polypeptide belongs to a non-human primate. In some embodiments, the non-human primate polypeptide is FXN of the crab-eating macaque long-tailed macaque (cynoFXN or cFXN) or the common macaque (rhesus macaque). In some embodiments, the non-human primate fastaxin polypeptide is at least partially humanized. In some embodiments, the payload delivered by the AAV particle is a polynucleotide encoding an FXN polypeptide that has at least 90% sequence identity to the sequence set forth in any of SEQ ID NOs: 1731-1733.

In some embodiments, delivery of AAV particles can halt or slow the progression of Friedrich's ataxia by 50% relative to a comparison group, as measured by mFARS/SARA. In certain embodiments, delivery of AAV particles increases the presence of functional FXN, improves and stabilizes gait, ameliorates ataxia-related cardiac conditions, reduces feelings of exhaustion, and treats metabolic conditions such as diabetes.

In some embodiments, the present invention contemplates delivery of a pharmaceutical, prophylactic, diagnostic or imaging composition in combination with an agent that improves its bioavailability, attenuates and/or modulates its metabolism, and/or modifies its distribution in the body.

In certain embodiments, pharmaceutical compositions described herein are used as research tools, particularly in in vitro studies using human cell lines such as HEK293T and in non-human primates that will precede human clinical trials. Under in vivo testing. CNS diseases

The present invention provides a method for treating a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject a viral particle, such as an AAV, AAV particle or AAV genome, that produces FXN as described herein (i.e., a viral genome or "VG"), or administering to an individual a particle comprising such AAV particle or AAV genome, or administering to an individual any of the compositions described (including pharmaceutical compositions) One.

In some embodiments, the AAV particles of the invention can modulate the content or function of gene products in the CNS via delivery as a functional payload for a therapeutic product comprising FXN or a variant thereof.

The functional payload can alleviate or reduce symptoms caused by abnormalities in the content and/or function of the gene product (such as the absence or defect of the protein) in an individual in need thereof, or otherwise provide CNS disorders in an individual in need thereof. Provide benefits.

As non-limiting examples, concomitant or combined therapeutic products delivered by the AAV particles of the invention may include, but are not limited to, growth and trophic factors, interleukins, hormones, neurotransmitters, enzymes, anti-apoptotic factors, Angiogenic factors, FXN polypeptides, and any protein known to be mutated in pathological conditions such as FA (eg, brain-specific Mir-128a, see Adlakha and Saini, Molecular cancer, 2014, 13:33, cited in full are incorporated into this article).

In some embodiments, the neurodegenerative disorder is Friedrich's ataxia caused by an expansion of the intronic GAA trinucleotide repeat sequence in the FXN gene, which reduces expression of the mitochondrial protein fataxin , thereby causing progressive damage to the nervous system.

In some embodiments, the AAV particles of the invention can be used to treat diseases associated with disorders of growth and development of the CNS, ie, neurodevelopmental disorders. In some forms, such neurodevelopmental disorders can be caused by genetic mutations.

In some embodiments, the neurological disorder may be a functional neurological disorder with motor and/or sensory symptoms that are neurologically derived from the CNS. As non-limiting examples, functional neurological disorders may be chronic pain, epilepsy, speech disorders, involuntary movements, and sleep disorders.

In some embodiments, the neurological or neuromuscular disease, disorder, and/or condition is Friedrich's ataxia. In some embodiments, delivery of AAV particles can halt or slow disease progression in Friedrich's ataxia by 10%, 20%, or 10% using known assays and comparison groups for Friedrich's ataxia. 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater than 95%. As a non-limiting example, delivery of AAV particles can halt or slow the progression of Friedrich's ataxia by 50% relative to a comparison group, as measured by mFARS/SARA.

In some embodiments, AAV particles encoding payloads can increase the amount of FXN in tissue by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or greater than 100%. In some embodiments, AAV particles encoding a payload can increase the amount of FXN in a tissue to a level comparable to (eg, approximately the same as) the amount of FXN in corresponding tissues of healthy individuals. In some embodiments, AAV particles encoding a payload can increase the amount of FXN in a tissue that is effective in alleviating one or more symptoms of a disease (eg, FA) associated with reduced FXN expression or deficiency in FXN quantity and/or function. VI. Administration and administration

In some aspects, the present invention provides methods of administration and/or delivery of vectors and viral particles (eg, AAV particles) encoding FXN or variants thereof for preventing, treating, or ameliorating diseases or disorders of the CNS. For example, administration of AAV particles encoding FXN prevents, treats, or ameliorates FA.

The AAV particles of the present invention can be administered by any route that produces a therapeutically effective result. Such routes include (but are not limited to) enteral (into the intestines), gastrointestinal, epidural (into the dura mater), oral (through the mouth), transcutaneous, epidural, intracerebral (into the dura mater) In the brain), intraventricular (into the ventricles of the brain), intracranial (into the skull), transdermal (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), administered through the nose with (through the nose), intravenously (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into a heart), intraosseous Infusion (into the bone marrow), intraparenchymal (into the brain substance), intrathecally (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye) ), intracavernous injection (into the pathological cavity), intracavity (into the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal ( via mucous membranes), vaginally, inhalation (inhaled through the nose), sublingual, sublipal, enema, eye drops (to the conjunctiva), ear drops, auricular (in or through the ear), buccal (for buccal), transconjunctival, transdermal, transdental (to one or more teeth), electroosmosis, intracervical, endosinusial, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial , intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic, intrachondral (in cartilage), intracaudal (in cauda equina), intracisternal (in cerebellomedullary cistern), Intracorneal (within the cornea), intradental crown, intracoronary (within the coronary arteries), intracorporus cavernosum (within the expandable space of the corpus cavernosum), intradiscal (within the intervertebral disc), tube Intraduodenal (in the duodenum), intradural (in or below the dura mater), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (into the esophagus) intragastric), intragingival (within the gums), intraileal (within the distal part of the small intestine), intralesional (within or directly introduced into a localized lesion), intraluminal (within the lumen of a tube), intralymphatic (in the lymph), intramedullary (in the marrow cavity of the bone), intrameningeal (in the meninges), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardium), pleura Intra (in the pleura), intraprostatic (in the prostate), intrapulmonary (in the lungs or their bronchi), intrasinus (in the paranasal or periorbital sinuses), intraspinal (in the spine), intrasynovial ( Within the synovial cavity of a joint), intratendonally (within the tendon), intratesticularly (within the testicles), intrathecally (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the chest cavity), Intratubular (within the small tubes of an organ), intratumoral (within a tumor), intratympanic (within the middle ear), intravascular (within one or more blood vessels), intraventricular (within a room), iontophoresis (with the help of electrical current, in which ions of soluble salts migrate into body tissues), perfusion (to flush or irrigate exposed wounds or body cavities), translaryngeal (directly on the throat), nasogastric feeding (through the nose to the stomach), occlusive dressing techniques (administered by local route, which is subsequently covered with an occlusive dressing to the area), transocular (to the external eye), transoropharyngeal (directly to the oropharynx), parenteral, transdermal, periarticular, epidural, Transperineural, transperiodontal, transrectal, transrespiratory (within the respiratory tract, by oral or nasal inhalation for local or systemic effects), retrobulbar (behind the pons or behind the eyeball), soft tissue, spider Submembranous, subconjunctival, submucosal, subpial, local, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (through or through the tympanic cavity), transureteral (to the ureter), trans Urethral (to the urethra), transvaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, and transspinal.

In some embodiments, AAV particles of the invention are administered for delivery to target cells or tissues. Without wishing to be bound by theory, delivery to target cells promotes FXN expression. The target cell can be any cell deemed necessary to increase the amount of FXN expression. The target cells may be CNS cells. Non-limiting examples of such cells and/or tissues include dorsal root ganglia and dorsal columns, proprioceptive sensory neurons, Clark's columns, nuclei of fasciculata and cuneatee, cerebellar dentate nucleus, corticospinal tracts and the like including the same cells, Bayesian cells and heart cells.

In some embodiments, the composition can be administered in a manner that allows it to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

In some embodiments, delivering FXN to central nervous system (eg, parenchymal) cells by adeno-associated virus (AAV) particles involves infusion into cerebrospinal fluid (CSF). CSF is produced by specialized ependymal cells contained in the choroid plexus located in the ventricles of the brain. CSF produced in the brain then circulates and surrounds the central nervous system including the brain and spinal cord. CSF continuously circulates around the central nervous system, including the ventricles and the subarachnoid space surrounding both the brain and spinal cord, while maintaining a constant balance of production and reabsorption into the vasculature. The entire volume of CSF is replaced approximately four to six times per day or approximately once every four hours, but individual values may vary.

In some embodiments, AAV particles can be delivered via systemic delivery. In some embodiments, systemic delivery can be by intravascular administration. In some embodiments, systemic delivery can be by intravenous administration.

In some embodiments, AAV particles can be delivered via intravenous delivery.

In some embodiments, AAV particles can be delivered by injection into the CSF pathway. Non-limiting examples of delivery routes to the CSF include intrathecal and intracerebroventricular administration.

In some embodiments, AAV particles can be delivered via thalamic delivery.

In some embodiments, AAV particles can be delivered via intracerebral delivery.

In some embodiments, AAV particles can be delivered by intracardiac delivery.

In some embodiments, AAV particles can be delivered via intracranial delivery.

In some embodiments, AAV particles can be delivered by direct (intraparenchymal) injection into an organ, such as the CNS (brain or spinal cord). In some embodiments, intraparenchymal delivery can be delivered to any region of the brain or CNS, such as within the striatum.

In some embodiments, AAV particles of the invention can be administered to the cerebral ventricles.

In some embodiments, AAV particles of the invention can be administered to the cerebral ventricles via intracerebroventricular delivery.

In some embodiments, AAV particles of the invention can be administered via intramuscular delivery.

In some embodiments, AAV particles of the invention are administered via more than one of the pathways described above. As a non-limiting example, AAV particles can be administered via intravenous delivery and thalamic delivery.

In some embodiments, AAV particles of the invention are administered via more than one of the pathways described above. As a non-limiting example, AAV particles can be administered via intravenous delivery and intracerebral delivery.

In some embodiments, AAV particles of the invention are administered via more than one of the pathways described above. As a non-limiting example, AAV particles can be administered via intravenous delivery and intracranial delivery.

In some embodiments, AAV particles of the invention are administered via more than one of the pathways described above. In some embodiments, AAV particles of the invention can be delivered by intrathecal and intracerebroventricular administration.

In some embodiments, AAV particles can be delivered to improve and/or correct mitochondrial dysfunction.

In some embodiments, AAV particles can be delivered to individuals to protect neurons. Neurons can be primary and/or secondary sensory neurons. In some embodiments, AAV particles are delivered to dorsal root ganglia and/or neurons thereof.

In some embodiments, administration of AAV particles may preserve and/or correct function in sensory pathways.

In some embodiments, AAV particles can be delivered via intravenous (IV), intracerebroventricular (ICV), intraparenchymal, and/or intrathecal (IT) infusion, and therapeutic agents can also be delivered via intramuscular (IM) limb infusion. individuals in order to deliver therapeutic agents to skeletal muscles. Gruntman and Flotte, Human Gene Therapy Clinical Development, 2015, 26(3), 159-164, the contents of which are incorporated by reference in their entirety, describe delivery of AAV encoding at least one FXN or variant thereof by intravascular limb infusion. in this article.

In some embodiments, delivery of a viral vector pharmaceutical composition according to the present invention to cells of the central nervous system (e.g., parenchyma) comprises a delivery rate defined by VG/hour=ml/hour*VG/mL, where VG is the viral genome , VG/mL is the composition concentration, and mL/hour is the infusion rate.

In some embodiments, delivering an AAV particle pharmaceutical composition according to the invention to cells of the central nervous system (eg, parenchyma) involves infusion of up to 1 mL. In some embodiments, delivering the viral vector pharmaceutical composition according to the invention to cells of the central nervous system (e.g. parenchyma) may comprise infusion of 0.0001, 0.0002, 0.001, 0.002, 0.003, 0.004, 0.005, 0.008, 0.010, 0.015, 0.020, 0.025, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mL.

In some embodiments, delivering an AAV particle pharmaceutical composition according to the invention to cells of the central nervous system (eg, parenchyma) includes infusion of about 1 mL to about 120 mL. In some embodiments, delivering a viral vector pharmaceutical composition according to the present invention to cells of the central nervous system (e.g., parenchyma) may comprise infusion of 0.1, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 mL. In some embodiments, delivering AAV particles to cells of the central nervous system (eg, parenchyma) includes infusion of at least 3 mL. In one embodiment, delivery of AAV particles to cells of the central nervous system (eg, parenchyma) consists of infusion of 3 mL. In one embodiment, delivering AAV particles to cells of the central nervous system (eg, parenchyma) includes infusion of at least 10 mL. In some embodiments, delivery of AAV particles to cells of the central nervous system (eg, parenchyma) consists of infusion of at least 10 mL.

In some embodiments, the volume of the AAV particle pharmaceutical composition delivered to cells of the central nervous system (e.g., parenchyma) of the subject is 2 µl, 20 µl, 50 µl, 80 µl, 100 µl, 200 µl, 300 µl, 400 µl, 500 µl, 600 µl, 700 µl, 800 µl, 900 µl, 1000 µl, 1100 µl, 1200 µl, 1300 µl, 1400 µl, 1500 µl, 1600 µl, 1700 µl, 1800 µl, 1900 µl, 200 0 µl or Greater than 2000 µl.

In some embodiments, the volume of the AAV particle pharmaceutical composition delivered to regions in both hemispheres of the subject's brain is 2 µl, 20 µl, 50 µl, 80 µl, 100 µl, 200 µl, 300 µl, 400 µl, 500 µl, 600 µl, 700 µl, 800 µl, 900 µl, 1000 µl, 1100 µl, 1200 µl, 1300 µl, 1400 µl, 1500 µl, 1600 µl, 1700 µl, 1800 µl, 1900 µl, 2000 µl l or greater than 2000 µl. In some embodiments, the volume delivered to the area in both hemispheres is 200 µl. As another non-limiting example, the volume delivered to the area in both hemispheres was 900 μl. As yet another non-limiting example, the volume delivered to the area in both hemispheres was 1800 μl.

In certain embodiments, the AAV particle or viral vector pharmaceutical composition according to the present invention can be used in a dosage of about 10 to about 600 microliters/site, about 50 to about 500 microliters/site, or about 100 to about 400 microliters. administered/site, about 120 to about 300 microliters/site, about 140 to about 200 microliters/site, or about 160 microliters/site.

In some embodiments, the total volume delivered to an individual may be split between one or more administration sites, such as 1, 2, 3, 4, 5, or greater than 5 sites. In some embodiments, the total volume is split between administration to the left and right hemispheres. Delivery of AAV particles

In some embodiments, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered using methods for treating disease described in U.S. Patent No. 8,999,948 or International Publication No. WO2014178863, among others. The content is incorporated by reference in its entirety.

In some embodiments, AAV particles or pharmaceutical compositions of the invention may be produced using methods as described in U.S. Application No. 20150126590 for delivering gene therapy in Alzheimer's disease or other neurodegenerative conditions. For submission or delivery, the contents of this application are incorporated herein by reference in their entirety.

In some embodiments, AAV particles or pharmaceutical compositions of the invention may be administered using methods for delivering CNS gene therapy as described in U.S. Patent Nos. 6,436,708 and 8,946,152 and International Publication No. WO2015168666 or delivered, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the AAV particles of the present invention may be administered or delivered using the methods for delivering AAV virions described in European Patent Application No. EP1857552, the contents of which are incorporated by reference in their entirety. into this article.

In some embodiments, the AAV particles or pharmaceutical compositions of the invention may be administered or delivered using methods for delivering proteins using AAV vectors as described in European Patent Application No. EP2678433, the contents of which are incorporated by reference in their entirety. are incorporated into this article.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods for delivering DNA molecules using AAV vectors as described in U.S. Pat. No. 5,858,351, the contents of which are incorporated herein by reference in their entirety. middle.

In some embodiments, AAV particles or pharmaceutical compositions of the present invention may be administered or delivered using methods for delivering DNA into the bloodstream as described in U.S. Patent No. 6,211,163, the contents of which are incorporated by reference in their entirety. method is incorporated into this article.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods for delivering AAV virions as described in U.S. Patent No. 6,325,998, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods for delivering DNA to muscle cells as described in U.S. Pat. No. 6,335,011, the contents of which are incorporated herein by reference in their entirety. middle.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods for delivering DNA to muscle cells and tissues as described in U.S. Pat. No. 6,610,290, the contents of which are incorporated by reference in their entirety. into this article.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods for delivering DNA to muscle cells as described in U.S. Pat. No. 7,704,492, the contents of which are incorporated herein by reference in their entirety. middle.

In some embodiments, viral vectors encoding FXN may be administered or delivered using methods for delivering payloads to skeletal muscle as described in U.S. Pat. No. 7,112,321, the contents of which are incorporated by reference in their entirety. in this article.

In some embodiments, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered using methods for delivering payloads to the central nervous system as described in U.S. Pat. No. 7,588,757, the contents of which are incorporated herein by reference in their entirety. Incorporated herein by reference.

In some embodiments, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered using methods of delivering payloads as described in U.S. Patent No. 8,283,151, the contents of which are incorporated by reference in their entirety. in this article.

In some embodiments, viral vectors encoding FXN may be administered or delivered using delivery vehicles as described in U.S. Pat. No. 8,318,687, the contents of which are incorporated by reference in their entirety, for the treatment of Alzheimer's disease. are incorporated into this article.

