Conjugate protein expression constructs with engineered promoters and methods of use thereof

Abstract

The present disclosure relates to compositions and methods for altering, for example, enhancing the expression of FXN in vitro and / or in vivo, including but not limited to the development of engineered promoters. Such compositions include delivery by administration of adeno-associated virus (AAV) particles. The compositions and methods of the present disclosure are used to treat subjects diagnosed with or suspected of having Friedreich's Ataxia or another neuromuscular or neurological condition caused by a deficiency in the amount and / or function of FXN or associated with decreased expression or protein levels of FXN.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 738,519, filed on September 28, 2018, entitled “Collamiin Compositions and Methods of Use Thereof,” and U.S. Provisional Patent Application No. 62 / 901,769, filed on September 17, 2019, entitled “Collamiin Expression Constructs Having Engineered Promoters and Methods of Use Thereof,” the contents of which are incorporated herein by reference in their entireties.

[0003] Reference Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing file is named 20571019PCTSEQLST.txt, was created on September 27, 2019, and is 6,732,273 bytes in size. The electronic format information of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to compositions and methods based on frataxin, which relate to enhancing the expression of frataxin (FXN) in vitro or in vivo at least in part by employing novel engineered promoters. Such frataxin-based compositions can be delivered in adeno-associated virus (AAV) vectors. In other embodiments, frataxin-based compositions, such as AAV-frataxin compositions, are used to treat subjects in need thereof, such as human subjects diagnosed with Friedreich's Ataxia or other neurological conditions caused by insufficient amount and / or function of frataxin, or as research tools for studying diseases or conditions in cell or animal models of such diseases or conditions. Background Art

[0006] Friedreich's ataxia, first described in the 1860s by German physician Nikolas Friedreich, is an autosomal recessive genetic disease 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 herein by reference in their entirety. Onset usually occurs during adolescence and is almost always at the age of 25. See Campuzano et al., Science, 271.5254 (March 8, 1996): 1423, the contents of which are incorporated herein by reference in their entirety. FA is usually caused by degeneration of nerve tissue in the spinal cord due to reduced expression of the mitochondrial protein frataxin (FXN) in sensory neurons that direct arm and leg muscle movement (through connections to the cerebellum). See Koeppen, Arnulf; J Neurol Sci., 2011, April 15; 303(1-2): 1–12, the contents of which are incorporated herein by reference in their entirety. The spinal cord thins, and peripheral nerve cells lose parts of their myelin sheath, which is an insulating covering on certain nerve cells that helps carry nerve impulses. The initial symptoms of FA include poor coordination, such as gait disturbance, poor balance, leg weakness, slowed walking, impaired coordination, dysarthria, 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 usually progresses until a wheelchair is required for mobility. The incidence of FA in the Caucasian population ranges from approximately 1 in 20,000 to approximately 1 in 50,000, and the estimated carrier frequency in the European population is approximately 1 in 120. See Nageshwaran and Festenstein, Frontiers in Neurology, Vol. 6, Art. 262 (2015); Campuzano et al., Science, 271.5254 (March 8, 1996): 1423, the contents of which are incorporated herein by reference in their entirety.

[0007] The expansion of the GAA triplet repeat inherent in the FXN gene is the genetic cause of reduced expression of the fraxin FA. See Parkinson et al., Journal of Neurochemistry, 2013, 126 (Suppl. 1), 103-117. Over time, deficiency leads to the aforementioned symptoms, as well as frequent fatigue due to its effects on cellular metabolism.

[0008] Sclerosis and degeneration are most common in the dorsal root ganglia, spinocerebellar tract, lateral corticospinal tract, and posterior column. See Sandi et al., Frontiers in Genetics, Vol. 5, Art. 165 (June 2014), the contents of which are incorporated herein by reference in their entirety.

[0009] Progressive destruction of the dorsal root ganglion leads to dorsal root thinning, dorsal column degeneration, transsynaptic atrophy of neurons in Clarke's columns and dorsal spinocerebellar fibers, atrophy of the gracile and cuneate nuclei, and sensory neuropathies. See Koeppen, Arnulf; J Neurol Sci., 2011, Apr 15;303(1-2):1–12, the contents of which are incorporated herein by reference in their entirety. Lesions in the dentate nucleus consist of progressive and selective atrophy of large glutamatergic neurons and grumose degeneration of gamma-aminobutyric acid (GABA)-containing corticonuclear synaptic terminals. Small GABAergic neurons and their projections in the dentato-olivary tract survive. Atrophy of Betz cells and the corticospinal tract constitutes a secondary lesion. Currently, there is no effective treatment for FA, and patients are typically only monitored for symptom management.

[0010] Therefore, there remains a long felt need in the art to develop pharmaceutical compositions and methods for treating FXN-related disorders and alleviating protein deficiency in patients suffering from FA.

[0011] Adeno-associated virus (AAV) has become one of the most widely studied and utilized viral particles for delivering therapeutically effective polypeptides to mammalian cells. See, for example, Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999), the contents of which are incorporated herein by reference in their entirety. Thus, this approach is well suited for developing treatments for FA and for the delivery of frataxin and frataxin-related proteins and peptides. Summary of the Invention

[0012] In some aspects, the present disclosure provides an AAV viral genome comprising at least one inverted terminal repeat (ITR) and a payload region, wherein the payload region encodes a comitatin. In some embodiments, the AAV viral genome comprises a 5'ITR, an engineered promoter, a payload region, and a 3'ITR. The encoded comitatin can be a human (Homo sapiens) comitatin, a cynomolgus monkey (Macaca fascicularis) comitatin, or a common macaque (rhesus monkey) (Macaca mulatta) comitatin, a synthetic (non-naturally occurring) comitatin, or a derivative thereof, for example, a variant that retains one or more functions of a wild-type comitatin. In some embodiments, the comitatin can be at least partially humanized.

[0013] The engineered promoter of the AAV viral genome is derived from the cytomegalovirus (CMV) promoter, the chicken β-actin (CBA) promoter, or the frataxin (FXN) promoter. In some embodiments, the engineered promoter is a promoter variant or a derivative of a parent promoter sequence.

[0014] In some embodiments, the engineered promoter is derived from a CMV promoter.

[0015] In some embodiments, the engineered promoter is derived from a CBA promoter.

[0016] In some embodiments, the engineered promoter is derived from the FXN promoter.

[0017] The engineered promoter of the AAV viral genome as described herein may comprise a sequence as set forth in any one of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter may consist of any one of SEQ ID NOs: 1734-1777. In some embodiments, the engineered promoter is derived from a CMV promoter and may comprise a sequence as set forth in any one 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 the CBA promoter and may comprise a sequence as set forth in any one 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 may comprise a sequence as set forth in any one of SEQ ID NOs: 1752-1759 and 1769-1770.

[0018] In some embodiments, the engineered promoter comprises a sequence as set forth in SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1738. In some embodiments, the engineered promoter consists of SEQ ID NO: 1738.

[0019] In some embodiments, the engineered promoter comprises a sequence as set forth in SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1740. In some embodiments, the engineered promoter consists of SEQ ID NO: 1740.

[0020] In some embodiments, the engineered promoter comprises a sequence as set forth in SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1742. In some embodiments, the engineered promoter consists of SEQ ID NO: 1742.

[0021] In some embodiments, the engineered promoter comprises a sequence as set forth in SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1750. In some embodiments, the engineered promoter consists of SEQ ID NO: 1750.

[0022] In some embodiments, the engineered promoter comprises a sequence as set forth in SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1756. In some embodiments, the engineered promoter consists of SEQ ID NO: 1756.