In some embodiments, viral vectors encoding FXN may be administered or delivered using methods of delivering payloads as described in International Patent Publication No. WO2012144446, the contents of which are incorporated herein by reference in their entirety. .

In some embodiments, the AAV particles or pharmaceutical compositions of the invention may be administered using the method described in International Patent Publication No. WO2001089583 using a glutamate decarboxylase (GAD) delivery vehicle to deliver the payload or As delivered, the contents of this publication are incorporated herein by reference in their entirety.

In some embodiments, AAV particles or pharmaceutical compositions of the invention may be administered or delivered using the methods for delivering payloads to neural cells described in International Patent Publication No. WO2012057363, The content is incorporated by reference in its entirety.

In some embodiments, viral vectors encoding FXN can be administered or delivered using methods of delivering payloads as described in International Patent Publication No. WO2001096587, the contents of which are incorporated herein by reference in their entirety. .

In some embodiments, viral vectors encoding FXN may be administered or delivered using methods for delivering payloads to muscle tissue as described in International Patent Publication No. WO2002014487, the contents of which are incorporated by reference in their entirety. incorporated herein.

In some embodiments, a catheter can be used to administer AAV particles. In certain embodiments, a catheter or cannula may be located at more than one site in the spine for multi-site delivery. Viral particles encoding FXN can be delivered as continuous infusion and/or bolus injection. Different dosing regimens can be used for each delivery site, or the same dosing regimen can be used for each delivery site. In some embodiments, delivery sites may be in the neck and lumbar areas. In some embodiments, the delivery site may be in the neck area. In some embodiments, the delivery site may be in the lumbar area.

In some embodiments, an individual's spinal anatomy and pathology can be analyzed prior to delivery of FXN-encoding AAV particles described herein. As a non-limiting example, the dosing regimen and/or catheter location may be different for individuals with scoliosis than for individuals without scoliosis.

In some embodiments, the delivery method and duration are selected to provide widespread transduction in the spinal cord. In some embodiments, intrathecal delivery is used to provide broad transduction along the rostral to caudal length of the spinal cord. In some embodiments, multisite infusion provides more uniform transduction along the rostral to caudal length of the spinal cord. Delivered to cells

In some aspects, the invention provides a method of delivering any of the AAV particles described above to a cell or tissue, comprising contacting the cell or tissue with the AAV particle or contacting the cell or tissue with the AAV particle comprising the AAV particle. The formulation of particles is contacted, or cells or tissues are contacted with any of the compositions described, including pharmaceutical compositions. Methods of delivering AAV particles to cells or tissues can be accomplished in vitro, ex vivo, or in vivo. delivered to individual

In some aspects, the present invention additionally provides a method of delivering any of the AAV particles described above to an individual, including a mammalian individual, comprising administering to the individual an AAV particle, or administering to the individual a particle comprising formulation of the AAV particles, or administration of any of the described compositions (including pharmaceutical compositions) to an individual.

In some embodiments, AAV particles can be delivered to bypass anatomical obstructions, such as (but not limited to) the blood-brain barrier.

In some embodiments, AAV particles can be formulated and delivered to an individual via a route that increases the rate of action of the drug compared to oral delivery.

In some embodiments, AAV particles can be delivered by a method that provides uniform transduction of the spinal cord and dorsal root ganglia (DRG). In some embodiments, AAV particles can be delivered using intrathecal infusion.

In some embodiments, AAV particles described herein can be administered to an individual using a bolus injection. As used herein, "bolus" means a single and rapid infusion of a substance or composition.

In some embodiments, FXN-encoding AAV particles may be delivered as a continuous infusion and/or bolus injection. Different dosing regimens can be used for each delivery site, or the same dosing regimen can be used for each delivery site. As a non-limiting example, delivery sites may be in the neck and lumbar areas. As another non-limiting example, the delivery site may be in the neck area. As another non-limiting example, the delivery site may be in the lumbar area.

In some embodiments, AAV particles can be delivered to an individual via a single route of administration.

In some embodiments, AAV particles can be delivered to an individual via a multi-site administration route. For example, AAV particles can be administered to an individual at 2, 3, 4, 5, or more than 5 sites.

In some embodiments, AAV particles described herein can be administered to an individual using sustained delivery over a period of minutes, hours, or days. The infusion rate may vary depending on the individual, distribution, formulation, or another delivery parameter known to those skilled in the art.

In some embodiments, if continuous delivery of AAV particles (continuous infusion) is used, the continuous infusion can be 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, 24 hours or more than 24 hours.

In some embodiments, intracranial pressure can be assessed prior to administration. Route, volume, AAV particle concentration, infusion duration, and/or vector titer may be optimized based on individual intracranial pressure.

In some embodiments, AAV particles can be delivered via systemic delivery. In some embodiments, systemic delivery can be by intravascular administration.

In some embodiments, AAV particles can be delivered by injection into the CSF pathway. Non-limiting examples of delivery routes to the CSF include intrathecal and intracerebroventricular administration.

In some embodiments, AAV particles can be delivered by direct (intraparenchymal) injection into the material of an organ, such as one or more regions of the brain.

In some embodiments, AAV particles can be delivered by subpial injection into the spinal cord. For example, the individual may be placed in a spinal immobilization device. A dorsal laminectomy may be performed to expose the spinal cord. Guide tubes and XYZ manipulators can be used to assist with catheter placement. A subpial catheter can be placed in the subpial space by advancing the catheter over the guide tube, and AAV particles can be injected through the catheter (Miyanohara et al., Mol Ther Methods Clin Dev. 2016; 3: 16046). In some cases, AAV particles may be injected into the subpial space of the neck. In some cases, AAV particles may be injected into the subpial space of the chest.

In some embodiments, AAV particles can be delivered to an individual such that FXN protein levels in the dorsal root ganglia (DRG) are increased relative to endogenous levels. The increase may be compared to endogenous content of 0.1× to 5×, 0.5× to 5×, 1× to 5×, 2× to 5×, 3× to 5×, 4× to 5×, 0.1× to 4 ×, 0.5× to 4×, 1× to 4×, 2× to 4×, 3× to 4×, 0.1× to 3×, 0.5× to 3×, 1× to 3×, 2× to 3×, 0.1× to 2×, 0.5× to 2×, 0.1× to 1×, 0.5× to 1×, 0.1× to 0.5×, 1× to 2×, 0.1×, 0.2×, 0.3×, 0.4×, 0.5× , 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.1×, 2.2 ×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3.0×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4.0×, 4.1×, 4.2×, 4.3×, 4.4×, 4.5×, 4.6×, 4.7×, 4.8×, 4.9× or greater than 5×. Increases may be seen in the cervical, thoracic and/or lumbar areas of the spine. As a non-limiting example, the increase in FXN in DRG can be greater than 0.5× the endogenous content. As a non-limiting example, the increase in FXN in DRG can range from 0.5× to 3× compared to endogenous content.

In some embodiments, AAV particles can be delivered to an individual to increase FXN protein content in the dorsal root ganglia (DRG) by transducing large DRG neurons in the cervical, thoracic, and/or lumbar regions. Transduction can also be referred to as the amount of positive DRG. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, the transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 15%. As a non-limiting example, the transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 20%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 25%. As a non-limiting example, the transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 30%. As a non-limiting example, the transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 35%. As a non-limiting example, the transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 40%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 45%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 50%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 55%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 60%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 65%. As a non-limiting example, transduction of large DRG neurons in the cervical, thoracic and/or lumbar regions may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual to increase FXN protein content in the dorsal root ganglia (DRG) by transducing large DRG neurons in the cervical, thoracic, and/or lumbar regions. Endogenous content increases. The FXN protein content in the DRG can be increased by 0.5× to 3× compared to the endogenous content in the neck, chest, and/or lumbar region, and large DRG neurons in the neck, chest, and/or lumbar region can Transduction is at least 20%.

In some embodiments, AAV particles can be delivered to an individual to transduce large neurons of the cerebellar dentate nucleus. Transduction can also be referred to as the number of positive large neurons. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 15%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 20%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 25%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 30%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 35%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 40%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 45%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 50%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 55%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 60%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 65%. As a non-limiting example, the transduction of large neurons in the cerebellar dentate nucleus may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual to transduce Clark's column neurons. Transduction can also be referred to as the number of positive neurons. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 15%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 20%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 25%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 30%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 35%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 40%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 45%. As a non-limiting example, the transduction of Clark's column neurons may be greater than or equal to 50%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 55%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 60%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 65%. As a non-limiting example, transduction of Clark's column neurons may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual to transduce neurons in the nucleus gracilis. Transduction can also be referred to as the number of positive neurons. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 15%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 20%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 25%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 30%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 35%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 40%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 45%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 50%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 55%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 60%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 65%. As a non-limiting example, the transduction of neurons in the nucleus gracilis may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual to transduce neurons in the nucleus cuneatee. Transduction can also be referred to as the number of positive neurons. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 15%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 20%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 25%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 30%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 35%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 40%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 45%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 50%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 55%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 60%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 65%. As a non-limiting example, the transduction of neurons in the nucleus cuneatee may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual such that FXN protein levels in the heart are increased relative to endogenous levels. The increase may be compared to endogenous content of 0.1× to 5×, 0.5× to 5×, 1× to 5×, 2× to 5×, 3× to 5×, 4× to 5×, 0.1× to 4 ×, 0.5× to 4×, 1× to 4×, 2× to 4×, 3× to 4×, 0.1× to 3×, 0.5× to 3×, 1× to 3×, 2× to 3×, 0.1× to 2×, 0.5× to 2×, 0.1× to 1×, 0.5× to 1×, 0.1× to 0.5×, 1× to 2×, 0.1×, 0.2×, 0.3×, 0.4×, 0.5× , 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.1×, 2.2 ×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3.0×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4.0×, 4.1×, 4.2×, 4.3×, 4.4×, 4.5×, 4.6×, 4.7×, 4.8×, 4.9× or greater than 5×. As a non-limiting example, the increase in FXN in the heart can be greater than 0.5× the endogenous level. As a non-limiting example, increases in FXN in the heart may range from 0.5× to 3× compared to endogenous levels.

In some embodiments, AAV particles can be delivered to an individual to increase FXN protein content by transducing cardiomyocytes. Transduction can also be referred to as the number of positive cardiomyocytes. Transduction can be greater than or equal to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or 99%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 15%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 20%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 25%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 30%. As a non-limiting example, cardiomyocyte transduction may be greater than or equal to 35%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 40%. As a non-limiting example, cardiomyocyte transduction may be greater than or equal to 45%. As a non-limiting example, transduction of cardiomyocytes may be greater than or equal to 50%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 55%. As a non-limiting example, the transduction of cardiomyocytes may be greater than or equal to 60%. As a non-limiting example, cardiomyocyte transduction may be greater than or equal to 65%. As a non-limiting example, transduction of cardiomyocytes may be greater than or equal to 70%.

In some embodiments, AAV particles can be delivered to an individual to increase FXN protein levels in the heart relative to endogenous levels and to transduce cardiomyocytes. FXN protein content in the heart can be increased by 0.5× to 3× compared to endogenous levels, and/or cardiomyocytes can be transduced by at least 30%.

In some embodiments, delivery of AAV particles comprising a viral genome encoding FXN described herein to sensory neurons in the dorsal root ganglia (DRG), ascending spinal sensory tracts, and cerebellum will increase the expression of FXN. Increased performance results in improved survival and function of various cell types.

Specifically, in some embodiments, increased expression of fastaxin results in improvements in ataxia (balance) and gait, sensory abilities, coordination of movement and strength, functional abilities, and/or quality of life. Give medication

In some aspects, the invention provides methods comprising administering to an individual in need thereof a viral vector according to the invention and its payload. Any amount and any route of administration that is effective in preventing, treating, diagnosing, or imaging diseases, disorders, and/or conditions, such as those associated with reduced expression of FXN or deficiency in the amount and/or function of FXN, may be used Viral vector pharmaceutical, imaging, diagnostic or prophylactic compositions are administered to an individual. In some embodiments, the disease, disorder and/or condition is FA. The precise amount required will vary from individual to individual depending on the individual's species, age and general condition, severity of disease, the particular composition, its mode of administration, its pattern of activity and similar factors. Compositions according to the present invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, it should be understood that the total daily dosage of the composition of the present invention can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound employed; the specific composition employed; and the age of the patient. , body weight, general health, gender and diet; administration time, route of administration and excretion rate of the specific FXN used; duration of treatment; drugs used in combination or concomitantly with the specific compound used; and similar ones well known in the medical arts factor.

In certain embodiments, AAV particle pharmaceutical compositions according to the present invention can be administered at a dosage level sufficient to deliver FXN to obtain the desired therapeutic, diagnostic, preventive or imaging effect: from about 0.0001 mg/kg to FXN per day based on the individual's body weight. About 100 mg/kg, about 0.001 mg/kg to about 0.05 mg/kg, about 0.005 mg/kg to about 0.05 mg/kg, about 0.001 mg/kg to about 0.005 mg/kg, about 0.05 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg kg, about 0.1 mg/kg to about 10 mg/kg or about 1 mg/kg to about 25 mg/kg, once or multiple times a day. It should be understood that the above dosage concentrations can be converted by those skilled in the art into the amount of VG or viral genome per kilogram administered or the total viral genome amount.

In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, Twelve, thirteen, fourteen or more throws). When multiple administrations are employed, divided dosing regimens such as those described herein may be used. As used herein, "fractionated dose" means dividing a single unit dose or total daily dose into two or more doses, such as two or more administrations of a single unit dose. As used herein, a "single unit dose" is a dose of any therapeutic composition administered in one dose/once-in/in a single route/single point of contact (i.e., a single administration event). In some embodiments, single unit doses are provided in discrete dosage forms (eg, lozenges, capsules, patches, loaded syringes, vials, etc.). As used herein, a "total daily dose" is the amount administered or prescribed in a 24-hour period. It can be administered in single unit dosage form. Viral particles can be formulated in buffer alone or in the formulations described herein.

Pharmaceutical compositions described herein may be formulated into dosage forms described herein, such as topical, intranasal, pulmonary, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and /or subcutaneous) dosage form.

In some embodiments, delivery of AAV particles described herein results in few serious adverse events (SAEs) resulting from delivery of the AAV particles.

In some embodiments, delivery of AAV particle pharmaceutical compositions according to the present invention to cells of the central nervous system (eg, parenchyma) may comprise a total concentration of between about 1×10 ⁶ VG/mL and about 1×10 ¹⁶ VG/mL. In some embodiments, the delivery may include about 1×10 ⁶ , 2×10 ⁶ , 3×10 6 , 4×10 ⁶ , 5×10 ^{6 ,} 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 9 , 6×10 ⁹ , 7×10 ⁹ , 8×10 ^{9 ,} 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 1.6×10 ¹¹ , 1.8×10 ¹¹ , 2×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 5×10 ¹¹ , 5.5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 0.8×10 ¹² , 0.83×10 ¹² , 1×10 ¹² , 1.1×10 ¹² , 1.2×10 12 , 1.3×10 ¹² , 1.4×10 ¹² , 1.5×10 ¹² , 1.6×10 ¹² , 1.7× ^{10 12 ,} 1.8×10 ¹² , 1.9×10 ¹² , 2×10 ¹² , 2.1×10 ¹² , 2.2×10 12 , 2.3×10 ¹² , 2.4×10 ¹² , 2.5×10 ¹² , 2.6×10 ¹² , 2.7× ^{10 12 ,} 2.8×10 ¹² , 2.9×10 ¹² , 3×10 ¹² , 3.1×10 ¹² , 3.2×10 12 , 3.3×10 ¹² , 3.4×10 ¹² , 3.5×10 ¹² , 3.6×10 ¹² , 3.7× ^{10 12 ,} 3.8×10 ¹² , 3.9×10 ¹² , 4×10 ¹² , 4.1×10 ¹² , 4.2×10 12 , 4.3×10 ¹² , 4.4×10 ¹² , 4.5×10 ¹² , 4.6×10 ¹² , 4.7× ^{10 12 ,} 4.8×10 ¹² , 4.9×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 12 , 8×10 ¹² , 9×10 ¹² , 1×10 ¹³ , 2×10 ¹³ , 2.3×10 ¹³ , 3×10 ^{13 ,} 4×10 ¹³ , 5×10 ^{13 ,} 6×10 ¹³ , 7×10 13 , 8×10 ¹³ , 9×10 ¹³ , 1×10 ¹⁴ , 1.9×10 ¹⁴ , 2×10 ¹⁴ , 3×10 ¹⁴ , 4×10 ¹⁴ , 5×10 ¹⁴ , 6×10 14 , 7×10 ¹⁴ , 8×10 ¹⁴ , 9×10 ¹⁴ , 1×10 ¹⁵ , 2×10 ¹⁵ , 3×10 ^{15 ,} 4×10 ¹⁵ , Composition concentration of 5×10 ¹⁵ , 6×10 ¹⁵ , 7×10 ¹⁵ , 8×10 ¹⁵ , 9×10 ¹⁵ or 1×10 ¹⁶ VG/mL. In some embodiments, the concentration of viral vector in the composition is 1×10 ¹³ VG/mL. In some embodiments, the concentration of viral vector in the composition is 1.1×10 ¹² VG/mL. In some embodiments, the concentration of viral vector in the composition is 3.7×10 ¹² VG/mL. In some embodiments, the concentration of viral vector in the composition is 8×10 ¹¹ VG/mL. In some embodiments, the concentration of viral vector in the composition is 2.6×10 ¹² VG/mL. In some embodiments, the concentration of viral vector in the composition is 4.9×10 ¹² VG/mL. In some embodiments, the concentration of viral vector in the composition is 0.8×10 ¹² VG/mL. In some embodiments, the concentration of viral vector in the composition is 0.83×10 ¹² VG/mL. In one embodiment, the concentration of viral vector in the composition is the maximum final dose that can be contained in the vial. In some embodiments, the concentration of viral vector in the composition is 1.6×10 ¹¹ VG/mL. In some embodiments, the concentration of viral vector in the composition is 5×10 ¹¹ VG/mL. In some embodiments, the concentration of viral vector in the composition is 2.3×10 ¹³ VG/mL. In some embodiments, the concentration of viral vector in the composition is 1.9×10 ¹⁴ VG/mL.

In some embodiments, delivery of AAV particle pharmaceutical compositions according to the invention to cells of the central nervous system (eg, parenchyma) may comprise a total concentration of between about 1×10 ⁶ VG and about 1×10 ¹⁶ VG per body. In some embodiments, the delivery may include about 1×10 ⁶ , 2×10 ⁶ , 3×10 6 , 4×10 ⁶ , 5×10 ^{6 ,} 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 9 , 6×10 ⁹ , 7×10 ⁹ , 8×10 ^{9 ,} 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 1.6×10 ¹¹ , 2×10 ¹¹ , 2.1×10 ¹¹ , 2.2×10 ¹¹ , 2.3×10 ¹¹ , 2.4×10 ¹¹ , 2.5×10 ¹¹ , 2.6×10 ¹¹ , 2.7×10 ¹¹ , 2.8×10 ¹¹ , 2.9×10 ¹¹ , 3×10 ¹¹ , 4×10 ¹¹ , 4.6×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 7.1×10 ¹¹ , 7.2×10 ¹¹ , 7.3×10 ¹¹ , 7.4×10 ¹¹ , 7.5×10 ¹¹ , 7.6×10 ¹¹ , 7.7×10 ¹¹ , 7.8×10 ¹¹ , 7.9×10 ¹¹ , 8×10 ¹¹ , 9×10 ¹¹ , 1×10 ¹² , 1.1×10 ¹² , 1.2×10 ¹² , 1.3×10 ¹² , 1.4×10 ¹² , 1.5×10 ¹² , 1.6×10 ¹² , 1.7×10 ¹² , 1.8×10 ^{12 , 1.9×10 12 ,} 2×10 ¹² , 2.3×10 ¹² , 3×10 ¹² , 4×10 ¹² , 4.1×10 ¹² , 4.2×10 ¹² , 4.3×10 ¹² , 4.4×10 ¹² , 4.5×10 ¹² , 4.6×10 ¹² , 4.7×10 ¹² , 4.8×10 ¹² , 4.9×10 ¹² , 5×10 ¹² , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 8.1×10 ¹² , 8.2×10 ¹² , 8.3×10 ¹² , 8.4×10 ¹² , 8.5×10 ¹² , 8.6×10 ¹² , 8.7×10 ¹² , 8.8×10 ¹² , 8.9×10 ¹² , 9×10 12 , 1×10 ^{13 , 2×10 13 , 3×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7 × 10 13 ,} 8×10 ¹³ , 9×10 ¹³ , 1×10 ¹⁴ , 2×10 14 , 3×10 ¹⁴ , 4×10 ¹⁴ , 5×10 ¹⁴ , 6×10 ¹⁴ , 7×10 ^{14 ,} 8×10 ¹⁴ , 9×10 ¹⁴ , 1×10 ¹⁵ , 2×10 ¹⁵ , 3×10 15 , 4×10 ¹⁵ , 5×10 ¹⁵ , 6×10 ¹⁵ , 7×10 ¹⁵ , 8×10 ^{15 ,} 9×10 ¹⁵ or Composition concentration of 1×10 ¹⁶ VG/individual. In some embodiments, the concentration of viral vector in the composition is 2.3×10 ¹¹ VG/individual. In some embodiments, the concentration of viral vector in the composition is 7.2×10 ¹¹ VG/individual. In some embodiments, the concentration of viral vector in the composition is 7.5×10 ¹¹ VG/individual. In some embodiments, the concentration of viral vector in the composition is 1.4×10 ¹² VG/individual. In some embodiments, the concentration of viral vector in the composition is 4.8×10 ¹² VG/individual. In some embodiments, the concentration of viral vector in the composition is 8.8×10 ¹² VG/individual. In some embodiments, the concentration of viral vector in the composition is 2.3×10 ¹² VG/individual. In some embodiments, the concentration of viral vector in the composition is 2×10 ¹⁰ VG/individual. In some embodiments, the concentration of viral vector in the composition is 1.6×10 ¹¹ VG/individual. In some embodiments, the concentration of viral vector in the composition is 4.6×10 ¹¹ VG/individual.

In some embodiments, delivery of AAV particles to cells of the central nervous system (eg, parenchyma) may comprise a total dose of between about 1×10 ⁶ VG and about 1×10 ¹⁶ VG. In some embodiments, the delivery may include about 1×10 ⁶ , 2×10 ⁶ , 3×10 6 , 4×10 ⁶ , 5×10 ^{6 ,} 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 9 , 7×10 ⁹ , 8× ^{10 9 ,} 9×10 ⁹ , 1×10 ¹⁰ , 1.9×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 3.73×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ , 1×10 ¹¹ , 2×10 ¹¹ , 2.5×10 ¹¹ , 3×10 11 , 4×10 ¹¹ , 5×10 ¹¹ , 6×10 ¹¹ , 7×10 ¹¹ , 8×10 ¹¹ , 9×10 ^{11 ,} 1×10 ¹² , 2×10 ¹² , 3×10 ¹² , 4×10 ¹² , 5×10 12 , 6×10 ¹² , 7×10 ¹² , 8×10 ¹² , 9×10 ¹² , 1 ^× 10 ¹³ , 2×10 ¹³ , 3×10 ¹³ , 4×10 ¹³ , 5×10 ¹³ , 6×10 13 , 7×10 ¹³ , 8×10 ¹³ , 9×10 ¹³ , 1×10 ¹⁴ , 2×10 ^{14 ,} 3×10 ¹⁴ , 4×10 ¹⁴ , 5×10 ¹⁴ , 6×10 14 , 7×10 ¹⁴ , 8×10 ¹⁴ , 9×10 ¹⁴ , 1×10 ¹⁵ , 2×10 ¹⁵ , 3×10 ^{15 ,} 4×10 ¹⁵ , Total dose of 5×10 ¹⁵ , 6×10 ¹⁵ , 7×10 ¹⁵ , 8×10 ¹⁵ , 9×10 ¹⁵ or 1×10 ¹⁶ VG. In some embodiments, the total dose is 1×10 ¹³ VG. In some embodiments, the total dose is 3×10 ¹³ VG. In some embodiments, the total dose is 3.73×10 ¹⁰ VG. In some embodiments, the total dose is 1.9×10 ¹⁰ VG. In some embodiments, the total dose is 2.5×10 ¹¹ VG. In some embodiments, the total dose is 5×10 ¹¹ VG. In some embodiments, the total dose is 1×10 ¹² VG. In some embodiments, the total dose is 5×10 ¹² VG. combination

AAV particles can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. Although such delivery methods are within the scope of the invention, the phrase "in combination with" is not intended to require that the agents must be administered simultaneously and/or formulated for delivery together. The compositions can be administered concurrently with, before or after one or more other desired treatments or medical procedures. Generally, each agent will be administered at a dose and/or schedule determined for that agent. In some embodiments, the present invention contemplates delivery of a pharmaceutical, prophylactic, diagnostic or imaging composition in combination with an agent that improves its bioavailability, attenuates and/or modulates its metabolism, and/or modifies its distribution in the body. performance measurement

The expression of FXN from viral genomes can be determined using various methods known in the art, such as (but not limited to) immunochemistry (e.g., IHC), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT, flow cytometry Metrology, immunocytology, surface plasmon resonance analysis, kinetic exclusion analysis, liquid chromatography mass spectrometry (LCMS), high performance liquid chromatography (HPLC), BCA analysis, immunoelectrophoresis, Western blot method, SDS -PAGE, protein immunoprecipitation, PCR and/or in situ hybridization (ISH). In some embodiments, transgenes encoding FXN delivered in different AAV capsids may have different expression levels in the dorsal root ganglion (DRG).

In certain embodiments, FXN polypeptides can be detected by Western blotting. VII. Sets and device sets

In some aspects, the invention provides kits for conveniently and/or efficiently performing the methods of the invention. Typically, a kit will contain an amount and/or number of components sufficient to allow the user to treat an individual multiple times and/or perform multiple experiments.

Any of the vectors, constructs or FXN of the invention may be included in the kit. In some embodiments, the kit may further include reagents and/or instructions for producing and/or synthesizing the compounds and/or compositions of the invention. In some embodiments, the kit may also include one or more buffering agents. In some embodiments, kits of the invention may include components for making protein or nucleic acid arrays or libraries, and thus may include, for example, solid supports.

In some embodiments, the kit components may be packaged in an aqueous medium or in a lyophilized form. The container components of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container component into which the components can be placed and appropriately aliquoted. Where more than one kit component is present (the labeling reagent and label may be packaged together), the kit may also generally contain second, third or other additional containers in which the additional components may be placed separately . In some embodiments, the kit may also include a second container component for containing a pharmaceutically acceptable sterile buffer and/or other diluent. In some embodiments, various combinations of components may be contained in one or more vials. Kits of the invention may also generally include components for containing compounds and/or compositions of the invention (eg, proteins, nucleic acids) and any other reagent containers that are sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers that hold the desired vials.

In some embodiments, the kit components are provided in one and/or more liquid solutions. In some embodiments, the liquid solution is an aqueous solution, with sterile aqueous solutions being particularly used. In some embodiments, the kit components may be provided in dry powder form. When reagents and/or components are provided as dry powders, such powders can be reconstituted by adding an appropriate volume of solvent. In some embodiments, it is contemplated that the solvent may also be provided in another container member. In some embodiments, the marking dye is provided in dry powder form. In some embodiments, it is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180 are provided in the kit of the present invention , 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most that amount of dry dye. In such embodiments, the dye can then be resuspended in any suitable solvent, such as DMSO.

In some embodiments, a kit may include instructions for employing the components of the kit as well as using any other reagents not included in the kit. The description may include variations that may be implemented. device

In some embodiments, the compounds and/or compositions of the invention can be combined with, coated onto, or embedded in a device. Devices may include, but are not limited to, dental implants, stents, bone substitutes, artificial joints, valves, pacemakers, and/or other implantable treatment devices.

The present invention provides devices that can incorporate viral vectors encoding one or more FXN molecules. These devices contain viral vectors in stable formulations that can be delivered immediately to individuals in need, such as human patients.

Devices for administration may be employed to deliver the FXN-encoding viral vectors of the invention according to single, multiple, or divided dosing regimens taught herein.

Methods and devices known in the art for multiple administration to cells are contemplated for use in conjunction with the methods and compositions disclosed herein in the form of embodiments of the invention. VIII.Definition

At various locations throughout this disclosure, substituents of the compounds of the present invention are disclosed in groups or ranges. It is specifically intended that this invention include each and every individual subcombination of members of such groups and ranges. The following is a non-limiting list of definitions of terms.

Adeno-associated virus : The term "adeno-associated virus" or "AAV" as used herein refers to a member of the genus AAV, including any particles, sequences, genes, proteins or components derived therefrom. The term "AAV particle" as used herein includes the capsid and the polynucleotide known as the AAV genome or viral (or vector) genome (VG). AAV particles may be derived from any serotype described herein or known in the art, including combinations of serotypes (i.e., "pseudotyped"AAV); or from various genomes (e.g., single-stranded or self-complementary). Additionally, AAV particles can be replication-deficient and/or targeted.

Active ingredient : As used herein, the term "active ingredient" refers to a molecule or complex thereof that is biologically active and responsible for producing a biological effect. The active ingredients in pharmaceutical compositions may be referred to as active pharmaceutical ingredients. For the purposes of this invention, the phrase "active ingredient" generally refers to a viral particle carrying a payload or a payload (or its gene product) delivered by a viral particle as described herein. In contrast, "inactive ingredients" refer to biologically inert substances. Excipients are examples of inactive ingredients.

Activity : As used herein, the term "activity" refers to a situation in which events are occurring or ongoing. The compositions of the invention can be active, and this activity can involve one or more biological events.

Administration in Combination : As used herein, the term "administration in combination" or "delivery in combination" or "combination administration" means the administration of two or more agents simultaneously or within a time interval. (e.g., AAV), such that the individual is exposed to both agents at the same time point and/or such that the effects of each agent on the patient are additive. In some embodiments, at least one dose of one or more agents is about 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes after at least one dose of one or more other agents. , invest within 5 minutes or 1 minute. In some embodiments, administration occurs in an overlapping dosage regimen. As used herein, the term "dose regimen" refers to a plurality of doses spaced apart in time. Such doses may occur at regular intervals or may include one or more dosing intervals. In some embodiments, administration of individual doses of one or more compounds and/or compositions of the invention as described herein are spaced in a sufficiently close manner such that a combined (eg, synergistic) effect is achieved.

Amelioration : As used herein, the term "amelioration or ameliorating" means a reduction in the severity of at least one indicator of a condition or disease. For example, in the context of neurodegenerative disorders, improvement includes reduction or stabilization of neuronal loss.

Animal : As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human being at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, or a purebred animal.

Approximately : As used herein, the terms "approximately" or "approximately" when applied to one or more values of interest refer to a value that is similar to the stated reference value. In certain embodiments, unless stated otherwise or otherwise apparent from the context, the term "approximately" or "approximately" means falling within 25%, greater or less, in either direction (greater or less) of the stated reference value. 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4% , 3%, 2%, 1%, 0.5%, 0.25%, 0.1% or less (except that such numbers would exceed 100% of possible values).

Biological Activity: As used herein, the phrase "biological activity" refers to any characteristic of any substance (eg, AAV) that is active in or on biological systems and/or organisms. For example, a substance is considered to be biologically active if it has a biological effect on an organism when administered to that organism. In certain embodiments, a compound and/or composition of the invention may be considered biologically active if even a portion of the compound and/or composition is biologically active or mimics an activity considered biologically relevant. In some embodiments, biological activity refers to the induction of syntaxin or a variant thereof. In some embodiments, biological activity refers to the prevention and/or treatment of diseases associated with reduced expression of fataxin or deficiency in the amount and/or function of fataxin. In some embodiments, biological activity refers to preventing and/or treating Friedrich's ataxia.

Biological System : As used herein, the term "biological system" refers to a system that functions together to perform a biological task within a cell membrane, cellular compartment, cell, tissue, organ, organ system, multicellular organism, or any biological entity. Groups of organs, tissues, cells, intracellular components, proteins, nucleic acids, molecules (including but not limited to biomolecules). In some embodiments, a biological system is a cell signaling pathway that includes intracellular and/or extracellular cell signaling biomolecules. In some embodiments, biological systems include growth factor signaling events within the extracellular/cellular matrix and/or niche.

Capsid : As used herein, the term "capsid" refers to the protein outer shell of a virus. It consists of several capsid proteins (eg VP1, VP2 and/or VP3 for AAV). The capsid encloses the genetic material of the virus. The capsid can be a wild-type capsid or a recombinant or engineered capsid.

Central Nervous System or CNS : As used herein, "central nervous system" or "CNS" refers to one of the two major divisions of the nervous system, which in vertebrate animals includes the brain and spinal cord. The central nervous system coordinates the activities of the entire nervous system.

Cervical Region : As used herein, "cervical region" refers to the region of the spinal cord that includes cervical vertebrae C1, C2, C3, C4, C5, C6, C7 and C8.

Ipsilateral element : As used herein, an ipsilateral element or the synonymous term "ipsilateral regulatory element" refers to a region of non-coding DNA that regulates the transcription of a nearby gene. The Latin prefix "on the same side" is translated as "on this side". Ipsilateral elements are found near one or more genes they regulate. Examples of ipsilateral elements include the Kozak sequence, the SV40 intron, or part of the backbone.

CNS tissue : As used herein, "CNS tissue" refers to the tissue of the central nervous system, which in vertebrates includes the brain and spinal cord and their substructures.

CNS Structure : As used herein, "CNS structure" refers to the structure of the central nervous system and its substructures. Non-limiting examples of structures in the spinal cord may include the ventral horn, dorsal horn, white matter, and nervous system pathways or nuclei therein. Non-limiting examples of structures in the brain include forebrain, midbrain, hindbrain, diencephalon, telencephalon, myelencephalon, metencephalon, mesencephalon, prosencephalon, Rhombencephalon, cortex, frontal lobe, parietal lobe, temporal lobe, occipital lobe, cerebrum, thalamus, hypothalamus, tectum, tegmentum, cerebellum, pons, medulla oblongata, amygdala, hippocampus, basal ganglia, corpus callosum , pituitary gland, tegument, striatum, ventricles and their substructures.

CNS cells : As used herein, "CNS cells" refers to cells of the central nervous system and its substructures. Non-limiting examples of CNS cells include neurons and subtypes thereof, glial cells, microglia, oligodendrocytes, ependymal cells, and astrocytes. Non-limiting examples of neurons include sensory neurons, motor neurons, interneurons, unipolar cells, bipolar cells, multipolar cells, pseudounipolar cells, pyramidal cells, basket cells, astrocytes, universal cells. Kings cells, Bayesian cells, axonal neurons, granule cells, oval cells, medium aspiny neurons and large spiny neurons.