[0023] As described herein, the engineered promoter can have a length of 50-1400 nucleotides (nt). In some embodiments, the engineered promoter is derived from the CMV promoter and is 50-700nt in length. In some embodiments, the engineered promoter is derived from the CMV promoter and is 109nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 100-700nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 100-400nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 100nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 200-350nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 260nt in length. In some embodiments, the engineered promoter is derived from the CBA promoter and is 332nt in length. In some embodiments, the engineered promoter is derived from the FXN promoter and is 200-1400 nt in length. In some embodiments, the engineered promoter is 950-1150 nt in length. In some embodiments, the engineered promoter is derived from the FXN promoter and is 1060 nt in length.

[0024] In some embodiments, the engineered promoter comprises an enhancer region.

[0025] An engineered promoter and a payload region encoding a frataxin protein can be incorporated into the AAV viral genome.

[0026] In some embodiments, in addition to the engineered promoter and payload region, the AAV viral genome comprises a 5'ITR, an enhancer, an intron, at least one miR binding site (e.g., one, two, or three miR binding sites), a polyA sequence, a stuffer sequence, and a 3'ITR. In some embodiments, the viral genome comprises multiple miR binding sites ("miR binding site array"), which may appear consecutively or separated by one or more nucleotides. In some embodiments, the 5'ITR and / or 3'ITR is an AAV2 ITR.

[0027] In some embodiments, the viral genome comprises at least one ITR sequence. In some embodiments, the ITR can 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' ITR 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.

[0028] In some embodiments, the sequence from ITR to ITR comprises an intron / exon region. In some embodiments, the intron / exon region can be an enhancer sequence. As a non-limiting example, the enhancer sequence may comprise two or more subcomponents, such as, but not limited to, ie1 exon (e.g., exon 1), ie1 intron (e.g., intron 1), human β-globin intron (e.g., intron 2) and / or human β-globin exon (e.g., 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 a sequence as given in any one 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 a sequence as given in any one 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 β-globin sequences, for example, a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1820 and / or 1821. In some embodiments, the intron may comprise a sequence as set forth in any one 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.

[0029] In some embodiments, the miR binding site series comprises at least one miR122 binding site sequence. In some embodiments, 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, 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, for example, three copies of SEQ ID NO: 1827 or a variant thereof having at least 90% sequence identity. In some embodiments, the miR binding site series may 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 miR binding site series may consist of SEQ ID NO: 1826.

[0030] 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.

[0031] In some embodiments, the AAV viral genome further comprises a filler sequence, for example, an albumin filler sequence. 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 a sequence as set forth in any one of SEQ ID NOs: 1829-1842. 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 SEQ ID NO: 1838. In some embodiments, the filler sequence may consist of SEQ ID NO: 1838. 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 SEQ ID NO: 1839. In some embodiments, the filler sequence may consist of SEQ ID NO: 1839. 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 SEQ ID NO: 1840. In some embodiments, the filler sequence may consist of SEQ ID NO: 1840. In some embodiments, the filler sequence may comprise a sequence 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.

[0032] In some embodiments, the AAV viral genome may comprise a sequence as set forth in any one of SEQ ID NOs: 1778-1810. In some embodiments, the AAV viral genome comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 1778-1810. In some embodiments, the AAV viral genome comprises a sequence having 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% sequence identity to any one of SEQ ID NOs: 1778-1810. The AAV viral genome wherein the encoded symtaxin is a cynomolgus sp. symtaxin may comprise a sequence as set forth in any one of SEQ ID NOs: 1778-1795. In some embodiments, the AAV viral genome comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 1778-1795. In some embodiments, the AAV viral genome comprises a sequence having 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% sequence identity to any one of SEQ ID NOs: 1778-1795. The AAV viral genome wherein the encoded frataxin is a human frataxin may comprise a sequence as set forth in any one of SEQ ID NOs: 1796-1810. In some embodiments, the AAV viral genome comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of SEQ ID NOs: 1796-1810. In some embodiments, the AAV viral genome comprises a sequence having 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% sequence identity to any one of SEQ ID NOs: 1796-1810.

[0033] 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: 1797. In some embodiments, the AAV viral genome may consist of SEQ ID NO: 1797. 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: 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 can consist of SEQ ID NO:1809.

[0034] In some embodiments, the payload region of the AAV vector genome encoding a symtaxin comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of the sequences set forth in SEQ ID NOs: 1822-1824. In some embodiments, the payload region of the AAV vector genome encoding a symtaxin comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 1822-1824. In some embodiments, the nucleic acid sequence encoding a symtaxin comprises SEQ ID NO: 1822. In some embodiments, the nucleic acid sequence encoding a symtaxin comprises SEQ ID NO: 1823. In some embodiments, the nucleic acid sequence encoding a symtaxin comprises SEQ ID NO: 1824. In some embodiments, the nucleic acid sequence encoding a symtaxin comprises a fragment of SEQ ID NOs: 1728, 1729, or 1730, or a variant thereof, having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In some embodiments, the nucleic acid sequence encoding a frataxin comprises a fragment of SEQ ID NO: 1728. In some embodiments, the nucleic acid sequence encoding a frataxin comprises nucleotides 221-853 of SEQ ID NO:1728.

[0035] In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding a frataxin polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1725, 1726, or 1727. In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding a frataxin polypeptide of SEQ ID NO: 1725, 1726, or 1727. In some embodiments, the AAV vector genome comprises a nucleic acid sequence encoding a frataxin polypeptide comprising SEQ ID NO: 1725. In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding a frataxin polypeptide having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1731, 1732, or 1733. In some embodiments, the payload region of the AAV vector genome comprises a nucleic acid sequence encoding a frataxin polypeptide of SEQ ID NO: 1731, 1732, or 1733.

[0036] The viral genome comprising an engineered promoter or promoter variant can be incorporated into an AAV particle, wherein the AAV particle comprises a viral genome and a capsid. In some embodiments, the capsid comprises a sequence as shown 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 an amino acid sequence of SEQ ID NO: 1 and / or a nucleic acid sequence of SEQ ID NO: 1722), and / or VOY201 (having an amino acid sequence of SEQ ID NO: 1724 and / or a nucleic acid sequence of SEQ ID NO: 1723). In some embodiments, the capsid is encoded by a nucleic acid sequence selected from SEQ ID NOs: 4, 135, 1722, and 1723. In some embodiments, the capsid may have an amino acid sequence as set forth in any one of SEQ ID NOs: 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 the 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 the 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 the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1722. In some embodiments, the capsid comprises the 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.

[0037] In some embodiments, the AAV particles described herein can be used in pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188. In some embodiments, the pharmaceutical composition comprises 192mM sodium chloride, 10mM sodium phosphate, 2.7mM potassium chloride, 2mM potassium phosphate, and 0.001% poloxamer 188 (v / v). In some embodiments, the sodium phosphate of the composition is dibasic. In some embodiments, the potassium phosphate of the composition is monobasic. In some embodiments, the pH of the pharmaceutical composition is between 7.3-7.7. In some embodiments, the pH of the pharmaceutical composition is 7.4.

[0038] In some embodiments, the AAV particles can comprise a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1797. In some embodiments, a pharmaceutical composition comprising AAV particles comprising a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1797 comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188; optionally, wherein the pharmaceutical composition comprises 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 pH of the composition is 7.4.

[0039] In some embodiments, the AAV particles can comprise a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1801. In some embodiments, a pharmaceutical composition comprising AAV particles comprising a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1801 comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188; optionally, wherein the pharmaceutical composition comprises 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 pH of the composition is 7.4.

[0040] In some embodiments, the AAV particles can comprise the vector genome set forth in SEQ ID NO: 1808 and a VOY101 capsid. In some embodiments, a pharmaceutical composition comprising AAV particles comprising the vector genome set forth in SEQ ID NO: 1808 and a VOY101 capsid comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188; optionally, wherein the pharmaceutical composition comprises 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 pH of the composition is 7.4.