Codon Optimization : As used herein, the term "codon optimization" refers to a method of changing the codons of a given gene in such a way that the polypeptide sequence encoded by the gene remains the same, while changing The codons will improve the expression of the polypeptide sequence. For example, if a polypeptide has a human protein sequence and is expressed in E. coli , performance will typically be achieved by performing codon optimization on the DNA sequence to change the human codons to codons that are more efficient for performance in E. coli Improvement.

Composition : As used herein, the term "composition" includes an AAV polynucleotide, an AAV genome or an AAV particle and at least one excipient.

Compound : As used herein, the term "compound" refers to a unique chemical entity. In some embodiments, a particular compound may exist in one or more isomeric or isotopic forms (including, but not limited to, stereoisomers, geometric isomers, and isotopes). In some embodiments, a compound is provided or utilized in only a single such form. In some embodiments, compounds are provided or utilized as mixtures of two or more such forms (including, but not limited to, racemic mixtures of stereoisomers). Those skilled in the art will appreciate that some compounds exist in different such forms, exhibiting different properties and/or activities (including but not limited to biological activity). In such cases, it is within the skill of the skilled person to select or avoid specific forms of the compounds for use in accordance with the invention. For example, compounds containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods of how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or stereoselective synthesis.

Conservative : As used herein, the term "conservative" means that the nucleotide or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, are unchanged at the same position in two or more sequences being compared. A relatively conserved nucleotide or amino acid is a conserved nucleotide or amino acid in a sequence that is more related to nucleotides or amino acids occurring elsewhere in the sequence.

In some embodiments, two or more sequences are said to be "completely conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are referred to as "if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98% or about 99% identical to each other. "Highly conservative." In some embodiments, if two or more sequences are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical to each other, or at least 95% consistent, it is called "conservative". In some embodiments, if two or more sequences are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical to each other, , about 95% agreement, about 98% agreement or about 99% agreement, it is called "conservative". Sequence conservation may apply to the entire length of an oligonucleotide or polypeptide or may apply to a portion, region, or feature thereof.

In one embodiment, the conserved sequences are not contiguous. One skilled in the art will understand how to achieve alignment when there are continuous alignment gaps between sequences and how to align corresponding residues without suffering the presence of insertions or deletions.

Delivery : As used herein, "delivery" refers to the act or manner of delivering parvovirus, such as an AAV compound, substance, entity, part, cargo, or payload, to a target. Such targets may be cells, tissues, organs, organisms or systems (whether biological or production systems).

Delivery Agent : As used herein, "delivery agent" refers to any agent that facilitates, at least in part, the delivery of one or more substances (including but not limited to the compounds and/or compositions of the invention, such as viral particles or AAV vectors) to a target Cell reagents.

Route of Delivery : As used herein, the term "route of delivery" and the synonymous term "route of administration" refers to any of the different methods used to provide a therapeutic agent to an individual. Routes of administration are generally classified by the location where the substance is applied and may also be classified based on the location of the target of action. Examples include, but are not limited to: intravenous administration, subcutaneous administration, oral administration, parenteral administration, enteral administration, topical administration, sublingual administration, inhalation administration, and injection administration, or other investment methods described in this article.

Derivative : As used herein, the term "derivative" refers to a composition (eg, sequence, compound, formulation, etc.) derived from or based on a parent composition. Non-limiting examples of parent compositions include wild-type or native amino acid or nucleic acid sequences, or undiluted formulations. In some embodiments, derivatives are variants of the parent composition. The derivative may differ from the parent composition by less than about 1%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, Less than about 40%, less than about 45%, or less than about 50%. In certain embodiments, the derivative may differ by greater than about 50% from the parent composition. In certain embodiments, the derivative may differ by greater than about 75% from the parent composition. In some embodiments, derivatives may be fragments or truncations of the parent amino acid or nucleotide sequence. As a non-limiting example, a derivative may be a sequence with a nucleotide or peptide insertion compared to the parent nucleic acid or amino acid sequence (eg AAVPHP.B compared to AAV9).

Effective Amount : As used herein, the term "effective amount" of an agent is an amount sufficient to achieve a beneficial or desired result, e.g., cure, alleviation, relief, when administered in single or multiple doses to an individual or cell. Or achieve these results by improving one or more symptoms of the disease, and therefore, the "effective amount" depends on the situation in which it is applied. For example, where an agent is administered to treat FA, an effective amount of the agent is an amount sufficient to achieve treatment of FA as defined herein, e.g., compared to the response obtained without administration of the agent. .

Engineered: As used herein, embodiments of the invention are "engineered" when they are designed to have characteristics or properties (whether structural or chemical) that differ depending on the starting point, wild-type or native molecule. change". Thus, engineered agents or entities are those for which the design and/or production of those agents or entities involves the actions of the human hand.

Representation : As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation and/or 3' end processing); (3) translating RNA into polypeptides or proteins; (4) folding polypeptides or proteins; and/or (5) translating polypeptides or proteins Grooming.

Excipient : As used herein, the term "excipient" refers to an inactive substance that serves as a vehicle or medium for an active pharmaceutical agent or other active substance.

Formulation : As used herein, "formulation" includes at least one compound and/or composition of the invention (eg, carrier, AAV particles, etc.) and a delivery agent.

Fragment : As used herein, "fragment" means a continuous part of a whole. For example, fragments of a protein may include polypeptides obtained by digesting full-length proteins isolated from cultured cells. In some embodiments, fragments of the protein include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. Fragment may also refer to a truncation of a protein (eg, N-terminal and/or C-terminal truncation) or a nucleic acid truncation (eg, at the 5' and/or 3' end). Protein fragments can be obtained by expressing truncated nucleic acids such that the nucleic acid encodes a portion of a full-length protein.

Gene Expression : The term "gene expression" refers to the process by which a nucleic acid sequence is successfully transcribed and, in most cases, translated to produce a protein or peptide. For the sake of clarity, when reference is made to measuring "gene expression", this should be understood to mean that the measurement may be on a nucleic acid transcription product, such as RNA or mRNA, or on an amino acid translation product, such as a polypeptide or peptide. Methods for measuring the amount or content of RNA, mRNA, polypeptides and peptides are well known in the art.

Homology : As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, such as between nucleic acid molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, if the sequence of the polymeric molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95% or 99% identical or similar, they are considered to be "homogeneous" to each other. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present invention, if for at least one sequence segment having at least about 20 amino acids, the polypeptide encoded by the two polynucleotide sequences is at least about 50%, 60%, 70%, 80%, 90%, 95%, or Even if they are 99% consistent, they are considered to have the same origin. In some embodiments, the homologous polynucleoside sequence is characterized by being able to encode a sequence segment having at least 4 to 5 specifically designated amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a sequence segment having at least 4 to 5 specifically designated amino acids. According to the present invention, two protein sequences are considered to be at least about 50%, 60%, 70%, 80% or 90% identical for at least one sequence segment of at least about 20 amino acids. Same origin. In many embodiments, homologous proteins may exhibit a greater degree of overall homology and have at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 in at least one , a higher degree of homology within a short sequence segment of 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In many embodiments, homologous proteins share one or more characteristic sequence elements. As used herein, the term "signature sequence element" refers to a motif present in the protein of interest. In some embodiments, the presence of such motifs is associated with a specific activity, such as biological activity.

Humanized : As used herein, the term "humanized" refers to a non-human sequence of a polynucleotide or polypeptide that has been altered to increase its similarity to its corresponding human sequence.

Identity : As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, such as between oligonucleotide molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. For example, calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into the first and second nucleic acid sequences). one or both for optimal alignment and discordant sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the length of the reference sequence. , at least 95% or 100%. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is related to the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Mathematical algorithms can be used to achieve sequence comparison and determination of percent identity between two sequences. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in: Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined, for example, using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the use of PAM120 weighted residues Table, gap length penalty 12 and gap penalty 4 in the ALIGN program (version 2.0). Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG suite of software using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated by reference are incorporated into this article. The techniques used to determine consistency are encoded in publicly available computer programs. Computer software used to determine homology between two sequences includes (but is not limited to) GCG suite (Devereux, J. et al., Nucleic Acids Research , 12(1), 387 (1984)), BLASTP, BLASTN and FASTA (Altschul, SF et al., J. Molecular Biol. , 215, 403 (1990)).

In vitro : As used herein, the term "ex vivo" refers to an occurrence that occurs in an artificial environment (e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.) rather than in an organism (e.g., an animal, plant or events within microorganisms).

In vivo : As used herein, the term "in vivo" refers to events that occur within an organism, such as an animal, plant or microorganism, or its cells or tissues.

Isolated : As used herein, the term "isolated" is synonymous with "separated," except that separation is an inference made by humans. The terms "isolated" and "substantially isolated" are used interchangeably herein. An isolated substance or entity is one that has been partially or completely separated from at least some components with which it was previously associated (whether in nature or in an experimental setting). Isolated substances can have different levels of purity relative to the substances with which they were associated. At least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% of other components with which the isolated substance and/or entity can be originally associated , approximately 90% or greater percent separation. In some embodiments, the purity of the isolated agent exceeds about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%.

Lumbar Region : As used herein, the term "lumbar region" refers to the region of the spinal cord that includes lumbar vertebrae L1, L2, L3, L4, and L5.

Modified : As used herein, the term "modified" refers to a change in the state or structure of a molecule or entity as compared to a parent or reference molecule or entity. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. In some embodiments, the compounds and/or compositions of the invention are modified by the introduction of non-natural amino acids or non-natural nucleotides.

Mutation : As used herein, the term "mutation" means change and/or change. In some embodiments, mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and/or alterations to proteins and/or nucleic acid sequences. Such changes and/or alterations may include the addition, substitution and or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and polynucleic acids) . In embodiments where the mutations comprise additions and/or substitutions of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues, and Modified amino acids and/or nucleotides may be included. One or more mutations may produce, for example, "mutants,""derivatives," or "variants" of a nucleic acid sequence or a polypeptide or protein sequence.

Naturally occurring : As used herein, "naturally occurring" or "wild type" means existing in nature without artificial aid or human intervention. "Naturally occurring" or "wild type" may refer to the native form of a biological molecule, sequence or entity.

Non-human vertebrates : As used herein, "non-human vertebrates" includes all vertebrates other than Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, banteng, bison, camels, cats, cattle, deer, dogs, donkeys, gayals, goats, guinea pigs, Horse, llama, mule, pig, rabbit, reindeer, sheep, buffalo and yak.

Nucleic acid : As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) or polyribonucleosides. Any nucleic acid polymer composed of acid (containing D-ribose) or any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base or a modified purine or pyrimidine base. There is no intended length distinction between the terms "nucleic acid,""polynucleotide," and "oligonucleotide," and these terms will be used interchangeably. These terms refer only to the primary structure of the molecule. Therefore, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA.

Operably linked : As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, portions, or the like.

Particle : As used herein, a "particle" is a virus that contains at least two components, a protein capsid and a polynucleotide sequence enclosed within the capsid.

Patient : As used herein, "patient" means an individual who may seek or need treatment, request treatment, be receiving treatment, be about to receive treatment, or be cared for by a professional trained (e.g., licensed) for a specific disease or condition individual.

Payload : As used herein, "payload" or "payload region" means one or more polynucleotides or polynucleotide regions or such polynucleotides encoded by or within the viral genome Or the expression product of a polynucleotide region, such as a transgenic gene or a polynucleotide encoding a polypeptide.

Payload Construct : As used herein, a "payload construct" is one or more polynucleotide regions that encode or contain a payload flanked on one or both sides by inverted terminal repeat (ITR) sequences. The payload construct is the template that is replicated in virus-producing cells to produce the viral genome.

Payload Construct Vector : As used herein, a "payload construct vector" is a vector that encodes or contains a payload construct and regulatory regions for replication and expression in bacterial cells. The payload construct vector may also contain components for viral expression in viral replicating cells.

Peptide : As used herein, the term "peptide" means less than or equal to about 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids in length. of amino acid chains.

Pharmaceutically Acceptable : The phrase "pharmaceutically acceptable" is used herein to mean, within the scope of sound medical judgment, suitable for contact with human and animal tissue without undue toxicity, irritation, allergic reaction, or other problems. or complications, with a reasonable benefit/risk ratio for those compounds, materials, compositions and/or dosage forms.

Pharmaceutically acceptable excipients : As used herein, the term "pharmaceutically acceptable excipients" as used herein means substances other than those present in a pharmaceutical composition that are substantially non-toxic in an individual and Any ingredients other than active agents (eg, as described herein) that do not have inflammatory properties. In some embodiments, a pharmaceutically acceptable excipient is a vehicle capable of suspending and/or dissolving the active agent. Excipients may include, for example: anti-adhesive agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents), film-forming agents Or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners and hydration water. Excipients include (but are not limited to): butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dicalcium phosphate), calcium stearate, croscarmellose, crospolyvinylpyrrolidone , citric acid, crosprovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, Methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, providone, pregelatinized starch, propylparaben, Retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A , vitamin E, vitamin C and/or xylitol.

Pharmaceutically Acceptable Salts : A pharmaceutically acceptable salt of a compound described herein is one in which the acid or base moiety of the disclosed compound is in the form of a salt thereof (e.g., by combining the free base with a suitable organic acid reaction to produce) form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and Analogues. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphor Acid salt, camphor sulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethane sulfonate, fumarate, glucoheptonate , glycerophosphate, hemisulfate, enanthate, caproate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethane sulfonate, lactobiate, lactate, lauric acid Salt, lauryl sulfate, malate, maleate, malonate, methane sulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate , palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, butyrate Diacidates, sulfates, tartrates, thiocyanates, tosylates, undecanoates, valerates and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, including (but not limited to) ammonium, quaternary ammonium and amine cations. Methylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and their analogs. Pharmaceutically acceptable salts include, for example, conventional nontoxic salts derived from nontoxic inorganic or organic acids. In some embodiments, pharmaceutically acceptable salts are prepared from parent compounds containing basic or acidic moieties by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, Use nonaqueous media such as diethyl ether, ethyl acetate, ethanol, isopropyl alcohol, or acetonitrile. A list of suitable salts can be found in: Remington's Pharmaceutical Science , 17th ed., Mack Publishing Company, Easton, Pa., 1985, page 1418; Pharmaceutical Salts: Properties, Selection, and Use , PH Stahl and CG Wermuth ( (Eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science , 66, 1-19 (1977), the contents of each of which are incorporated herein by reference in their entirety.

Pharmaceutical composition : As used herein, the term "pharmaceutical composition" or "pharmaceutically acceptable composition" includes an AAV polynucleotide, an AAV genome or an AAV particle and one or more pharmaceutically acceptable excipients , solvents, adjuvants and/or the like.

Polypeptide : As used herein, the term "polypeptide" refers to an organic polymer consisting of a large number of amino acid residues bonded together in a chain. Monomeric protein molecules are polypeptides.

Prevention : As used herein, the term "prevention" means to partially or completely delay the onset of an infection, disease, disorder and/or condition; to partially or completely delay one or more symptoms of a particular infection, disease, disorder and/or condition, The onset of a characteristic or clinical manifestation; a partial or complete delay in the onset of one or more symptoms, features or manifestations of a particular infection, disease, disorder and/or condition; a partial or complete delay in the onset of an infection, a particular disease, disorder and/or condition progression; and/or reduce the risk of developing pathologies associated with infections, diseases, disorders and/or conditions.

Promoter : As used herein, the term "promoter" refers to the nucleic acid site to which a polymerase will bind to initiate transcription (DNA to RNA) or reverse transcription (RNA to DNA).

Protein of Interest : As used herein, the term "protein of interest" or "protein of interest" includes the proteins provided herein and fragments, mutants, variants and altered forms thereof.

Purified : As used herein, "purified" means becoming substantially pure or clean from one or more undesirable components, material contaminants, contaminants, or defects. "Purified" refers to the pure state. "Purification" means the process of becoming pure. As used herein, a substance is "pure" if it is substantially free of one or more components, eg, as found in its native context (substantially separated therefrom).

Region : As used herein, the term "region" refers to a zone or general area. In some embodiments, when referring to a protein or protein module, a region may comprise a linear amino acid sequence along the protein or protein module, or may comprise a three-dimensional region, an epitope and/or an epitope cluster. In some embodiments, the region includes a terminal region. As used herein, the term "terminal region" refers to the region located at the end or ends of a given agent. When referring to a protein, the terminal region may include the N-terminus and/or the C-terminus. The N-terminus refers to the end of a protein that contains amino acids with free amine groups. The C-terminus refers to the end of a protein containing an amino acid with a free carboxyl group. The N-terminal and/or C-terminal region may include the N-terminal and/or C-terminal and surrounding amino acids. In some embodiments, the N-terminal and/or C-terminal region includes about 3 amino acids to about 30 amino acids, about 5 amino acids to about 40 amino acids, about 10 amino acids to About 50 amino acids, about 20 amino acids to about 100 amino acids, and/or at least 100 amino acids. In some embodiments, the N-terminal region may comprise any length of amino acid, including the N-terminus but excluding the C-terminus. In some embodiments, the C-terminal region may comprise any length of amino acid, including the C-terminus but excluding the N-terminus.

In some embodiments, when referring to a polynucleotide, a region may comprise a linear nucleic acid sequence along the polynucleotide, or may comprise a three-dimensional region, secondary structure or tertiary structure. In some embodiments, the region includes a terminal region. As used herein, the term "terminal region" refers to the region located at the end or ends of a given agent. When referring to polynucleotides, the terminal region may include a 5' end and a 3' end. The 5' end refers to the end of a polynucleotide containing a nucleic acid having a free phosphate group. The 3' end refers to the end of the polynucleotide containing the nucleic acid having a free hydroxyl group. The 5' and 3' regions may thus include the 5' and 3' ends as well as surrounding nucleic acids. In some embodiments, the 5' end region and the 3' end region include about 9 nucleic acids to about 90 nucleic acids, about 15 nucleic acids to about 120 nucleic acids, about 30 nucleic acids to about 150 nucleic acids, about 60 nucleic acids. to about 300 nucleic acids and/or at least 300 nucleic acids. In some embodiments, the 5' region may comprise any length of nucleic acid including the 5' end but excluding the 3' end. In some embodiments, the 3' region may comprise any length of nucleic acid including the 3' end but not the 5' end.

RNA or RNA molecule : As used herein, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides; the term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" ” refers to the polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally, for example by DNA replication and DNA transcription, respectively; or chemically synthesized. DNA and RNA can be single-stranded (ie, ssRNA or ssDNA, respectively) or multiple strands (eg, double-stranded, ie, dsRNA and dsDNA, respectively). The term "mRNA" or "messenger RNA" as used herein refers to single-stranded RNA encoding the amino acid sequence of one or more polypeptide chains.

Sample : As used herein, the term "sample" means an aliquot or portion obtained from a source and/or made available for analysis or processing. In some embodiments, the sample is from a biological source, such as a tissue, cell, or component portion (e.g., body fluid, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic fluid, umbilical cord blood, urine, vaginal fluid and semen). In some embodiments, a sample may be or comprise a homogenate, lysate, or extract prepared from an intact organism or a subset of its tissues, cells, or components, or fractions or portions thereof, including (But not limited to) For example, plasma, serum, spinal fluid, lymph, external sections of skin, respiratory tract, intestine and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the sample is or contains a culture medium, such as a nutrient solution or nutrient gel, which may contain cellular components, such as proteins or nucleic acid molecules. In some embodiments, a "primary" sample is an aliquot of the source. In some embodiments, a primary sample is subjected to one or more processing (eg, isolation, purification, etc.) steps to prepare the sample for analysis or other uses.

Serotype : As used herein, the term "serotype" refers to the different variations in the capsid of AAV based on surface antigens, which allows epidemiological classification of AAV at the subspecies level.

Signal sequence : As used herein, the phrase "signal sequence" refers to a sequence that can direct transport or localization.

Single Unit Dose : As used herein, a "single unit dose" is a dose of any therapeutic agent administered in one dose/once-in/in a single route/single point of contact (i.e., a single administration event). In some embodiments, single unit doses are provided in discrete dosage forms (eg, lozenges, capsules, patches, loaded syringes, vials, etc.).

Similarity : As used herein, the term "similarity" refers to the overall relatedness between polymeric molecules, such as between polynucleotide molecules (eg, DNA molecules and/or RNA molecules) and/or between polypeptide molecules. The calculation of the percent similarity of polymeric molecules to one another can be performed in the same manner as the calculation of the percent identity, except that the calculation of the percent similarity takes into account conservative substitutions as understood in the art.

Stable : As used herein, "stable" means that a compound or entity is sufficiently stable to withstand isolation from a reaction mixture to a suitable purity and to be formulated into an effective therapeutic agent.

Stabilization : As used herein, the terms "stabilize,""stabilize," and "stabilizing zone" mean to stabilize or become stable. In some embodiments, stability is measured relative to absolute values. In some embodiments, stability is measured relative to a reference compound or entity.

Subject : As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present invention may be administered, for example, for experimental, diagnostic, prophylactic and/or therapeutic purposes. Similarly, "individual" or "patient" means an organism that may seek, may need, be receiving or about to receive treatment, or be cared for by a professional trained for a particular disease or condition. Typical individuals include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans). In certain embodiments, an individual or patient may be susceptible to or suspected of having Friedrich's ataxia. In certain embodiments, an individual or patient may be diagnosed with Friedrich's ataxia.

Substantially : As used herein, the term "substantially" refers to a qualitative condition that exhibits an overall or near-total degree or grade of the characteristic or characteristic of interest. Those of ordinary skill in biotechnology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and/or continue to completion or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Substantially equal : As used herein, this term means plus/minus 2% as it relates to the time difference between doses.

Substantially simultaneously : As used herein and when it relates to a plurality of doses, the term generally means within about 2 seconds.

Suffering : An individual "having" a disease, disorder and/or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder and/or condition.

Vulnerable : An individual who is "vulnerable" to a disease, disorder, and/or condition has not been diagnosed with the disease, disorder, and/or condition and/or may not exhibit symptoms thereof, but has a tendency to develop the disease, disorder, and/or condition or develop its symptoms. In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., cancer) may be characterized by one or more of the following: (1) associated with having the disease, disorder, and/or condition; Gene mutation; (2) Genetic polymorphism associated with the disease, disorder and/or condition; (3) Increased expression and/or activity of proteins and/or nucleic acids associated with the disease, disorder and/or condition and/or reduced; (4) habits and/or lifestyle associated with suffering from the disease, disease and/or condition; (5) family history of the disease, disease and/or condition; and (6) exposure to and/or infection with microorganisms associated with the disease, disease and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, individuals susceptible to a disease, disorder, and/or condition do not develop the disease, disorder, and/or condition.

Synthetic : The term "synthetic" means produced, prepared and/or manufactured by human hands. The synthesis of polynucleotides, polypeptides or other molecules of the present invention may be chemical synthesis or enzymatic synthesis.

Targeting : As used herein, "targeting" means the process of designing and selecting nucleic acid sequences that will hybridize to a target nucleic acid and induce a desired effect.

Targeted Cell: As used herein, "target cell" or "targeted cell" refers to any one or more cells of interest. Cells can be found in vitro, in vivo, in situ, or in tissues or organs of an organism. The organism may be an animal, a mammal, a human, and/or a patient. The target cells may be CNS cells or cells in CNS tissue.

Therapeutic Agent : The term "therapeutic agent" refers to any agent that, when administered to an individual, has therapeutic, diagnostic and/or prophylactic effects and/or causes a desired biological and/or pharmacological effect.

Therapeutically Effective Amount : As used herein, the term "therapeutically effective amount" means an amount sufficient to treat an infection, disease, disorder, and/or condition when administered to an individual suffering from or susceptible to the infection, disease, disorder, and/or condition. , the amount of the agent to be delivered (e.g., nucleic acids, drugs, therapeutic agents, diagnostic agents, preventive agents, etc.) that ameliorates its symptoms, diagnoses it, prevents it, and/or delays its onset. In some embodiments, the therapeutically effective amount will be provided in a single dosage form. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising multiple doses. It will be understood by those skilled in the art that, in some embodiments, a unit dosage form is considered to contain a therapeutically effective amount of a particular agent or entity if the dosage unit form contains an amount that is effective when administered as part of such a dosage regimen. .

Therapeutically Effective Result : As used herein, the term "therapeutically effective result" means sufficient to treat, or ameliorate the symptoms of, an infection, disease, disorder, and/or condition in an individual suffering from or susceptible to the infection, disease, disorder, and/or condition. , diagnose, prevent and/or delay its onset.

Thoracic Region: As used herein, "thoracic region" refers to the region of the spinal cord that includes the thoracic vertebrae T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12.

Treatment : As used herein, the term "treatment" means to partially or completely alleviate, slow down, ameliorate, alleviate, reverse, delay the onset of, inhibit the progression of, reduce the severity of, and/or a particular infection, disease, disorder and/or condition. Reduce the incidence of one or more of its symptoms or characteristics. For the purpose of reducing the risk of developing pathology associated with a disease, disorder and/or condition, individuals exhibiting no symptoms of the disease, disorder and/or condition and/or individuals exhibiting only the disease, disorder and/or condition may be Individuals with early symptoms of the condition are treated.

Unmodified : As used herein, "unmodified" refers to any substance, compound, or molecule that has been altered in any way. Unmodified may refer to, but does not always refer to, the wild-type or native form of a biological molecule or entity. A molecule or entity can undergo a series of modifications, whereby each modified product can serve as the "unmodified" starting molecule or entity for a subsequent modification.

Vector : As used herein, a "vector" is any molecule or moiety that transports, transduces, or otherwise serves as a carrier for a heterologous molecule. Vectors of the invention may be produced recombinantly and may be based on and/or may comprise adeno-associated virus (AAV) parental or reference sequences. Such parental or reference AAV sequences may serve as original, second, third or subsequent sequences for engineering the vector. In non-limiting examples, such parent or reference AAV sequences may comprise any one or more of the following sequences: encoding a polypeptide or polypeptide, having a sequence that may be wild-type or modified from wild-type, and the sequence A polynucleotide sequence that can encode the full or partial sequence of a protein, a protein domain, or one or more subunits of FXN and its variants; a polynucleotide sequence that encodes FXN and its variants, which can be wild-type or modified from the wild-type polynucleotides of the sequence; and transgenic genes encoding FXN and variants thereof that may or may not be modified from the wild-type sequence.

Viral construct vector : As used herein, a "viral construct vector" is a vector that contains one or more polynucleotide regions encoding or containing Rep and/or Cap proteins. The viral construct vector may also contain one or more polynucleotide regions that encode or contain components for expression of the virus in cells where the virus replicates.

Viral genome : As used herein, a "viral genome" or "vector genome" is a polynucleotide comprising at least one inverted terminal repeat (ITR) and at least one encoded payload. At least one copy of the viral genome encoding the payload.

Wild type : As used herein, "wild type" is the native form of a biological molecule, sequence or entity.

Described herein are compositions, methods, processes, kits, and devices for designing, preparing, manufacturing, and/or formulating AAV particles.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, these methods and materials are now described.

The invention is further illustrated by the following non-limiting examples. IX. Examples Example 1. Design of promoter variants A. Promoter

Previous studies have shown that the CMV promoter drives the highest amounts of syntaxin expression, with CBA showing less efficacy in driving expression. The PGK and FXN promoters have been shown to be weaker promoters against driver proteins. Variants of the CMV, CBA and FXN promoters were generated to determine which promoters would result in the highest expression of the payload (eg, cotaxin or luciferase). The promoter is inserted into a vector expressing luciferase, which is constructed using standard molecular selection techniques.

The designed promoters are described in Table 3 above. In Table 3, CMV stands for "Cellular Giant Virus"; CBA stands for "Chicken β-actin", which can have CMV IE enhancer region and promoter region; CAG stands for CMV enhancer, CBA promoter and rabbit β-actin hemoglobulin splice acceptor site; FXN stands for "taxin"; and mCBA stands for a variant of the CBA promoter generated using PCR.

Each of the engineered promoter variants was analyzed by gel electrophoresis, digested (Sac1 and HindIII), verified and sequenced. B. In vitro evaluation of promoters

Seven cotaxin promoter variants, 15 CBA promoter variants, and 8 CMV promoter variants were evaluated in vitro for expression activity. HEK293 cells were plated in 96-well culture plates (3.0 × 10 ⁴ cells/well with 0.3 μl of FuGENE® HD transfection reagent per well) and grown in duplicate or triplicate with the primers containing starter from Table 3 Plasmid transfection (5 transfections) of subtype and luciferase payload (firefly (approximately 75 ng) or renilla (25 ng)). 48 hours after transfection, use the DUAL-GLO® Luciferase Assay System to measure luciferase activity and performance. Briefly, an equal volume of Dual-Glo® Luciferase Reagent was added directly to cells in growth medium, incubated for 10 min, and then firefly luciferase performance was measured. The same volume of reagent was then added to quench firefly fluorescence, and Renilla luciferase activity was measured 10 min later. A control of the pGL3-basic promoterless vector was used as a control.

The activities of the promoter variants as determined by firefly and Renilla luciferase performance are shown in Tables 18 to 20, quantified in relative light units (RLU). Table 18 shows data for the original FXN, CBA and CMV promoter variants, while Table 19 shows data for the second generation of PCR-generated CBA promoter variants. Table 20 shows data on all promoter variants measured together in the children. Table 18. Luciferase activity; FXN, CBA and CMV promoters promoter name Promoter SEQ ID NO Firefly luciferase activity Renilla luciferase activity Relative luciferase activity Control (pGL3-basic) - 5818.1 22280.2 0.3 FXNproN1336 1759 39598.9 22747.0 2.6 FXNpro1226 1757 34531.6 27263.4 2.6 FXNpro1060 1756 41490.3 21445.3 2.5 FXNpro907 1755 41025.0 20960.2 1.6 FXNpro534 1754 29035.6 25628.5 2.2 FXNpro363 1753 18861.3 25370.3 1.6 FXNpro223 1752 36665.0 22399.8 1.4 CBA 1734 1.9 x 10 ⁶ 29991.5 90.0 CBA-D1 1735 1.9 x 10 ⁶ 30389.3 76.7 CBA-D2 1736 1.4 x 10 ⁶ 24396.2 68.3 CBA-D3 1737 1.7 x 10 ⁶ 21590.0 41.7 CBA-D4 1738 1.1 x 10 ⁶ 21131.4 24.8 CBA-D5 1739 528925.0 33152.1 29.3 CBA-D6 1740 701503.0 31690.0 28.0 CBA-D7 1741 87118.2 29718.0 13.4 CBA-D8 1742 142306.0 25404.8 14.9 CMV 1743 1.7 x 10 ⁶ 27365.4 158.8 CMV-D1 1744 2.0 x 10 ⁶ 29305.7 83.1 CMV-D2 1745 1.7 x 10 ⁶ 22804.9 80.6 CMV-D3 1746 1.6 x 10 ⁶ 25367.4 71.5 CMV-D4 1747 823269.0 31143.1 68.1 CMV-D5 1748 656529.0 27455.9 26.5 CMV-D6 1749 745160.0 24906.0 56.1 CMV-D7 1750 455119.0 20416.5 14.0 CMV-D8 1751 265574.0 26751.1 14.3 Table 19. Luciferase activity: CBA promoter promoter name Promoter SEQ ID NO Firefly luciferase activity Renilla luciferase activity Relative luciferase activity Control (pGL3-basic) - 7805.3 21204.0 0.7 mCBA 1760 1.1 x 10 ⁶ 24315.1 70.2 CBA 1734 1.2 x 10 ⁶ 19496.6 80.4 mCBA-D1 1761 1.2 x 10 ⁶ 21795.7 50.8 CBA-D1 1735 1.2 x 10 ⁶ 23643.8 57.2 mCBA-D2 1762 866445.0 24847.4 37.3 CBA-D2 1736 1.1 x 10 ⁶ 26002.7 54.9 mCBA-D3 1763 483938.0 22957.8 20.1 CBA-D3 1737 394159.0 24134.9 20.1 mCBA-D4 1764 378331.0 21767.5 20.0 CBA-D4 1738 423313.0 25556.2 17.3 mCBA-D5 1765 481034.0 21420.2 20.0 CBA-D5 1739 512204.0 23063.8 22.6 mCBA-D6 1766 600066.0 25365.9 21.1 CBA-D6 1740 499860.0 28509.2 22.2 CBA-D7 1741 397064.0 24788.7 17.0 CBA-D8 1742 193978.0 19823.0 10.6 CMV 1743 1.2 x 10 ⁶ 21271.2 103.2 CMV-D1 1744 1.5 x 10 ⁶ 18371.5 64.1 CMV-D2 1745 1.3 x 10 ⁶ 20514.7 68.4 CMV-D3 1746 753070.0 25375.3 52.8 CMV-D4 1747 860089.0 21706.6 48.6 CMV-D5 1748 607362.0 18944.0 44.7 CMV-D6 1749 835859.0 22311.5 48.5 CMV-D7 1750 257050.0 23927.6 14.2 CMV-D8 1751 223323.0 18691.3 12.6 Table 20. Luciferase activity: FXN, CBA and CMV promoters promoter name Promoter SEQ ID NO Firefly luciferase activity Renilla luciferase activity Relative luciferase activity Control (pGL3-basic) - 7921.8 20459.1 0.4 FXNproN1336 1759 63926.2 25025.5 2.6 FXNpro1226 1757 36579.9 17960.0 2.0 FXNpro1060 1756 46231.1 20049.1 2.3 FXNpro907 1755 40450.8 20082.3 2.0 FXNpro534 1754 40052.5 21713.7 1.9 FXNpro363 1753 39193.8 22916.8 1.7 FXNpro223 1752 32047.5 22625.3 1.4 mCBA 1760 1.8 x 10 ⁶ 23690.8 73.9 CBA 1734 1.8 x 10 ⁶ 19496.6 70.4 mCBA-D1 1761 989155.0 19090.6 50.8 CBA-D1 1735 1.2 x 10 ⁶ 23643.8 57.2 mCBA-D2 1762 580926.0 24847.4 30.6 CBA-D2 1736 1.1 x 10 ⁶ 26002.7 54.9 mCBA-D3 1763 786194.0 22957.8 33.4 CBA-D3 1737 410735.0 24134.9 20.1 mCBA-D4 1764 495034.0 21767.5 20.0 CBA-D4 1738 385259.0 25556.2 17.3 mCBA-D5 1765 525189.0 21420.2 20.0 CBA-D5 1739 472985.0 23062.8 22.6 mCBA-D6 1766 481465.0 25365.9 21.1 CBA-D6 1740 568296.0 25175.8 22.2 CBA-D7 1741 407952.0 24788.7 17.0 CBA-D8 1742 229847.0 19823.0 10.6 CMV 1743 3.2 x 10 ⁶ 19365.4 124.2 CMV-D1 1744 1.4 x 10 ⁶ 23521.7 64.1 CMV-D2 1745 1.6 x 10 ⁶ 25113.0 68.4 CMV-D3 1746 1.0 x 10 ⁶ 22383.5 39.5 CMV-D4 1747 1.2 x 10 ⁶ 19553.7 48.6 CMV-D5 1748 1.1 x 10 ⁶ 20376.2 44.7 CMV-D6 1749 1.0 x 10 ⁶ 22311.5 51.8 CMV-D7 1750 274083.0 20989.1 14.2 CMV-D8 1751 296686.0 25589.8 12.6