[0041] In some embodiments, the AAV particles can comprise a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1809. In some embodiments, a pharmaceutical composition comprising AAV particles comprising a vector genome and a VOY101 capsid as set forth in SEQ ID NO: 1809 comprises sodium chloride, sodium phosphate, potassium chloride, potassium phosphate, and poloxamer 188; optionally, wherein the pharmaceutical composition comprises 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 pH of the composition is 7.4.

[0042] The pharmaceutical compositions and / or AAV particles of the present disclosure can be used to treat neurological or neuromuscular disorders, such as, but not limited to, Friedreich's ataxia.

[0043] In some embodiments, the AAV particles of the present disclosure are used to treat disorders or conditions associated with reduced commissurin expression or protein levels. In some embodiments, the disorders or conditions associated with reduced commissurin expression or protein levels are neurological or neuromuscular disorders. In some embodiments, the disorders or conditions associated with reduced commissurin protein levels are FA or commissurin deficiency. In some embodiments, the administration of AAV particles can result in enhanced commissurin expression in target cells to 0.5-3 times (0.5-3x) (e.g., 0.5-1 times, 1-1.5 times, 1.5-2 times, 2-2.5 times, 2.5-3 times) of the commissurin expression in equivalent target cells of normal subjects not suffering from disorders associated with reduced commissurin levels. In some embodiments, the administration of AAV particles can result in commissurin expression of approximately 5.5-32.8 ng / mg protein in target cells.

[0044] The details of various aspects or embodiments of the present disclosure are set forth below. Other features, objects, and advantages of the present disclosure will be apparent from the specification and claims. In the specification, the singular also includes the plural, unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. In the event of a conflict, the present specification shall prevail. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments presented herein, as illustrated in the accompanying drawings, which are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the various embodiments described herein.

[0046] Figure 1A Graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA and AAV biodistribution (VG / DC) measured by quantitative PCR for cardiac tissue are presented. Figure 1B Graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA and AAV biodistribution (VG / DC) measured by quantitative PCR for cerebellar tissue are presented. Figure 1C Presented are graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA and AAV biodistribution (VG / DC) measured by quantitative PCR for dorsal root ganglia (DRG). Figure 1D Graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA and AAV biodistribution (VG / DC) measured by quantitative PCR for liver tissue are presented.

[0047] Figure 2 Presented are graphs showing the quantification of striatal cFXN protein levels as measured by ELISA for certain promoter constructs of the present disclosure.

[0048] Figure 3A Shown are graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA for lumbar DRG tissues. Figure 3B Shown are graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA for cerebellar tissues.

[0049] Figure 4Shown are graphs showing electromyographic (H-wave intensity) measurements of Pvalb cKO animals treated intravenously with VOY101-CMV-D7-hFXN or with VOY101-CBA-D8-hFXN AAV particles compared to Pvalb cKO mice and wild type (WT).

[0050] Figure 5 Graphs are presented showing behavioral analysis by notched-bar test 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).

[0051] Figure 6A Presented are graphs showing the quantitative results of frataxin expression levels (ng / mg) measured by ELISA for certain DRG tissues of the present disclosure. Figure 6B Shown Figure 6A Expanded graph of the quantification results for hFXN13 (CBA.D4) having SEQ ID NO: 1808, hFXN14 (CBA.D6) having SEQ ID NO: 1809, and hFXN2 (CBA.D8) having SEQ ID NO: 1797.

[0052] Figure 6C Presented are graphs showing the quantitative results of frataxin expression levels (ng / mg) as measured by ELISA for certain cardiac ventricular tissues of the present disclosure. Figure 6D Shown Figure 6C Expanded graph of the quantification results for hFXN13 (CBA.D4) having SEQ ID NO: 1808, hFXN14 (CBA.D6) having SEQ ID NO: 1809, and hFXN2 (CBA.D8) having SEQ ID NO: 1797.

[0053] Figure 7 Presented are graphs showing quantification of frataxin expression levels as measured by luciferase expression (FXN:luciferase ratio) for promoter constructs of the present disclosure. DETAILED DESCRIPTION

[0054] I. Composition

[0055] Adeno-associated virus (AAV) vectors

[0056] The virus of the Parvoviridae family is a small non-enveloped icosahedral capsid virus characterized by a single-stranded DNA genome. The Parvoviridae family virus consists of two subfamilies: the Parvovirinae subfamily that infects vertebrates, and the Densovirinae subfamily that infects invertebrates. Due to the relatively simple structure that can be manipulated with standard molecular biology techniques, this virus family can be used as a biological tool. The genome of the virus can be modified to contain the minimum components for assembling functional recombinant viruses or viral particles, which are loaded with or engineered to express or deliver the desired nucleic acid construct or payload, for example, transgenic, polynucleotides encoding polypeptides or FXN, which can be delivered to target cells, tissues or organisms. In some embodiments, the target cell is a CNS cell. In some embodiments, the target tissue is a CNS tissue.

[0057] Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," FIELDS VIROLOGY (3rd ed. 1996), Chapter 69, the contents of which are incorporated herein by reference in their entirety.

[0058] The Parvoviridae family contains the Dependovirus genus, which includes adeno-associated viruses (AAVs) capable of replicating in vertebrate hosts, including but not limited to humans, primates, bovine, canine, equine, and ovine species.

[0059] Adeno-associated virus (AAV) is a kind of parvovirus (like other parvoviruses) that relies on, is a kind of single-stranded non-enveloped DNA virus, it has a genome of about 5000 nucleotides in length, and the genome contains two open reading frames encoding the protein (Rep) responsible for replication and the structural protein (Cap) of capsid. The flank of open reading frame is two inverted terminal repeat (ITR) sequences, which serve as the replication origin of viral genome. Wild-type AAV viral genome comprises the nucleotide sequence of two open reading frames, one for four kinds of non-structural Rep proteins (Rep78, Rep68, Rep52, Rep40 encoded by Rep gene), and one for three kinds of capsid or structural proteins (VP1, VP2, VP3 encoded by capsid gene or Cap gene). Rep albumen is important for replication and packaging, and capsid protein is then assembled to produce the protein shell or AAV capsid of AAV. Variable splicing and optional start codon and promoter result in producing four different Rep albumen from a single open reading frame, and produce three kinds of capsid proteins from a single open reading frame. Although it varies with the 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 herein by reference in their entirety), VP1 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. As a result, changes in sequence in the VP3 region are also changes in VP1 and VP2, however, the percentage difference compared to the parent sequence will be the largest for VP3 because it is the shortest of the three sequences. Although described herein with respect to amino acid sequences, the nucleic acid sequences encoding these proteins can be similarly described. These three capsid proteins assemble together to produce the AAV capsid proteins. Although not wishing to be bound by theory, the AAV capsid proteins typically comprise a molar ratio of VP1:VP2:VP3 of 1:1:10. As used herein, "AAV serotype" is primarily defined by the AAV capsid. In some cases, the ITRs are also specifically described by AAV serotype (eg, AAV2 / 9).

[0060] AAV vectors typically require a co-helper (e.g., adenovirus) to produce infection in infected cells. In the absence of such helper functions, AAV virions actually enter host cells but are not integrated into the genome of the cell. As used herein, the term "AAV vector" or "AAV particle" comprises a capsid and a viral genome comprising a polynucleotide payload. As used herein, "payload" or "payload region" refers to one or more polynucleotides or polynucleotide regions encoded by or within the viral genome or the expression product of such polynucleotides or polynucleotide regions, such as transgenes, polynucleotides encoding polypeptides or multiple polypeptides, such as FXN.