Compared to the control, the increase in promoter activity ranged from 0.4 to 125-fold. The difference in activity between the promoter with the lowest performance and the promoter causing the highest performance was 321-fold. The CMV (SEQ ID NO: 1743) promoter provides the greatest activity, among which CBA (SEQ ID NO: 1734), mCBA (SEQ ID NO: 1760), CMV-D2 (SEQ ID NO: 1745), CBA-D1 (SEQ ID NO: 1745) NO: 1735), CBA-D2 (SEQ ID NO: 1736), CMV-D6 (SEQ ID NO: 1749), mCBA-D1 (SEQ ID NO: 1761) and CMV-D4 (SEQ ID NO: 1747) promoters Each showed reduced efficacy in inducing luciferase expression. The overall activity ranking of promoter variants is as follows, listed in order from the promoter with the highest performance to the promoter with the lowest activity: CMV (SEQ ID NO: 1743), mCBA (SEQ ID NO: 1760), CBA (SEQ ID NO: 1760) NO: 1734), CMV-D2 (SEQ ID NO: 1745), CMV-D1 (SEQ ID NO: 1744), CBA-D1 (SEQ ID NO: 1735), CBA-D2 (SEQ ID NO: 1736), CMV -D6 (SEQ ID NO: 1749), mCBA-D1 (SEQ ID NO; 1761), CMV-D4 (SEQ ID NO: 1747), CMV-D5 (SEQ ID NO: 1748), CMV-D3 (SEQ ID NO : 1746), mCBA-D3 (SEQ ID NO: 1763), mCBA-D2 (SEQ ID NO: 1762), CBA-D5 (SEQ ID NO: 1739), CBA-D6 (SEQ ID NO: 1740), mCBA- D6 (SEQ ID NO: 1766), CBA-D3 (SEQ ID NO: 1737), mCBA-D5 (SEQ ID NO: 1765), mCBA-D4 (SEQ ID NO: 1764), CBA-D4 (SEQ ID NO: 1738), CBA-D7 (SEQ ID NO: 1741), CMV-D7 (SEQ ID NO: 1750), CMV-D8 (SEQ ID NO: 1751), CBA-D8 (SEQ ID NO: 1742), pGL3-FXNpro1336 (SEQ ID NO: 1759), pGL3-FXNpro1060 (SEQ ID NO: 1756), pGL3-FXNpro1226 (SEQ ID NO: 1757), pGL3-FXNpro534 (SEQ ID NO: 1754), pGL3-FXNpro363 (SEQ ID NO: 1753 ), pGL3-FXNpro223 (SEQ ID NO: 1752) and pGL3-basic.

Based on these findings, 12 promoter variants were selected for further study. Such variants include CMV (SEQ ID NO: 1743), CBA (SEQ ID NO: 1734), CMV-D1 (SEQ ID NO: 1744), mCBA-D1 (SEQ ID NO: 1761), CMV-D3 (SEQ ID NO: 1746), mCBA-D2 (SEQ ID NO: 1762), CBA-D6 (SEQ ID NO: 1740), CBA-D4 (SEQ ID NO: 1738), CMV-D7 (SEQ ID NO: 1750), CBA-D8 (SEQ ID NO: 1742), pGL3-FXNpro1060 (SEQ ID NO: 1756) and pGL3-FXNpro534 (SEQ ID NO: 1754). Promoters exhibiting low, medium and high performance were selected. Example 2. Design of payload construct encoding syntaxin

The payload construct is designed to include at least a nucleic acid sequence encoding a cotaxin protein. Payload constructs were constructed using standard molecular selection techniques. The FXN-tag transgenic gene was selected into the AAV expression vector, and the resulting pure line was further sequenced to confirm the correctness of all elements such as ITR, promoter and tag.

To construct the cynomolgus macaque cotaxin (cFXN) payload construct, a hemagglutinin (HA)-tagged cFXN transgene was selected to contain the 5' and 3' ITR sequences (141 nucleotides) derived from the AAV2 genome. acid), CBA, CMV or FXN promoter, hβglobin intronic region, hGH poly(A) signal and three miR-122 binding sites (miR-122 BS) for liver off-targeting. Assess deletion variants of CBA, CMV or FXN promoters. A 450 bp human albumin sequence (Alb450) was tested as a filler sequence in three constructs (see Table 4; ITR to ITR sequence).

To construct human fascin (hFXN) payload constructs with different configurations, the 5' and 3' ITR sequences (141 nucleotides) derived from the AAV2 genome, the CBA promoter, and the hβglobin intron region were used , hFXN gene and the starting construct of hGH poly(A) signal. Subsequent payload constructs derived from the starting construct contained one of four promoter deletion variants: CBA-D8, CMV, CMV-D7, and FXNpro1060. In order to have full genome size, human albumin was selected to serve as a filler (Alb1384, Alb1856, Alb450, Alb2266, Alb2335, Alb1785, Alb2264 or Alb1313) and added to subsequent vehicle constructs. Human fascin constructs containing stuffer sequences were constructed with or without a miR-122 binding site (miR-122 BS) for liver off-targeting (see Table 4).

The plastids containing the payload construct are described in Table 4. These payload constructs include the vector backbone, 5' and 3' ITR sequences (141 nucleotides) derived from the AAV2 genome, human beta-hemoglobin (hβglobin) intronic region, human growth hormone (hGH) ) poly(A) signal, and may also include the following components: CBA, mCBA, CMV or FXN promoters or deletion variants thereof; cynomolgus monkey fascin (cFXN) or human fascin (hFXN) genes; blood cells Lectin (HA) tag; three miR-122 binding sites (BS); and stuffer sequences of varying lengths derived from the human albumin gene. The hβglobin intron region includes immediate early protein 1 (ie1) exon 1, part of the ie1 intron, hβglobin intron 2 and hβglobin exon 3. The components between ITR sequences are presented in Table 4 from 5' end to 3' end.

The construct ITR to ITR sequences were confirmed by sequencing and are given as SEQ ID NOs: 1778-1804. Example 3. Validation of cFXN construct and its components A. Identification of promoter variants and hβglobin intron

To verify the promoter and hβglobin intronic regions in the engineered cFXN construct driven by CMV, CBA and FXN promoter variants, the constructs were digested with high-fidelity restriction enzymes MluI-HF and AgeI-HF. Double digestion produced a cleavage product consisting of the promoter and hβglobin intronic regions. Digested samples were analyzed by agarose gel electrophoresis. Variants tested included cFXN1-cFXN18 (SEQ ID NO: 1778-1795). The gel reveals bands with a pattern corresponding to the size of the promoter variant. B. Identification of miR-122 and hGH poly(A)

To verify the miR-122 and hGH poly(A) regions in engineered cFXN constructs driven by CMV, CBA and FXN promoter variants, the constructs were digested with restriction enzymes XhoI and ClaI. Double digestion produces a cleavage product consisting of the miR-122 binding site, hGH poly(A) sequence, and in some cases a stuffer sequence. Digested samples were analyzed by agarose gel electrophoresis. Variants tested included cFXN1-cFXN18 (SEQ ID NO: 1778-1795). Bands corresponding to miR-122 and hGH poly(A) regions were detected in most constructs. Variants lacking the miR-122 sequence exhibited lower color banding compared to constructs with the miR-122 sequence. Variants with filler sequences exhibit higher color bands, confirming the presence of fillers. C. Identification of ITR

To verify the inverted terminal repeats (ITRs) in the engineered cFXN constructs driven by CMV, CBA and FXN promoter variants, the constructs were individually digested with restriction enzymes XmaI or MscI. Both XmaI and MscI are cleaved within the ITR and at an additional site in the construct. Digested samples were analyzed by agarose gel electrophoresis. Variants tested included cFXN1-cFXN18 (SEQ ID NO: 1778-1795). Both gels revealed three cleavage products in all constructs, confirming the presence of ITR. Example 4. Generation of cFXN constructs in mammalian cells

HEK-293T or Huh7 cells were transfected using cFXN construct (SEQ ID NO: 1778-1795). Transfections were performed at approximately 1.5 × 10 ⁵ cells per well in 12-well culture dishes. Cells were grown with 1.0 µg of cFXN construct, 1.0 µg of control plasmid containing the EGFP gene, and 4.0 µl of FUGENE® HD transfection reagent (Promega, Madison, WI). 72 hours after transfection, cells were analyzed for EGFP expression by fluorescence microscopy. Cells transfected with the EGFP plasmid alone exhibited fluorescence and served as a positive control, whereas untransfected cells served as a negative control and did not exhibit any fluorescence. Various constructs exhibited similar amounts of EGFP expression, indicating that the transfection was efficient. The cells were then subjected to RNA and protein extraction for subsequent analysis. Example 5. Detection of hβglobin intron splicing

The forward and reverse primers were used to amplify a fragment covering the hβglobin intron and cFXN-HA transgene sequence from the cFXN construct. PCR reactions were performed, and products were analyzed by agarose gel electrophoresis. All constructs showed the same color band corresponding to the fragment retaining the hβglobin intron.

cDNA was synthesized by reverse transcription from RNA extracted from transfected HEK-293T or Huh7 cells. The PCR reaction was repeated using the cDNA template, and the products were analyzed by agarose gel electrophoresis. The cDNA reaction produces smaller fragments than those amplified directly from the construct. The size difference is consistent with the length of the ie1 intron and hβglobin intron 2. The results demonstrated that the hβglobin intron was successfully spliced after transfection. Example 6. Expression of cFXN protein in HEK-293T and Huh7 cells A. HEK-293T cells

Expression of cFXN (SEQ ID NO: 1778-1795) in HEK293T cells after introduction of different cFXN constructs was detected by Western blotting. Total protein was extracted from HEK-293T cells transfected with different cFXN constructs including cFXN1-cFXN18 (SEQ ID NO: 1778-1795). Each sample was loaded with a total of 10 µg protein. GAPDH serves as an internal control. Western blotting revealed three different cFXN protein species, namely precursor protein (approximately 25 KDa), intermediate protein (approximately 20 KDa) and mature protein (approximately 15 KDa). No cFXN protein was detected in cells transfected with EGFP alone. Almost all constructs showed robust expression of cFXN protein, except cFXN17 (SEQ ID NO: 1794) and cFXN18 (SEQ ID NO: 1795). cFXN11 (SEQ ID NO: 1788), cFXN12 (SEQ ID NO: 1789), cFXN13 (SEQ ID NO: 1790), cFXN14 (SEQ ID NO: 1791) had the highest performance.

The amount of cFXN protein expressed was quantified via enzyme-linked immunosorbent assay (ELISA). A total of 20 ng of protein was used per group. The cFXN construct expresses cFXN in different amounts, and the expression amount of cFXN is highly consistent with the promoter activity of the CBA/CMV/FXN promoter variant. Table 21 lists the cFXN protein concentrations expressed by different cFXN constructs in HEK-293T cells. Table 21. cFXN protein concentration in HEK-293T cells cFXN construct SEQ ID NO: Average cFXN concentration (ng/µg total protein ) EGFP control - 0 cFXN1 1778 9 cFXN2 1779 14 cFXN8 1785 16.5 cFXN9 1786 5 cFXN3 1780 2 cFXN4 1781 1 cFXN6 1783 0.5 cFXN11 1788 17.5 cFXN12 1789 20 cFXN13 1790 9.5 cFXN14 1791 3.5 cFXN15 1792 1 cFXN18 1795 0.2 cFXN17 1794 0.1 cFXN5 1782 2.5 cFXN7 1784 2 cFXN16 1793 2 B. Huh7 cells

The expression of cFXN in Huh7 cells after the introduction of different cFXN constructs was detected by Western blotting. Total protein was extracted from Huh7 cells transfected with different cFXN constructs including cFXN1-cFXN18 (SEQ ID NO: 1778-1795). Each sample was loaded with a total of 10 µg protein. GAPDH serves as an internal control. Western blotting revealed three different cFXN protein species, namely precursor protein (25 KDa), intermediate protein (20 KDa) and mature protein (15 KDa). No cFXN protein was detected in cells transfected with EGFP alone. Bright bands were seen in cells transfected with constructs containing miR-122 binding sites, indicating that miR-122 effectively reduced cFXN protein expression in hepatocytes.

The amount of cFXN protein expressed was quantified via enzyme-linked immunosorbent assay (ELISA). A total of 20 ng of protein was used per construct. The cFXN construct expresses cFXN in different amounts, and the expression amount of cFXN is highly consistent with the promoter activity of the CBA/CMV/FXN promoter variant. Cells transfected with a construct containing a miR-122 binding site showed significantly lower levels of cFXN protein than cells transfected with a non-miR-122 construct. Table 22 lists the cFXN protein concentrations expressed by different cFXN constructs in Huh7 cells. Table 22. cFXN protein concentration in Huh7 cells cFXN construct SEQ ID NO: Average cFXN concentration (ng/µg total protein ) EGFP control - 0.1 cFXN1 1778 4.8 cFXN2 1779 1 cFXN8 1785 0.8 cFXN9 1786 0.8 cFXN3 1780 0.2 cFXN4 1781 0.2 cFXN6 1783 0.2 cFXN11 1788 7.8 cFXN12 1789 0.8 cFXN13 1790 0.6 cFXN14 1791 0.3 cFXN15 1792 0.3 cFXN18 1795 0.3 cFXN17 1794 0.2 cFXN5 1782 0.3 cFXN7 1784 0.2 cFXN16 1793 0.3 Example 7. In vitro testing of hFXN1 constructs

hFXN1 (SEQ ID NO: 1796) was co-transfected into HEK-293T cells via AAV PHP.B encapsulating plasmid. AAV particles were purified using four culture dishes of transfected cells. AAV particles were purified by iodixanol gradient ultracentrifugation. The purified sample contained 1×10 ¹² vg/ml AAV particles. Samples were run on a sodium dodecyl sulfate (SDS)-gel, followed by silver staining to visualize transgene bands and check for impurities. The gel revealed co-purified hFXN protein, confirming successful encapsulation of the transgene into the AAV.

HEK-293T cells were transfected with three different AAV multiplicity of infection (MOI), namely 5×10 ⁵ , 1×10 ⁵ and 2×10 ⁴ . After transfection, proteins were extracted from the cells and detected by Western blotting. The expression of hFXN is directly proportional to the MOI of transfection. Cells infected at the highest MOI of 5×10 ⁵ were found to have robust hFXN expression. hFXN protein was not detected against HEK-293T cells transfected with MOI 2×10 ⁴ , whereas hFXN protein was detected against HEK-293T cells transfected with MOI 1×10 ⁵ .

Denaturing agarose gel electrophoresis was used to analyze the size of the AAV viral genome. A single band corresponding to the expected genome size (2881 bp) was detected. Example 8. Generation of human hFXN construct A. Alternative promoter design of construct encoding hFXN1

The hFXN1 (SEQ ID NO: 1796) construct was used as the starting template to generate four intermediate plastids by exchanging promoters. Each of the four intermediate plasmids generated contained one of the following promoters: FXNpro1060 (SEQ ID NO: 1756), CMV (SEQ ID NO: 1743), CMV-D7 (SEQ ID NO: 1750), and CBA-D8 (SEQ ID NO: 1742). Intermediate plastids were generated by subcloning the four promoter sequences into the hFXN1 template using MluI and Hind3 restriction sites. The ligation products were transformed into bacterial cells, and the plasmids were purified using standard small-scale purification protocols. Purified plasmids were verified by restriction digestion with MluI/Hind3 or MluI/Agel, followed by agarose gel electrophoresis. B. Insertion of human albumin (Alb) stuffer sequence

To ensure full genome size, human albumin DNA was chosen to serve as filler. Genomic DNA of human albumin was isolated from HEK-297T and HeLa cells. PCR reaction was performed to amplify 8 different sizes of albumin DNA, namely 1313 bp (SEQ ID NO: 1831), 1384 bp (SEQ ID NO: 1832), 1785 bp (SEQ ID NO: 1833), 1856 bp ( SEQ ID NO: 1835), 2264 bp (SEQ ID NO: 1840), 2266 bp (SEQ ID NO: 1841) and 2335 bp (SEQ ID NO: 1842). The four intermediate plastids were digested with either restriction enzyme AvrII that cleaves between the miR-122 binding site and the 3' ITR or two restriction enzymes AvrII and BspEI that cleave the fragment containing the miR-122 binding site. Eight albumin PCR products were ligated via Gibson assembly to enzymatically digested plastids with or without miR-122 binding sites. The ligation products were transformed into bacterial cells, and the plasmids were purified using standard small-scale purification protocols. Purified plasmids were verified by restriction digestion with XbaI/KpnI followed by agarose gel electrophoresis.

Eight final constructs with a genome size of 4.6 kb were sequenced from inverted terminal repeat (ITR) to ITR to confirm sequence integrity. Example 9. Expression of hFXN protein in HEK-293T cells

The hFXN construct was transfected into HEK-293T cells, and hFXN protein expression was evaluated by Western blotting and ELISA. Transfections were performed as described previously. GFP plasmids were used as internal controls. A total of 10 µg protein was loaded per sample. Western blotting detected three forms of hFXN protein: precursor hFXN, intermediate hFXN and mature hFXN. Constructs hFXN4 and hFXN5 (SEQ ID NO: 1799 and 1800) had the strongest hFXN expression, while hFXN2, hFXN3, hFXN6 and hFXN7 (SEQ ID NO: 1797, 1798, 1801 and 1802) showed limited expression, and hFXN8-hFXN9 (SEQ ID NO: 1803-1804) has weak to undetectable performance.