[0061] AAV vectors have been studied for delivery due to several unique characteristics. Non-limiting examples of characteristics include: (i) the ability to infect both dividing and non-dividing cells; (ii) a broad host range of infection, including human cells; (iii) wild-type AAV has not been associated with any disease and has not been shown to replicate in infected cells; (iv) the lack of a cell-mediated immune response against the vector, and (v) the non-integrative nature of AAV vectors in the host chromosome, thereby reducing the potential for long-term genetic changes. Furthermore, infection with AAV vectors has minimal effect on altering the pattern of cellular gene expression (Stilwell and Samulski et al., Biotechniques, 2003, 34, 148, the contents of which are incorporated herein by reference in their entirety).

[0062] Typically, AAV vectors used for FXN delivery can be replication-defective recombinant viral vectors, as they lack sequences encoding functional Rep and Cap proteins within the viral genome. In some cases, defective AAV vectors may lack most or all coding sequences and contain 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.

[0063] In one embodiment, the AAV particles of the present disclosure can be introduced into mammalian cells.

[0064] AAV vectors can be modified to enhance delivery efficiency. Such modified AAV vectors of the present disclosure can be efficiently packaged and used to successfully infect target cells with high frequency and minimal toxicity.

[0065] In other embodiments, the AAV particles of the present disclosure can be used to deliver FXN to the central nervous system (see, e.g., U.S. Patent No. 6,180,613; the contents of which are herein incorporated by reference in their entirety).

[0066] AAV serotypes

[0067] The AAV particles of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, the AAV particles can utilize or be based on a serotype or include a peptide selected from any of the following: 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- P.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-SG N. 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, AAV 9.47, 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、AA V223.7、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、AA V3-11 / 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、AAV2 9.3 / 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.5 3、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、AAVr h.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, AAVhEr!1.1!, AAVhEr1!1.18!, 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-LK1:8, 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 Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 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, AAV5!4.4!R / hu.27, AAV46.2 / hu.28 It should be noted that there seem to be some unclear or potentially incorrect notations in the original text (such as "AAVhEr!1.1!" and "AAV5!4.4!"), which are maintained as they are in the translation for the purpose of following the rules.AAV46.6 / hu.29, AAV128.1 / hu.43, True-to-form 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, AAVCBr-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, AAVCHt-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, AAVCKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAVCKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAVCKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAVCLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAVCLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAVCLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2AAV 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-AAV-AAV6 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-8 CLv-M9、AAVCLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAVCLv-R8、AAV-RAVCLv-AAV-1 CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAVCSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-2、AAV. CSp-8.4、AAVCSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、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, AAVF9 / HSC9, AAVF9 / HSC9

[0068] In some embodiments, the AAV serotype used in the compositions disclosed herein can be or comprise a sequence 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 NOs: 6 and 64 of US20030138772), AAV2 (SEQ ID NOs: 7 and 70 of US20030138772), AAV3 (SEQ ID NOs: 8 and 71 of US20030138772), AAV4 (SEQ ID NO: 63 of US20030138772), AAV5 (SEQ ID NO: 114 of US20030138772), AAV6 (SEQ ID NO: 65 of US20030138772), AAV7 (SEQ ID NOs: 1-3 of US20030138772), AAV8 (SEQ ID NOs: 1-4 of US20030138772), AAV9 (SEQ ID NOs: 2-3 of US20030138772), AAV10 (SEQ ID NOs: 3-4 of US20030138772), AAV11 (SEQ ID NOs: 4-5 of US20030138772), AAV12 (SEQ ID NOs: 5-6 of US20030138772), AAV13 (SEQ ID NOs: 6-7 of US20030138772), AAV14 (SEQ ID NO: 6 ID NO: 4 and 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). 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(US20030138772SEQ 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(US20030138772SEQ ID NO:25)、AAVH2(US20030138772 SEQ ID NO:26)、AAV42-8(US20030138772 SEQ ID NO:27)、AAV42-15(US20030138772 SEQ ID NO:28)、AAV42-5b(US20030138772 SEQ ID NO:29)、AAV42-1b(US20030138772SEQ ID NO:30)、AAV42-13(US20030138772 SEQ ID NO:31)、AAV42-3a(US20030138772 SEQ ID NO:32)、AAV42-4(US20030138772 SEQ ID NO:33)、AAV42-5a(US20030138772 SEQ ID NO:34)、AAV42-10(US20030138772SEQ ID NO:35)、AAV42-3b(US20030138772 SEQ ID NO:36)、AAV42-11(US20030138772 SEQ ID NO:37)、AAV42-6b(US20030138772 SEQ ID NO:38)、AAV43-1(US20030138772 SEQ ID NO:39)、AAV43-5(US20030138772SEQ 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(US20030138772SEQ 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), AAVA3.5 (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.

[0069] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Patent Application Publication No. US20150159173, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV2 (SEQ ID NOs: 7 and 23 of US20150159173), rh20 (SEQ ID NO: 1 of US20150159173), rh32 / 33 (SEQ ID NO: 2 of US20150159173), rh39 (SEQ ID NOs: 3, 20, and 36 of US20150159173), rh46 (SEQ ID NOs: 4 and 22 of US20150159173), rh73 (SEQ ID NO: 5 of US20150159173), rh74 (SEQ ID NO: 6 of US20150159173), rh82 (SEQ ID NO: 7 of US20150159173), rh91 (SEQ ID NO: 8 of US20150159173), rh92 (SEQ ID NO: 9 of US20150159173), rh93 (SEQ ID NO: 10 of US20150159173), rh94 (SEQ ID NO: 11 of US20150159173), rh95 (SEQ ID NO: 12 of US20150159173), rh96 (SEQ ID NO: 13 of US20150159173), rh97 (SEQ ID NO: 14 of US20150159173), rh98 (SEQ ID NO: 15 of US20150159173), rh99 (SEQ ID NO: 16 of US2015 NO:6), AAV6.1 (SEQ ID NO:29 of US20150159173), rh.8 (SEQ ID NO:41 of US20150159173), 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:41 of US20150159173), NO: 8 and 24), 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 28 of US20150159173), 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 NO: 16 and 32 of US20150159173), hu.26 (SEQ ID NO: 17 and 18 of US20150159173), ID NO: 17 and 33), 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: 43 of US20150159173), 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 NO: 49 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.

[0070] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Patent No. US 7198951, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV9 (SEQ ID NOs: 1-3 of US 7198951), AAV2 (SEQ ID NO: 4 of US 7198951), AAV1 (SEQ ID NO: 5 of US 7198951), AAV3 (SEQ ID NO: 6 of US 7198951), or AAV8 (SEQ ID NO: 7 of US 7198951), or variants or hybrids / chimeras / combinations thereof.

[0071] In some embodiments, the AAV serotype can be the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), the contents of which are incorporated herein by reference in their entirety), or can be a variant 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.

[0072] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Pat. No. 6,156,303, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV3B (SEQ ID NOs: 1 and 10 of U.S. 6,156,303), AAV6 (SEQ ID NOs: 2, 7, and 11 of U.S. 6,156,303), AAV2 (SEQ ID NOs: 3 and 8 of U.S. 6,156,303), AAV3A (SEQ ID NOs: 4 and 9 of U.S. 6,156,303), or derivatives or variants or hybrids / chimeras / combinations thereof.

[0073] In some embodiments, the AAV serotype can be or comprise a sequence as described in U.S. Patent Application Publication No. US20140359799, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV8 (SEQ ID NO: 1 of US20140359799), AAVDJ (SEQ ID NO: 2 and 3 of US20140359799), or variants thereof.

[0074] In some embodiments, the serotype can 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), which is incorporated herein by reference in its entirety). The amino acid sequence of AAVDJ8 can comprise two or more mutations to effectively 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 their entirety) can comprise two mutations: (1) R587Q, wherein the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (2) R590T, wherein the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr). As another non-limiting example, the AAV-DJ described in U.S. Pat. No. 7,588,772 may comprise three mutations: (1) K406R, in which the lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q, in which the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, in which the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0075] In some embodiments, the AAV serotype may be or comprise an AAV4 sequence as described in International Publication No. WO1998011244 (the contents of which are herein incorporated by reference in their entirety), such as but not limited to AAV4 (SEQ ID NOs: 1-20 of WO1998011244).

[0076] In some embodiments, the AAV serotype may be or comprise a mutation in the AAV2 sequence to generate AAV2G9 as described in International Publication No. WO2014144229, which is herein incorporated by reference in its entirety.

[0077] In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2005033321 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV3-3 (SEQ ID NO: 217 of WO2005033321), 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: 11 of WO2005033321), AAV127.2 / hu.41 (SEQ ID NO: 6 and 8 of WO2005033321), AAV128.3 / hu.44 (SEQ ID NO: 129 of WO2005033321), AAV129.1 / hu.50 (SEQ ID NO: 230 of WO2005033321), AAV130.2 / hu.51 (SEQ ID NO: 231 of WO2005033321), AAV131.3 / hu.52 (SEQ ID NO: 233 of WO2005033321), AAV132.4 / hu.53 (SEQ ID NO: 234 of WO2005033321), AAV133.5 / hu.54 (SEQ ID NO: 235 of WO2005033321), AAV134.6 / hu.55 (SEQ ID NO: 236 of WO2005033321), AAV135.7 / hu.56 (SEQ ID NO: 237 of WO2005033321), AAV136.8 / 3321), AAV145.1 / hu.53 (SEQ ID NOs: 176 and 177 of WO2005033321), AAV145.6 / hu.56 (SEQ ID NOs: 168 and 192 of WO2005033321), AAV16.12 / hu.11 (SEQ ID NOs: 153 and 57 of WO2005033321), AAV16.8 / hu.10 (SEQ ID NOs: 156 and 56 of WO2005033321), AAV161.10 / hu.60 (SEQ ID NO: 170 of WO2005033321), AAV161.6 / hu.61 (SEQ ID NOs: 168 and 192 of WO2005033321), AAV16.12 / hu.11 (SEQ ID NOs: 153 and 57 of WO2005033321), AAV16.8 / hu.10 (SEQ ID NOs: 156 and 56 of WO2005033321), AAV161.10 / hu.60 (SEQ ID NO: 170 of WO2005033321), AAV161.6 / hu.61 (SEQ ID NOs: 168 and 192 of WO2005033321), No: 174), 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 NOs: 211 and 221 of WO2005033321), AAV2-15 / rh.62 (SEQ ID NOs: 33 and 114 of WO2005033321), AAV2-3 / rh.61 (SEQ ID NO: 21 of WO2005033321), AAV2-4 / rh.50 (SEQ ID NOs: 23 and 108 of WO2005033321), AAV2-5 / rh.51 (SEQ ID NOs: 211 and 221 of WO2005033321), NO: 104 and 22), AAV3.1 / hu.6 (SEQ ID NO: 5 and 84 of WO2005033321), AAV3.1 / hu.9 (SEQ ID NOs: 155 and 58 of WO2005033321), AAV3-11 / rh.53 (SEQ ID NOs: 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 NOs: 96 and 18 of WO2005033321), AAV4-19 / rh.55 (SEQ ID NO: 190 of WO2005033321), AAV4-23 / rh.56 (SEQ ID NO: 231 of WO2005033321), AAV4-33 / hu.57 (SEQ ID NO: 233 of WO2005033321), AAV4-43 / hu.58 (SEQ ID NO: 234 of WO2005033321), AAV4-5 NO: 117), AAV4-4 (SEQ ID NOs: 201 and 218 of WO2005033321), AAV4-9 / rh.54 (SEQ ID NO: 116 of WO2005033321), AAV5 (SEQ ID NOs: 199 and 216 of WO2005033321), AAV52.1 / hu.20 (SEQ ID NO: 63 of WO2005033321), 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: 105 of WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 116 of WO2005033321), AAV5 3321), AAV58.2 / hu.25 (SEQ ID No: 49 of WO2005033321), AAV6 (SEQ ID NOs: 203 and 220 of WO2005033321), AAV7 (SEQ ID NOs: 222 and 213 of WO2005033321), AAV7.3 / hu.7 (SEQ ID No: 55 of WO2005033321), AAV8 (SEQ ID NOs: 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 NOs: 203 and 220 of WO2005033321), AAV7 (SEQ ID NOs: 222 and 213 of WO2005033321), AAV NO: 144), AAVhu.10 (SEQ ID NO: 156 of WO2005033321), AAVhu.11 (SEQ ID NO:153 of WO2005033321), AAVhu.12 (WO2005033321 SEQ ID NO:59), 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:134 of WO2005033321), 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:136 of WO2005033321), 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:145 of WO2005033321), 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 WO200503332), AAVhu.52 (SEQ ID NO: of WO2005033321), 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:192 of WO2005033321), 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 (SEQ ID NO:184 of WO2005033321), AAVhu.61 (SEQ ID NO:185 of WO2005033321), AAVhu. (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 (SEQ ID NO:28 of WO2005033321), AAVpi.2 (SEQ ID NO:30 of WO2005033321), AAVpi.3 (SEQ ID NO:29 of WO2005033321), AAVrh.38 (SEQ ID NO:86 of WO2005033321), AAVrh.40 (SEQ ID NO:92 of WO2005033321), AAVrh.43 (SEQ ID NO:163 of WO200503332), AAVrh.44 (SEQ ID NO:34 of WO2005033321), AAVrh.45 (SEQ ID NO:41 of WO2005033321), AAVrh.47 (SEQ ID NO:38 of WO2005033321), 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 (SEQ ID NO:104 of WO2005033321), AAVrh.52 (SEQ ID NO:96 of WO2005033321), AAVrh.53 (SEQ ID NO:97 of WO2005033321), AAVrh.55 (SEQ ID NO:37 of WO2005033321), 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 (SEQ ID NO:31 of WO2005033321), AAVrh.61 (SEQ ID NO:107 of WO2005033321), AAVrh.62 (SEQ ID NO: 114 of WO2005033321), AAVrh.64 (SEQ ID NO: 99 of WO2005033321), AAVrh.65 (SEQ ID NO: 35 of WO2005033321), AAVrh.68 (SEQ ID NO: WO2005033321 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 AAVrh 14. Non-limiting examples of variants include 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 AAVrh 14. Non-limiting examples of variants include SEQ ID NO:9) or variants thereof, including but not limited to AAVrh.1 NO:13,15,17,19,24,36,40,45,47,48,51,52,53,54,60,61,62,64,65,66,67,68,69,70,71,72,73,74,75,76,7 7, 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, 15 4, 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.

[0078] In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2015168666 (the contents of which are herein incorporated by reference in their entirety), such as, but not limited to, AAVrh8R (SEQ ID NO: 9 of WO2015168666), AAVrh8R A586R mutant (SEQ ID NO: 10 of WO2015168666), AAVrh8R R533A mutant (SEQ ID NO: 11 of WO2015168666), or variants thereof.