The amount of hFXN protein and GFP expression was quantified via ELISA. Among the hFXN constructs, hFXN4 (SEQ ID NO: 1799) and hFXN5 (SEQ ID NO: 1800) exhibited the highest hFXN protein concentration, with relative hFXN performance >20 times higher than other proteins. Table 23 lists hFXN protein concentration and GFP expression in HEK-293T cells. Relative hFXN performance was calculated as hFXN/GFP ratio, showing fold change relative to endogenous hFXN control. Table 23. hFXN protein concentration hFXN construct SEQ ID NO: Average hFXN concentration (ng/ml) Average GFP concentration (ng/ml) Average fold change relative to internal controls AHr 1803 794.0 2354.7 1.6 AHr 1804 2187.4 2331.4 4.5 AHr 1799 73010.1 2133.7 166.0 ikB 1800 87718.4 1777.6 236.1 hFXN6 1801 8533.8 1864.9 22.6 htK 1802 7594.3 2186.5 16.3 hFXN2 1797 6424.0 2364.7 13.0 hFXN3 1798 6375.4 2234.9 13.7 GFP control - 204.3 977.3 1.0 Example 10. In vivo promoter selection studies

The ITR-to-ITR sequence containing the promoter was encapsulated into AAV6 capsids and delivered to Sprague Dawley rats by intrastriatal injection at a dose of 1×10 ¹⁰ VG. Tissue samples were collected at 3 or 10 weeks, and fataxin content was quantified. Constructs cFXN2 (SEQ ID NO: 1779), cFXN3 (SEQ ID NO: 1780), cFXN4 (SEQ ID NO: 1781), cFXN13 (SEQ ID NO: 1790), cFXN14 (SEQ ID NO: 1791), cFXN17 ( SEQ ID NO: 1794), cFXN18 (SEQ ID NO: 1795), hFXN2 (SEQ ID NO: 1797), hFXN4 (SEQ ID NO: 1799), hFXN5 (SEQ ID NO: 1800) and hFXN6 (SEQ ID NO: 1801 ) conduct such research.

Based on the quantification of syntaxin expression, the expressions displayed were selected to be one-third (hFXN4; SEQ ID NO: 1799), one-fifth (hFXN8; SEQ ID NO: 1803), and one-eighth (hFXN2; SEQ ID NO: 1797) and one-twenty-fifth construct (hFXN6; SEQ ID NO: 1801) for further study. A construct containing wild-type CBA and CMV promoters (eg, cFXN1; SEQ ID NO: 1778) was used as a control. The viral genome was encapsulated into capsid VOY201 (SEQ ID NO: 1724) and administered to Spoggery rats via intravenous delivery at a dose of 6.3×10 ¹² or 2×10 ¹³ VG/kg. After 28 or 90 days, tissue samples were collected and processed to quantify fataxin expression (ng/mg).

Promoters CMV-D7 (SEQ ID NO: 1750) and CBA-D8 (SEQ ID NO: 1742) exhibited moderate syntaxin performance of interest compared to other constructs and were therefore selected for further study.

To test the durability and persistence of syntaxin expression driven by the CMV-D7 (SEQ ID NO: 1750) and CBA-D8 (SEQ ID NO: 1742) promoters, a time course study was performed. Will contain CMV-D7 (SEQ ID NO: 1750), CBA-D8 (SEQ ID NO: 1742), CBA (SEQ ID NO: 1734) or CMV (SEQ ID NO: 1743) promoter and cotaxin payload sequence The viral genome was encapsulated into VOY101 (SEQ ID NO: 1) capsid to produce AAV particles. These AAV particles were administered via intravenous delivery to male Spurgdoodle rats via the tail vein at one of two doses (6.3×10 ¹² or 2×10 ¹³ ). Tissue samples (heart, liver, cerebellum, thoracic and lumbar DRG) were collected at 28, 90 or 180 days post-dose and processed for expression of vector genome/diploid cells and fataxin based on anti-Fataxin SimpleStep ELISA Quantify the quantity. The data are presented in Table 24 and Table 25, Figure 1A, Figure 1B, Figure 1C and Figure 1D. Table 24. Performance of cotaxin (ng/mg) organization CMV-D7 CBA-D8 6.3e12 2.3e13 6.3e12 2.3e13 28d 90d 180d 28d 90d 180d 28d 90d 180d 28d 90d 180d heart 14.9 168.7 98.5 37.4 254.9 234.7 20.4 112.1 164.6 77.5 713.8 304.8 cerebellum 1.0 1.9 4.3 5.8 13.6 23.6 0.9 2.2 3.1 6.9 11.5 21.4 WaistDRG 75.2 144.7 64.7 249.0 250 152.1 45.0 98.3 53.2 121.3 212.8 95.9 Table 25. Vector genome/diploid cells (VG/dc) organization CMV-D7 CBA-D8 6.3e12 2.3e13 6.3e12 2.3e13 28d 90d 180d 28d 90d 180d 28d 90d 180d 28d 90d 180d heart 1.8 2.1 1.8 3.3 8.11 7.2 1.1 2.2 1.8 3.6 9.6 6.1 Liver 0.7 0.6 0.6 4.2 2.4 2.9 0.7 0.5 0.5 3.2 1.4 0.9 cerebellum 0.0 0.0 0.00 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 ChestDRG 0.1 0.1 0.1 0.5 0.4 0.1 0.1 0.1 0.1 0.5 0.4 0.3 CBA ( without miR-122) CMV 6.3e12 2.3e13 6.3e12 2.3e13 28d 90d 180d 28d 90d 180d 28d 90d 180d 28d 90d 180d heart 4.3 7.0 7.0 - - - - - - 4.8 - - Liver 0.8 0.4 0.6 - - - - - - 5.7 - - cerebellum 0.1 0.0 0.0 - - - - - - 0.1 - - ChestDRG 0.5 0.4 0.1 - - - - - - 2.2 - -

In tissue collected from the ventricle, driving fataxin expression using the CMV-D7 (SEQ ID NO: 1750) promoter enhanced fataxin expression by 0.2-2.5×, whereas using CBA-D8 (SEQ ID NO: 1742) Promoter-driven fataxin expression enhanced fataxin expression by 0.3-7.8×. For comparison, the CBA (SEQ ID NO: 1734) promoter enhances the expression of Fataxin by 41.2-70× compared to the normal expression of Fataxin, and the CMV (SEQ ID NO: 1743) promoter enhances the expression of Fataxin 297×.

In tissue collected from the cerebellum, using the CMV-D7 (SEQ ID NO: 1750) promoter to drive Fataxin expression enhanced Fataxin expression by 0.01-0.31×, while using CBA-D8 (SEQ ID NO: 1742) Promoter-driven fataxin expression enhanced fataxin expression by 0.01-0.28×. For comparison, the CBA (SEQ ID NO: 1734) promoter enhances the expression of Fataxin by 0.0-0.9× compared to the normal expression of Fataxin, and the CMV (SEQ ID NO: 1743) promoter enhances the expression of Fataxin 0.21×.

In tissue collected from the lumbar DRG, driving fataxin expression using the CMV-D7 (SEQ ID NO: 1750) promoter enhanced fataxin expression by 1.6-6.2×, whereas using CBA-D8 (SEQ ID NO: 1742 ) promoter drives the expression of Fataxin, which enhances the expression of Fataxin by 1.1-5.2×. For comparison, the CBA (SEQ ID NO: 1734) promoter enhances the expression of Fataxin by 0.0-5.4× compared to the normal expression of Fataxin, and the CMV (SEQ ID NO: 1743) promoter enhances the expression of Fataxin 0.5×.

Immunohistochemical analysis was performed on 30 µm tissue samples collected 28 days after AAV particle administration. Anti-hFXN antibody (1/50,000) was used. Fataxin expression driven by the CMV-D7 (SEQ ID NO: 1750) and CBA-D8 (SEQ ID NO: 1742) promoters was detected in the dentate nucleus of treated rats.

Each of the CMV-D7 (SEQ ID NO: 1750), CBA-D8 (SEQ ID NO: 1742) and CBA (SEQ ID NO: 1734) promoters showed similar distribution and expression patterns in the DRG and brain. In the heart, the CMV-D7 and CBA-D8 promoters produce FXN expression that is approximately one-fiftieth to one-hundred-sixtieth of that of CBA-driven syntaxin expression.

Both the CMV-D7 (SEQ ID NO: 1750) and CBA-D8 (SEQ ID NO: 1742) promoters drive fataxin expression in the cerebellum, heart, and DRG 180 days after administration of AAV particles. At this time, performance in the cerebellum was approximately 3-fold that achieved using the reference CBA promoter, indicating that the CMV-D7 and CBA-D8 promoters are active in target cells in the cerebellum. Example 11. In vivo cFXN expression following intrastriatum administration of viral genomes comprising alternative promoter variants in rats .

To test fascin expression driven by alternative promoter variants, a promoter selected from any of the following promoters, AAV2 wild-type ITR, HA-tagged long-tailed macaque fascin (cynoFXN or cFXN ), the viral genome of the miR-122 target trinucleotide repeat sequence and the human growth hormone polyadenylation sequence were encapsulated into AAV6 capsids and administered via intrastriatal injection at a dose of 1×10 ¹⁰ VG/kg (unless (otherwise mentioned) delivered to Spodoptera rats: CMV-D1 (SEQ ID NO: 1744), CMV-D3 (SEQ ID NO: 1746), CBA-D4 (SEQ ID NO: 1738) and CBA-D6 (SEQ ID NO: 1740) (as taught in Table 3). As a control, FXNpro534 (SEQ ID NO: 1754), FXNpro1060 (SEQ ID NO: 1756), CMV (SEQ ID NO: 1743), CBA (SEQ ID NO: 1734) promoters or variants thereof were used to drive cFXN from the viral genome Expressed and encapsulated into AAV6 capsids. Tissue samples were collected 3 weeks after injection, and striatal cFXN protein content was quantified by ELISA. The data is presented below in Table 26 and Figure 2. Table 26. In vivo cFXN performance (ng/mg) in rats driven by alternative promoters. promoter Promoter SEQ ID NO: Dosage(VG/kg) cFXN (ng/mg) Fold change relative to endogenous FXN FXNpro534 1754 1e10 62.2 1 CMV-D7 1750 6e9 87.5 1.4 CBA-D8 1742 1e10 260 4.2 FXNpro1060 1756 1e10. 406 6.6 CMV (no miR122) 1743 1e10 640 10.4 CBA-D4 1738 1e10 643.3 10.4 CMV-D3 1746 1e10 657.5 10.7 CMV-D1 1744 1e10 1076 17.5 CBA-D6 1740 1e10 1496 24.3 CMV 1743 6e9 1504 24.4 CBA 1734 1e10 2142 34.7 CBA (without miR122) 1734 1e10 5302 86

Striatal cFXN driven by the FXNpro534 (SEQ ID NO: 1754) promoter showed equivalent levels of endogenous syntaxin, while the FXNpro1060 (SEQ ID NO: 1756) promoter produced 6.6× the endogenous FXN level. As a control, high cFXN expression was achieved through CMV.miR122 (24.4×), CBA.miR122 (34.7×) or CBA (86×) promoters respectively. Intermediate expression amounts of 1.4× and 4.2× were observed for cFXN driven by CMV-D7 (SEQ ID NO: 1750) or CBA-D8 (SEQ ID NO: 1742) promoters, respectively, which was observed in cFXN driven by two FXN promoters. Within the range of those manifestations driven by the variant. Generation of alternative promoter variants CMV-D1 (SEQ ID NO: 1744), CMV-D3 (SEQ ID NO: 1746), CBA-D4 (SEQ ID NO: 1738) and CBA-D6 (SEQ ID NO: 1749) cFXN performance was higher than CMV-D7 (SEQ ID NO: 1750) or CBA-D8 (SEQ ID NO: 1742) but still much lower than CMV.miR122. Taken together, these data indicate that additional promoter variants are effective in inducing cFXN expression in the rat striatum. Example 12. In vivo cFXN expression and vector genome biodistribution after intrathalamic administration of VOY101-cFXN-HA AAV vector

To achieve widespread expression of cFXN in the deep cerebellar nuclei and associated cFXN levels in the spinal cord and dorsal root ganglia, the VOY101-cFXN-HA (SEQ ID NO: 1778) vector was administered to SP by bilateral intrathalamic injection. Gedori rat.

The single-stranded viral genome containing the CBA promoter and cFXN-HA sequence was encapsulated into VOY101 particles and injected intrathalamicly at a dose of 5×10 ¹⁰ vg/injection. The injection volume and injection rate were 12 µl/injection and 0.5 µl/min, respectively. One rat was treated with vehicle control. Six weeks after treatment, immunohistochemical analysis using anti-HA immunostaining was performed to assess the expression of cFXN-HA in the dentate nucleus of the cerebellum and spinal cord. Stronger HA staining was observed throughout the dentate nucleus of the cerebellum in all treated animals compared to vehicle controls. In addition, widespread cFXN manifestations were evident in the dorsal columns, central canal, ventral horn, and Clark's column of the cervical, thoracic, and lumbar spinal cord remote from the injection site. Together, these results demonstrate that intrathalamic injection of VOY101-CBA-cFXN vector can effectively transduce neurons in the deep cerebellar nuclei and spinal cord and drive the expression of FXN protein. Example 13. Transgenic expression of hFXN after intravenous administration of VOY101-CMV-D7-hFXN or VOY101-CBA-D8-hFXN AAV particles to Pvalb cKO mice

Friedrich's ataxia (FA) is caused by an amplification of the intronic GAA in the fastaxin gene that significantly reduces FXN expression in mitochondria. In the early stages of Friedrich's ataxia, patients experience difficulty walking and loss of balance due to the loss of large proprioceptive neurons in the peripheral dorsal root ganglia (DRG). Subsequently, trunk and arm function deteriorates due to increased spinocerebellar neuronal dysfunction. Patients become wheelchair bound and incapacitated, resulting in a shortened average life span to approximately 40 years. To model the selective nature of neuronal loss in FA, transgenic mice were generated in which FXN expression was abolished in parvalbumin-expressing cells (Pvalb cKO mice) via the Cre Lox system ( et al. 2018 ). Pvalb cKO mice display loss of proprioceptive sensory function and progressive ataxia within weeks of life.

AAV particles with the capsid serotype of VOY101 (SEQ ID NO: 1) were generated using viral genomes containing CMV-D7-hFXN (SEQ ID NO: 1801) or CBA-D8-hFXN (SEQ ID NO: 1797). By triple transfection of HEK293 cells, a polyadenylation sequence containing the promoter (CMV-D7 or CBA-D8), two AAV2 ITRs, hFXN, the trinucleotide repeats of the miR-122 target, and the human growth hormone polyadenylation sequence were The viral genome is encapsulated into VOY101 AAV particles. AAV particles were purified and formulated in phosphate buffered saline (PBS) containing 0.001% F-68, and then administered intravenously to mice at a dose of 2×10 ¹³ VG/kg.

To test the expression of hFXN in Pvalb cKO animals administered intravenously a single strand of VOY101-CMV-D7-hFXN or VOY101-CBA-D8-hFXN AAV particles, three doses of 6.32×10 ¹² VG/kg or 2.0× Pellets were administered intravenously to Pvalb cKO mice aged 7.5 weeks at one of 10 ¹³ VG/kg. A group of Pvalb cKO mice were treated intravenously with AAV9-hFXN particles at 7.0×10 ¹² VG/kg. WT or Pvalb cKO mice were also dosed with vehicle controls.

Four weeks after administration, 11.5-week-old mice were euthanized. Cerebellar and lumbar DRG tissues were collected and processed to quantify the amount of hFXN expression. hFXN protein content was measured by ELISA and reported as ng hFXN/mg total protein. Fold changes in hFXN expression in cerebellar or lumbar DRG relative to WT endogenous FXN levels were calculated. The data are presented in Table 27, Figure 3A and Figure 3B. Table 27. Expression of hFXN in the cerebellum and lumbar DRG of Pvalb cKO mice WT KO CMV-D7 6.3e12 VG/kg CMV-D7 2e13 VG/kg CBA-D8 6.3e12 VG/kg CBA-D8 2e13 VG/kg AAV9 7e12 VG/kg L-DRG (ng/mg) 0.4 0.4 27.4 63.1 8.2 25.9 1.7 Fold changes in L-DRG 1.0 0 2.8 6.4 0.8 2.6 0.1 Cerebellum(ng/mg) 0.7 0.7 472.2 1114.0 293.6 861.0 3.8 Fold changes in cerebellum 1.0 0 10.3 24.3 6.4 18.8 0.1

Intravenous administration of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) particles in Pvalb cKO animals resulted in hFXN expression compared to WT endogenous FXN levels 4 weeks after delivery in tissue collected from the lumbar DRG. Increase 2.8-6.4×. Intravenous administration of VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles in Pvalb cKO animals increased hFXN performance by 0.8-2.6× 4 weeks after delivery.

Intravenous administration of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) particles in Pvalb cKO animals demonstrated increased endogenous FXN content compared to WT at 4 weeks post-delivery in tissue collected from the cerebellum. 10.3-24.3×. Intravenous administration of VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles in Pvalb cKO animals resulted in a 6.4-18.8× increase in endogenous FXN content compared to WT at 4 weeks post-delivery.

Taken together, the high expression of hFXN in the cerebellar and lumbar DRG of Pvalb cKO animals indicates that VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles are capable of transduction Such organizations. Example 14. Correction of proprioceptive deficiency and rescue of motor function and muscle function in Pvalb cKO mice after intravenous administration of VOY101-CMV-D7-hFXN or VOY101-CBA-D8-hFXN AAV particles .