[0079] In some embodiments, the AAV serotype can be or comprise a sequence as described in U.S. Pat. No. 9,233,131 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAVhE1.1 (SEQ ID NO: 44 of U.S. Pat. No. 9,233,131), AAVhEr1.5 (SEQ ID NO: 45 of U.S. Pat. No. 9,233,131), AAVhER1.14 (SEQ ID NO: 46 of U.S. Pat. No. 9,233,131), AAVhEr1.8 (SEQ ID NO: 47 of U.S. Pat. No. 9,233,131), AAVhEr1.16 (SEQ ID NO: 48 of U.S. Pat. No. 9,233,131), AAVhEr1.18 (SEQ ID NO: 49 of U.S. Pat. No. 9,233,131), AAVhEr1.35 (SEQ ID NO: 50 of U.S. Pat. No. 9,233,131), AAVhEr1.7 (SEQ ID NO: 51 of U.S. Pat. No. 9,233,131), NO: 51), 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: 54 of US9233131), NO:59), AAV2.5T (SEQ ID NO:42 of US9233131) or variants thereof.

[0080] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Patent Application Publication No. US20150376607 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV-PAEC (SEQ ID NO: 1 of US20150376607), 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), NO:7), 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 of US20150376607 NO:11), 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 of US20150376607 NO:15), AAV-LK15 (SEQ ID NO:16 of US20150376607), AAV-LK16 (SEQ ID NO:17 of US20150376607), AAV-LK17 (SEQ ID NO:18 of US20150376607), AAV-LK18 (SEQ ID NO: US20150376607 NO: 19), AAV-LK19 (SEQ ID NO: 20 of US20150376607), AAV-PAEC2 (SEQ ID NO: 21 of US20150376607), AAV-PAEC4 (SEQ ID NO: 22 of US20150376607), AAV-PAEC6 (SEQ ID of US20150376607) NO: 23), AAV-PAEC7 (SEQ ID NO: 24 of US20150376607), AAV-PAEC8 (SEQ ID NO: 25 of US20150376607),NO: 25), AAV-PAEC11 (SEQ ID NO: 26 of US20150376607), AAV-PAEC12 (SEQ ID NO: 27 of US20150376607), or variants thereof.

[0081] In some embodiments, the AAV serotype can be or comprise a sequence as described in U.S. Pat. No. 9,163,261, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV-2-pre-miRNA-101 (SEQ ID NO: 1 US9163261 ), or a variant thereof.

[0082] In some embodiments, the AAV serotype can be or have a sequence as described in U.S. Patent Application Publication No. US20150376240 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV-8h (SEQ ID NO: 6 of US20150376240), 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.

[0083] In some embodiments, the AAV serotype can be or have a sequence as described in U.S. Patent Application Publication No. US20160017295 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV SM 10-2 (SEQ ID NO: 22 of US20160017295), AAV shuffled 100-1 (SEQ ID NO: 23 of US20160017295), AAV shuffled 100-3 (SEQ ID NO: 24 of US20160017295), AAV shuffled 100-7 (SEQ ID NO: 25 of US20160017295), AAV shuffled 10-2 (SEQ ID NO: 34 of US20160017295), AAV shuffled 10-6 (SEQ ID NO: 35 of US20160017295), AAV shuffled 10-8 (SEQ ID NO: 40 of US20160017295), AAV shuffled 10-9 (SEQ ID NO: 41 of US20160017295), AAV shuffled 10-10 (SEQ ID NO: 42 of US20160017295), AAV shuffled 10-11 (SEQ ID NO: 43 of US20160017295), AAV shuffled 10-12 (SEQ ID NO: 44 of US20160017295), AAV shuffled 10-13 (SEQ ID NO: 45 of US20160017295), AAV shuffled 10-14 (SEQ ID NO: 46 of US20160017295), AAV shuffled 10-1 ID NO: 36), AAV shuffled 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), AAVSM 100-3 (SEQ ID NO: 40 of US20160017295), AAV SM100-10 (SEQ ID NO: 41 of US20160017295), or variants thereof.

[0084] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Patent Publication No. US20150238550 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, BNP61 AAV (SEQ ID NO: 1 of US20150238550), BNP62 AAV (SEQ ID NO: 3 of US20150238550), BNP63 AAV (SEQ ID NO: 4 of US20150238550), or variants thereof.

[0085] In some embodiments, the AAV serotype may be or may comprise a sequence as described in U.S. Patent Publication No. US20150315612 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAVrh.50 (SEQ ID NO: 108 of US20150315612), 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: 164 of US20150315612), AAVrh.64 (SEQ ID NO: 165 of US20150315612), AAVrh.65 (SEQ ID NO: 166 of US20150315612), AAVrh.67 (SEQ ID NO: 167 of US20150315612), AAVrh.68 (SEQ ID NO: 169 of US20150315612), AAVhu.19 (SEQ ID NO: 134 of US20150315612), AAVhu.11 (SEQ ID NO: 168 of US20150315612), AAVhu.11 (SEQ ID NO: 169 of US20150315612), AAVhu.11 (SEQ ID NO: 167 of US20150315612), AAVhu NO: 186), AAV4-8 / rh.64 (SEQ ID NO: 15 of US20150315612), 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: 67 of US20150315612), 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 NO: 70 of US20150315612), AAV54.5 / hu.23 (SEQ ID NO: 60 of US20150315612), AAV54.2 / hu.22 (SEQ ID NO: 67 of US20150315612), AAV54.7 / hu.24 (SEQ ID NO: 66 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 ID NO:80 of US20150315612), or variants thereof.

[0086] In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2015121501 (the contents of which are herein incorporated by reference in their entirety), such as, but not limited to, authentic 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.

[0087] According to the present disclosure, the AAV capsid serotype selection or use can be from a variety of species. In one embodiment, the AAV can be an avian AAV (AAAV). The AAAV serotype can be or have a sequence as described in U.S. Patent No. US 9238800 (the contents of which are incorporated herein by reference in their entirety), such as but not limited to AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12 or 14 of US 9238800) or a variant thereof.

[0088] In one embodiment, AAV can be bovine AAV (BAAV). The BAAV serotype can be or have a sequence as described in U.S. Patent No. US 9,193,769 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, BAAV (SEQ ID NOs: 1 and 6 of US9193769) or a variant thereof. The BAAV serotype can be or have a sequence as described in U.S. Patent No. US7427396 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, BAAV (SEQ ID NOs: 5 and 6 of US7427396) or a variant thereof.

[0089] In some embodiments, the AAV may be a goat AAV. The goat AAV serotype may be or have a sequence as described in U.S. Pat. No. 7,427,396 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, goat AAV (SEQ ID NO: 3 of US7,427,396) or a variant thereof.

[0090] In some embodiments, the AAV can be a hybrid AAV engineered from two or more parental serotypes. In one embodiment, the AAV can be AAV2G9, which comprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype can be or have a sequence as described in U.S. Patent Application Publication No. US20160017005 (the contents of which are incorporated herein by reference in their entirety).

[0091] In certain embodiments, the AAV may be a serotype generated from an AAV9 capsid library having mutations in amino acids 390-627 (VP1 numbering), as described by 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 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, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T; Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.3 5(A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T4 50S), AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G48 1R, W509R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65 (C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), 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, T582S, D611V), AAV9.94 (A1675T; M559L) or AAV9.95 (T1605A; F535L).

[0092] In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2016049230 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAVF1 / HSC1 (SEQ ID NOs: 2 and 20 of WO2016049230), AAVF2 / HSC2 (SEQ ID NOs: 3 and 21 of WO2016049230), AAVF3 / HSC3 (SEQ ID NOs: 5 and 22 of WO2016049230), AAVF4 / HSC4 (SEQ ID NOs: 6 and 23 of WO2016049230), AAVF5 / HSC5 (SEQ ID NOs: 11 and 25 of WO2016049230), AAVF6 / HSC6 (SEQ ID NOs: 7 and 24 of WO2016049230), AAVF7 / HSC7 (SEQ ID NOs: 11 and 26 of WO2016049230), AAVF8 / HSC9 (SEQ ID NOs: 11 and 12 of WO2016049230), AAVF9 / HSC10 (SEQ ID NOs: 11 and 12 of WO2016049230), AAVF11 / HSC12 (SEQ ID NOs: 11 and 12 of WO2016049230), AAVF12 / HSC13 (SEQ ID NOs: 11 and 12 of WO2016049230), AAVF13 / HSC14 (SEQ ID NOs: 11 and 12 of WO2016049230), AAVF14 / HSC15 (SEQ ID NOs: 11 and 12 of WO201 8 and 27 of WO2016049230), AAVF8 / HSC8 (SEQ ID NOs: 9 and 28 of WO2016049230), AAVF9 / HSC9 (SEQ ID NOs: 10 and 29 of WO2016049230), AAVF11 / HSC11 (SEQ ID NOs: 4 and 26 of WO2016049230), AAVF12 / HSC12 (SEQ ID NOs: 12 and 30 of WO2016049230), AAVF13 / HSC13 (SEQ ID NOs: 14 and 31 of WO2016049230), AAVF14 / HSC14 (SEQ ID NOs: 15 and 32 of WO2016049230), AAVF15 / HSC15 (SEQ ID NOs: 16 and 17 of WO2016049230), AAVF16 / HSC17 (SEQ ID NOs: 17 and 18 of WO2016049230), AAVF17 / HSC18 (SEQ ID NOs: 19 and 20 of WO2016049230), NO: 16 and 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.

[0093] In some embodiments, the AAV serotype may be or comprise a sequence as described in U.S. Pat. No. 8,734,809 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV CBr-E1 (SEQ ID NOs: 13 and 87 of U.S. Pat. No. 8,734,809), AAV CBr-E2 (SEQ ID NOs: 14 and 88 of U.S. Pat. No. 8,734,809), AAV CBr-E3 (SEQ ID NOs: 15 and 89 of U.S. Pat. No. 8,734,809), AAV CBr-E4 (SEQ ID NOs: 16 and 90 of U.S. Pat. No. 8,734,809), AAV CBr-E5 (SEQ ID NOs: 17 and 91 of U.S. Pat. No. 8,734,809), AAV CBr-e5 (SEQ ID NOs: 18 and 92 of U.S. Pat. No. 8,734,809), AAV CBr-E6 (SEQ ID NOs: 19 and 93 of U.S. Pat. No. 8,734,809), AAV CBr-E7 (SEQ ID NOs: 19 and 104 of U.S. Pat. No. 8,734,809), or AAV CBr-E8 (SEQ ID NOs: 20 and 21 of U.S. Pat. No. 8,734,809). ID NO: 20 and 94), AAV CBr-E8 (SEQ ID NO: 21 and 95 of US8734809), AAV CLv-D1 (SEQ ID NO: 22 and 96 of US8734809), 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 NOs: 29 and 103 of US8734809), AAV CLv-E1 (SEQ ID NOs: 13 and 87 of US8734809), AAV CLv-R1 (SEQ ID NOs: 30 and 104 of US8734809), AAV CLv-R2 (SEQ ID NOs: 31 and 105 of US8734809), AAV CLv-R3 (SEQ ID NOs: 32 and 106 of US8734809), AAV CLv-R4 (SEQ ID NOs: 33 and 107 of US8734809), AAV CLv-R5 (SEQ ID NOs: 34 and 108 of US8734809), AAV CLv-R6 (SEQ ID NOs: 35 and 109 of US8734809), AAVCLv-R7 (SEQ ID NOs: 36 and 110 of US8734809), AAVCLv-R8 (SEQ ID NOs: 37 and 111 of US8734809), AAVCLv-R9 (SEQ ID NOs: 38 and 112 of US8734809), AAVCLg-F1 (SEQ ID NOs: 39 and 113 of US8734809), AAVCLg-F2 (SEQ ID NOs: 40 and 114 of US8734809), AAVCLg-F3 (SEQ ID NOs: 41 and 115 of US8734809), AAVCLg-F4 (SEQ ID NOs: 42 and 116 of US8734809), AAVCLg-F5 (SEQ ID NOs: 43 and 117 of US8734809), AAVCLg-F6 (SEQ ID NOs: 44 and 118 of US8734809), AAVCLg-F7 (SEQ ID NOs: 45 and 119 of US8734809), AAVCLg-F8 (SEQ ID NOs: 46 and 119 of US8734809), AAVCLg-F9 (SEQ ID NOs: 47 and 110 of US8734809), AAVCLg-F10 (SEQ ID NOs: 48 and 111 of US8734809), AAVCLg-F20 (SEQ ID NOs: 49 and 120 of US8734809), AAVCLg-F31 (SEQ ID NOs: 41 and 121 of US8734809), AAVCLg-F42 (SEQ ID NOs: 49 and 122 of US8734809), AAVCLg-F5 NO: 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 117 of US8734809), 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 809), AAV CSp-4 (SEQ ID NOs: 50 and 124 of US8734809), AAV CSp-6 (SEQ ID NOs: 51 and 125 of US8734809), AAV CSp-7 (SEQ ID NOs: 52 and 126 of US8734809), AAV CSp-8 (SEQ ID NOs: 53 and 127 of US8734809), AAV CSp-9 (SEQ ID NOs: 54 and 128 of US8734809), AAV CSp-2 (SEQ ID NOs: 55 and 129 of US8734809), AAV CSp-3 (SEQ ID NOs: 56 and 130 of US8734809), AAV CKd-1 (SEQ ID NOs: 57 and 131 of US8734809), AAV CKd-10 (SEQ ID NOs: 58 and 132 of US8734809), AAV CSp-2 (SEQ ID NOs: 59 and 140 of US8734809), AAV CSp-3 (SEQ ID NOs: 51 and 141 of US8734809), AAV CSp-4 (SEQ ID NOs: 50 and 124 of US8734809), AAV CSp-6 (SEQ ID NOs: 51 and 125 of US8734809), AAV CSp-7 (SEQ ID NOs: 52 and 126 of US8734809), AAV CSp-8 (SEQ ID NOs: 53 and 127 of US8734809), AAV CSp-9 (SEQ ID NOs: 54 and 128 of US8734809), NO: 58 and 132), AAVCKd-2 (SEQ ID NO: 59 and 133 of US8734809), AAVCKd-3 (SEQ ID NOs: 60 and 134 of US8734809), AAV CKd-4 (SEQ ID NOs: 61 and 135 of US8734809), AAV CKd-6 (SEQ ID NOs: 62 and 136 of US8734809), AAV CKd-7 (SEQ ID NOs: 63 and 137 of US8734809), AAV CKd-8 (SEQ ID NOs: 64 and 138 of US8734809), AAV CLv-1 (SEQ ID NOs: 35 and 139 of US8734809), AAV CLv-12 (SEQ ID NOs: 66 and 140 of US8734809), AAV CLv-13 (SEQ ID NOs: 67 and 141 of US8734809), AAV CLv-2 (SEQ ID NOs: 68 and 142 of US8734809), AAV CLv-3 (SEQ ID NOs: 69 and 150 of US8734809), AAV CLv-4 (SEQ ID NOs: 61 and 151 of US8734809), AAV CLv-5 (SEQ ID NOs: 69 and 152 of US8734809), AAV CLv-6 (SEQ ID NOs: 61 and 153 of US8734809), AAV CLv-7 (SEQ ID NOs: 63 and 137 of US8734809), AAV CLv-8 (SEQ ID NOs: 64 and 138 of US8734809), AAV CLv-1 (SEQ ID NOs: 35 and 139 of US8734809), AAV CLv-1 NO: 68 and 142), AAV CLv-3 (SEQ ID NO: 69 and 143 of US8734809), 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 NOs: 77 and 151 of US8734809), AAV CKd-B6 (SEQ ID NOs: 78 and 152 of US8734809), AAV CKd-B7 (SEQ ID NOs: 79 and 153 of US8734809), AAV CKd-B8 (SEQ ID NOs: 80 and 154 of US8734809), AAV CKd-H1 (SEQ ID NOs: 81 and 155 of US8734809), AAV CKd-H2 (SEQ ID NOs: 82 and 156 of US8734809), AAV CKd-H3 (SEQ ID NOs: 83 and 157 of US8734809), AAV CKd-H4 (SEQ ID NOs: 84 and 158 of US8734809), AAV CKd-H5 (SEQ ID NO: 85 and 159 of US8734809), AAVCKd-H6 (SEQ ID NOs: 77 and 151 of US8734809), AAV CHt-1 (SEQ ID NOs: 86 and 160 of US8734809), AAV CLv1-1 (SEQ ID NO: 171 of US8734809), 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: 178 of US8734809), AAV.VR-355 (SEQ ID NO: 181 of US8734809), AAV.hu.48R3 (SEQ ID NO: 183 of US8734809), or variants or derivatives thereof.