In order to evaluate the therapeutic efficacy of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) vector, 6.32×10 ¹² VG/kg or 2.0×10 ¹³ Rescue of proprioceptive deficiency in Pvalb cKO mice was tested by electromyography analysis after intravenous (IV) delivery of these vectors at doses of VG/kg.

Single-stranded VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles are purified and formulated in a solution containing 0.001% Pluronic acid (Pluronic F- 68) and 192mM sodium chloride, 2.7mM potassium chloride, 2mM potassium phosphate (potassium dihydrogenphosphate) and 10mM sodium phosphate (disodium hydrogenphosphate) at pH 7.4, and administered via intravenous injection at 6.32× A single dose of 10 ¹² VG/kg or 2.0×10 ¹³ VG/kg was administered to 7.5-week-old adult Pvalb cKO mice. The control group received intravenous injection of AAV9-hFXN particles at a dose of 7×10 ¹² VG/kg.

Electromyography analysis was performed using a Natus UltraProS100 device (Mag2Health, France). Pvalb cKO mice were anesthetized using IP injection of ketamine/xylazine (130/13 mg/kg). Animals were maintained at 37°C throughout electrophysiological assessments. After sciatic nerve stimulation (0.1 ms and 8 mA intensity), the latency and amplitude of spinal somatosensory evoked responses (H waves) in the hindpaw plantar muscles were recorded. Depending on the age of the mice, EMG analysis was performed by recording spinal somatosensory evoked responses (H waves) every one, two or three weeks starting at 6.5 weeks of age according to Piguet et al. 2018.

As shown in Figure 4, the H-wave intensity in 6.5-week-old Pvalb cKO mice was significantly reduced compared to WT mice. In 8.5-week-old cKO mice, H-wave levels were no longer measurable one week after injection. In contrast, intravenous injection of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles within one week of administration resulted in almost complete recovery of H-wave intensity to levels comparable to those seen in WT animals, indicating reversal of the proprioceptive deficit in Pvalb cKO mice. Complete rescue was observed after 4 weeks of administration with either AAV particle at a high dose of 2.0×10 ¹³ VG/kg.

These results demonstrate the dose-dependent effects of IV VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY-CBA-D8-hFXN (SEQ ID NO: 1797) on spinal somatosensory evoked responses, especially at 11.5 weeks IV doses ranging from 6.32×10 ¹² VG/kg to 2.00×10 ¹³ VG/kg in aged mice.

To test the rescue of locomotor and muscle function in Pvalb cKO animals following intravenous delivery of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) particles, 7.5 , 8.5, 9.5 and 11.5 weeks old cKO animals were subjected to the notched stick test (scoring the number of upper or lower limbs that slide to “drop”) as previously described (Piguet et al. 2018). VOY101 particles were administered by IV injection into cKO animals (7.5 weeks old) as described above.

As shown in Figure 5, Pvalb cKO mice rapidly developed ataxia defects from 7.5 to 11.5 weeks. In comparison, VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101CBA-D8-hFXN (SEQ ID NO: 1797) was administered intravenously at a dose of 6.3×10 ¹² or 2.0×10 ¹³ VG/kg. ) particles rapidly reversed the ataxia phenotype one week after injection. The rescue effect persisted in cKO animals 4 weeks after treatment. Example 15. In vivo hFXN expression and vector genome biodistribution following intravenous administration of VOY101-CMV-D7 or VOY101-CBA-D8-hFXN AAV particles in non-human primates (NHP)

Fragments containing two AAV2 ITRs, promoter (CMV-D7 or CBA-D8), hFXN cDNA sequence, miR-122 target trinucleotide repeat sequence, human growth hormone polyA sequence, and human albumin as filler sequences Single-stranded viral genomes were encapsulated into VOY101 capsids, generated by triple transfection, purified and formulated in phosphate-buffered saline (PBS) containing 0.001% F-68.

Three groups of adult cynomolgus macaques (3 animals per group), approximately 2.7 to 5.8 years old, prescreened for low anti-AAVVoy101 antibodies using LEC2 nAb in vitro assay and IgG ELISA will be administered at a dose of 5×10 ¹³ VG/kg (titer 1.0×10 ¹³ VG/ml) received vehicle (0.001% F-68 in PBS), VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797). The injection rate will be 3 ml/min, and the dose volume will be 5 mL/kg (approximately 17.5 ml/animal).

To test the distribution and performance of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) in NHPs, any test known in the art can be utilized. hFXN protein and mRNA expression will be evaluated by ELISA, PCR, immunohistochemistry, in situ hybridization (ISH) and liquid chromatography combined with mass spectrometry (LC-MS/MS). PCR and ISH will be used to quantify the vector genome content in different tissues.

The following samples can be collected: cervical, thoracic and/or lumbar DRG, cervical, thoracic and/or lumbar spinal cord, frontal cortex, motor cortex, hippocampus, striatum, cerebellum, brainstem, liver, heart, atrium, Ventricles. Tissues from the DRG, ventricles, lower lumbar spinal cord, upper lumbar spinal cord, frontal cortex, cerebellum, and liver will be used to determine hFXN expression in target tissues using ELISA, LC-MS, and digital droplet PCR to evaluate the vector genome/diploidy Cells were quantified. The expression and distribution of hFXN will be measured by in situ hybridization (ISH) in cells of the DRG, cerebellar dentate nucleus, Clark's column, gracilis fasciculus and cuneate nucleus, which are cells of the heart. The data obtained will be used to confirm the expected tissue biodistribution of VOY101-CMV-D7-hFXN (SEQ ID NO: 1801) or VOY101-CBA-D8-hFXN (SEQ ID NO: 1797) AAV particles in non-human primates and Expression of transgenic gene hFXN. Example 16. In vivo promoter selection studies in non-human primates (NHP)

Selected AAV particles with viral genomes containing promoter variant sequences will be tested in non-human primates to determine syntaxin expression in target cells and tissues. hFXN ITR to ITR constructs selected from Table 4 will be used, including hFXN2 (SEQ ID NO: 1797), hFXN3 (SEQ ID NO: 1798), hFXN6 (SEQ ID NO: 1801), hFXN7 (SEQ ID NO: 1802) , hFXN13 (SEQ ID NO: 1808) and hFXN14 (SEQ ID NO: 1809).

AAV particles including selected hFXN ITR to ITR constructs in the VOY101 capsid will be produced by triple transfection using mammalian HEK293 cells according to the present invention. The resulting AAV particles will be purified and subsequently formulated into Formulation 1 (192 mM sodium chloride, 2.7 mM potassium chloride, 2 mM potassium phosphate (potassium dihydrogen phosphate), and 10 mM sodium phosphate (disodium hydrogen phosphate), containing 0.001 % Plonic acid (Plonic F-68) and pH 7.4).

A group of juvenile cynomolgus macaques (Chinese origin, pre-screened for low anti-AAVVoy101 antibodies using NAb assay) will be selected for each hFXN ITR to ITR construct tested (two females and one male per group). Cynomolgus macaques will receive AAV particles via intravenous injection (saphenous vein) according to the test parameters of Table 28. Table 28. Study design for testing hFXN promoter variants hFXN ITR to ITR construct SEQ ID NO: AAV dose (vg/kg) Duration ( days) Dosing regimen (ml/kg/h) hFXN2 1797 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 hFXN3 1798 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 hFXN6 1801 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 htK 1802 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 hFXN13 1808 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 hFXN14 1809 2x10 ¹³ 30 5 ml/kg over 1 hour 90 6.32x10 ¹² 30 90 Preparation 1 (enzyme) 0 90 5 ml/kg over 1 hour

The resulting distribution and performance of selected hFXN constructs will be tested in a variety of tissues, including one or more of the following: cervical DRG, thoracic DRG, lumbar DRG, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, frontal spinal cord Lobe cortex, motor cortex, hippocampus, striatum, cerebellum, brainstem, liver, heart, atria and ventricles.

The following primary reads will be measured and collected from test individuals: complete blood count (CBC), serum chemistry, serum interleukin protein, cage side observation, body weight, AAV vector genome (VG) distribution, hFXN protein distribution and hFXN protein expression. These primary reads will be tested and measured according to various methods known in the art, including ELISA, PCR, immunohistochemistry, in situ hybridization (ISH) and liquid chromatography coupled with mass spectrometry (LC). -MS/MS). Example 17. Testing of promoter constructs with alternative capsids

Viral genomes containing selected promoter variant constructs from Table 4 (e.g., hFXN2; SEQ ID NO: 1797, hFXN6; SEQ ID NO: 1801) will be incorporated into AAV particles with alternative capsids selected from Table 1 . Such capsids may have suboptimal or atypical initiation codons for translation of VP1, such as (but not limited to) CTG. Such AAV particles will be used to deliver fataxin to target cells in mice via intravenous injection via the tail vein. Twenty-three male C57BL/6J mice will be administered the AAV particle composition at a dose of 2×10 ¹³ VG/kg. Fourteen days after delivery, mice will be transcardially perfused with cold 1×PBS and tissue samples will be collected.

Any or all of the following tissue samples will be collected. Cervical, thoracic and/or lumbar DRG, cervical, thoracic and/or lumbar spinal cord, frontal cortex, motor cortex, hippocampus, striatum, cerebellum, brainstem, liver, heart, atrium, ventricle and/or gastrocnemius . ELISA will be performed using tissues from the lumbar DRG, ventricles, lower lumbar spinal cord, frontal cortex, and cerebellum to determine fataxin expression in target tissues. Digital droplet PCR will be performed using thoracic DRG, ventricles, upper lumbar spinal cord, frontal cortex, cerebellum and/or liver to quantify vector genomes/diploid cells. The resulting data will be used to confirm expected tissue biodistribution and transduction. It is expected that use of alternative capsids will result in similar, if not better, biodistribution and transduction as VOY101, VOY201 and/or AAV9 or variants thereof. Example 18. In vivo promoter selection studies in rats

Selected AAV particles with viral genomes containing promoter variant sequences were tested in rats to determine syntaxin expression in target cells and tissues. The following hFXN ITR to ITR constructs selected from Table 4 were used: hFXN2 (SEQ ID NO: 1797), hFXN10 (SEQ ID NO: 1805), hFXN11 (SEQ ID NO: 1806), hFXN12 (SEQ ID NO: 1807), hFXN13 (SEQ ID NO: 1808), hFXN14 (SEQ ID NO: 1809) and hFXN15 (SEQ ID NO: 1810). AAV particles including selected hFXN ITR to ITR constructs in the VOY101 capsid were produced by triple transfection using mammalian HEK293 cells in accordance with the present invention. The resulting AAV particles were purified and subsequently formulated to contain 0.001% Plonic acid (Plonic F-68) and a pH of 7.4 in 192 mM sodium chloride, 2.7 mM potassium chloride, and 10 mM sodium phosphate (disodium hydrogen phosphate )middle.

AAV particles were administered via intravenous delivery via the tail vein to male Spoogdock rats (5 per group) at one of two doses: 6.3×10 ¹² vg/kg or 2×10 ¹³ vg/ kg (5 ml/kg over 1 hour). At 28 days after administration, tissue samples (ventricular and DRG) were collected and analyzed for the amount of fataxin expression based on the anti-fataxin SimpleStep ELISA. The data are presented in Figures 6A, 6B, 6C and 6D.

hFXN13 (CBA.D4), hFXN14 (CBA.D6) and hFXN2 (CBA.D8) are well tolerated at 6.3×10 ¹² vg/kg or 2×10 ¹³ vg/kg, compared with hFXN10, hFXN11, hFXN12 and hFXN15 have low expression levels in DRG and ventricular tissue. Example 19. In vitro assessment of promoter variants

Seven promoter variant constructs, including hFXN2 (SEQ ID NO: 1797), hFXN6 (SEQ ID NO: 1801), hFXN10 (SEQ ID NO: 1805), hFXN11 (SEQ ID NO: 1806), hFXN12 (SEQ ID NO: 1807), hFXN13 (SEQ ID NO: 1808) and hFXN14 (SEQ ID NO: 1809). CMV and CBA promoter constructs were used as controls. Cells were transfected with plasmids containing one of the hFXN constructs or a control and a luciferase payload. Determine luciferase activity and performance using a luciferase assay system. 239T cells were used as negative control.

The activity of the promoter variants as determined by luciferase performance is shown in Figure 7. X. Equivalents and Scope

Using no more than routine experimentation, those skilled in the art will be able to ascertain many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the above description, but rather as set forth in the appended claims.

In the scope of the claim, articles such as "a/an" and "the" may mean one or more than one unless indicated to the contrary or otherwise apparent from the context. Unless indicated to the contrary or otherwise apparent from the context, a term is included in a group if one, more than one, or all of the group members are present in, used in, or otherwise associated with a given product or process. Claims or descriptions that include "or" between or among multiple members are deemed to be satisfied. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The invention includes embodiments in which more than one or all of the group members are present in, used in, or otherwise associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and permits, but does not require, the inclusion of additional elements or steps. When the term "comprises" is used herein, it also encompasses and discloses the term "consisting of."

When a range is given, include the endpoints. Furthermore, it is to be understood that values expressed as ranges may be employed in various embodiments of the invention at any particular value within the stated ranges, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art. or subrange, unless the context clearly requires otherwise, one-tenth of the unit up to the lower end of that range.

In addition, any specific embodiment of the present invention that belongs to the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are believed to be known to those of ordinary skill in the art, they are excluded, even if such exclusion is not expressly set forth herein. For any reason, whether or not related to the existence of prior art, any particular embodiment of a composition of the invention (e.g., any antibiotic, therapeutic, or active ingredient; any method of production; any method of use; etc.) may be derived from any one or more excluded from requests.

It is to be understood that the words used are words of description rather than limitation and that they may be used within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects. Make changes.

Although the invention has been described at some length and with some particularity with respect to several embodiments described, it is not intended that the invention be limited to any such details or embodiments or to any particular embodiment, but instead reference should be made to The accompanying claims are interpreted so as to provide the broadest possible interpretation of such claims taking into account the prior art and thereby effectively cover the intended scope of the invention.

The foregoing and other objects, features and advantages will be apparent from the following description of specific embodiments presented herein, as illustrated in the accompanying drawings. The drawings are not necessarily to scale but rather serve to illustrate the principles of the various embodiments described herein.

Figure 1A presents a graph showing the quantitative results of the expression amount of fataxin (ng/mg) obtained by ELISA and the AAV biodistribution (VG/DC) obtained by quantitative PCR for cardiac tissue. Figure 1B presents a graph showing quantitative results of fataxin expression amount (ng/mg) obtained by ELISA and AAV biodistribution (VG/DC) obtained by quantitative PCR for cerebellar tissue. Figure 1C presents a graph showing quantitative results of fataxin expression (ng/mg) by ELISA and AAV biodistribution (VG/DC) by quantitative PCR for dorsal root ganglia (DRG). Figure 1D presents a graph showing the quantitative results of the expression amount of fataxin (ng/mg) obtained by ELISA and the AAV biodistribution (VG/DC) obtained by quantitative PCR for liver tissue.

Figure 2 presents a graph showing quantitative results of striatal cFXN protein content by ELISA for certain promoter constructs of the invention.

Figure 3A presents a graph showing the quantitative results of the expression amount (ng/mg) of fataxel protein obtained by ELISA for lumbar DRG tissue. Figure 3B presents a graph showing the quantitative results of the expression amount (ng/mg) of fataxel protein obtained by ELISA for cerebellar tissue.

Figure 4 presents electromyograms ( H-wave intensity) measurement chart.

Figure 5 presents a presentation showing the trans-notch performance of Pvalb cKO mice treated intravenously with VOY101-CMV-D7-hFXN or with VOY101-CBA-D8-hFXN AAV particles compared to Pvalb cKO mice and wild-type (WT) mice. Chart of behavioral analysis of strip test.

Figure 6A presents a graph showing the quantitative results of the expression amount (ng/mg) of syntaxin obtained by ELISA for a certain DRG tissue of the present invention. Figure 6B presents the results of Figure 6A for hFXN13 (CBA.D4) with SEQ ID NO: 1808, hFXN14 (CBA.D6) with SEQ ID NO: 1809 and hFXN2 (CBA.D8) with SEQ ID NO: 1797 Expanded plot of quantitative results.

Figure 6C presents a graph showing the quantitative results of the expression amount (ng/mg) of fataxel protein obtained by ELISA for a certain ventricular tissue of the present invention. Figure 6D presents the results of Figure 6C for hFXN13 (CBA.D4) with SEQ ID NO: 1808, hFXN14 (CBA.D6) with SEQ ID NO: 1809 and hFXN2 (CBA.D8) with SEQ ID NO: 1797 Expanded plot of quantitative results.

Figure 7 presents a graph showing the quantitative results of syntaxin expression by luciferase expression (FXN:luciferase ratio) for the promoter construct of the present invention.

Claims (7)

An adeno-associated virus (AAV) vector genome comprising the nucleotide sequence of SEQ ID NO: 1797. The AAV vector genome of claim 1, wherein the AAV vector genome consists of SEQ ID NO: 1797. An adeno-associated virus (AAV) comprising the AAV vector genome and capsid of claim 1 or 2. Such as the AAV of claim 3, wherein the capsid is an AAV5 variant capsid. Such as the AAV of claim 3, wherein the capsid is an AAV9 variant capsid. A pharmaceutical composition comprising the AAV according to any one of claims 3 to 5. A use of the pharmaceutical composition of claim 6 for the manufacture of a medicament for the treatment of Friedreich's ataxia or a disorder associated with reduced fataxin content in an individual.