[0094] In some embodiments, the AAV serotype may be or comprise a sequence as described in International Publication No. WO2016065001 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV CHt-P2 (SEQ ID NOs: 1 and 51 of WO2016065001), AAV CHt-P5 (SEQ ID NOs: 2 and 52 of WO2016065001), AAV CHt-P9 (SEQ ID NOs: 3 and 53 of WO2016065001), AAV CBr-7.1 (SEQ ID NOs: 4 and 54 of WO2016065001), AAV CBr-7.2 (SEQ ID NOs: 5 and 55 of WO2016065001), AAV CBr-7.3 (SEQ ID NOs: 6 and 56 of WO2016065001), AAV CBr-7.4 (SEQ ID NOs: 7 and 8 of WO2016065001), NO: 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 60 of WO2016065001), AAV CBr-7.10 (SEQ ID NOs: 11 and 61 of WO2016065001), AAV CKd-N3 (SEQ ID NOs: 12 and 62 of WO2016065001), AAV CKd-N4 (SEQ ID NOs: 13 and 63 of WO2016065001), AAV CKd-N9 (SEQ ID NOs: 14 and 64 of WO2016065001), AAV CLv-L4 (SEQ ID NOs: 15 and 65 of WO2016065001), AAV CLv-L5 (SEQ ID NOs: 16 and 66 of WO2016065001), AAV CLv-L6 (SEQ ID NOs: 17 and 67 of WO2016065001), AAV CLv-K1 (SEQ ID NOs: 18 and 68 of WO2016065001), AAV CLv-K3 (SEQ ID NOs: 19 and 69 of WO2016065001), AAV CLv-K6 (SEQ ID NOs: 20 and 70 of WO2016065001), AAV CLv-M1 (SEQ ID NOs: 21 and 71 of WO2016065001), AAV CLv-M11 (SEQ ID NOs: 22 and 72 of WO2016065001), AAV CLv-M2 (SEQ ID NOs: 23 and 73 of WO2016065001), AAV CLv-M5 (SEQ ID NOs: 24 and 74 of WO2016065001), AAV CLv-M6 (SEQ ID NOs: 25 and 75 of WO2016065001), AAV CLv-M7 (SEQ ID NOs: 26 and 27 of WO2016065001), 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: 29 and 79 of WO2016065001), 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: 32 and 82 of WO2016065001), AAV CHt-6.10 (SEQ ID NO: 33 and 83 of WO2016065001), AAV CHt-6.5 (SEQ ID NO: 34 and 35 of WO2016065001), AAV CHt-6.7 (SEQ ID NO: 35 and 36 of WO2016065001), AAV CHt-6.8 (SEQ ID NO: 36 and 37 of WO2016065001), AAV CHt-6.9 (SEQ ID NO: 37 and 38 of WO2016065001), AAV CHt-7.10 (SEQ ID NO: 38 and 39 of WO2016065001), AAV CHt-7.11 (SEQ ID NO: 39 and 40 of WO2016065001), AAV CHt-7.12 (SEQ ID NO: 39 and 41 of WO2016065001), AAV CHt-7.13 (SEQ ID NO: 39 and 42 of WO NO:34 and 84), AAV CHt-6.6 (SEQ ID NOs: 35 and 85 of WO2016065001), AAV CHt-6.7 (SEQ ID NOs: 36 and 86 of WO2016065001), AAV CHt-6.8 (SEQ ID NOs: 37 and 87 of WO2016065001), AAV CSp-8.10 (SEQ ID NOs: 38 and 88 of WO2016065001), AAV CSp-8.2 (SEQ ID NOs: 39 and 89 of WO2016065001), AAV CSp-8.4 (SEQ ID NOs: 40 and 90 of WO2016065001), AAV CSp-8.5 (SEQ ID NOs: 41 and 91 of WO2016065001), AAV CSp-8.6 (SEQ ID NOs: 42 and 92 of WO2016065001), AAV CSp-8.7 (SEQ ID NOs: 43 and 93 of WO2016065001), AAV CSp-8.8 (SEQ ID NOs: 44 and 94 of WO2016065001), AAV CSp-8.9 (SEQ ID NOs: 45 and 95 of WO2016065001), AAV CBr-B7.3 (SEQ ID NOs: 46 and 96 of WO2016065001), AAV CBr-B7.4 (SEQ ID NOs: 47 and 97 of WO2016065001), AAV3B (SEQ ID NOs: 48 and 98 of WO2016065001), AAV4 (SEQ ID NOs: 49 and 99 of WO2016065001), AAV5 (SEQ ID NOs: 50 and 100 of WO2016065001), or variants or derivatives thereof.

[0095] In some embodiments, the AAV particle can be a serotype comprising any one selected from those listed in Table 1.

[0096] In some embodiments, the AAV particle can comprise a sequence, fragment, or variant of any of the sequences in Table 1.

[0097] In some embodiments, the AAV particle can be encoded by a sequence, fragment, or variant of any of the sequences in Table 1.

[0098] In the DNA and RNA sequences cited and / or described herein, the single-letter symbols have the following descriptions: A is adenine; C is cytosine; G is guanine; T is thymine; U is uracil; W is a weak base. base), such as adenine or thymine; S is a strong nucleotide, such as cytosine and guanine; M is an amino nucleotide, such as adenine and cytosine; K is a keto nucleotide, such as guanine and thymine; R is the purines adenine and guanine; Y is the pyrimidines cytosine and thymine; B is any non-A base (such as cytosine, guanine and thymine); D is any non-C base (such as adenine, guanine and thymine); H is any non-G base (such as adenine, cytosine and thymine); V is any non-T base (such as adenine, cytosine and guanine); N is any nucleotide (non-vacant); Z is zero.

[0099] In any amino acid sequence cited and / or described herein, the single-letter symbols have the following descriptions: G (Gly) is glycine; A (Ala) is alanine; L (Leu) is leucine; M (Met) is methionine; F (Phe) is phenylalanine; W (Trp) is tryptophan; K (Lys) is lysine; Q (Gln) is glutamine; E (Glu) is glutamic acid; S (Ser) is serine; P (Pro) is proline; V (Val) is valine; I (Ile) is isoleucine; C (C ys) is cysteine; Y (Tyr) is tyrosine; H (His) is histidine; R (Arg) is arginine; N (Asn) is asparagine; D (Asp) is aspartic acid; T (Thr) is threonine; B (Asx) is aspartic acid or asparagine; J (Xle) is leucine or isoleucine; O (Pyl) is pyrrolysine; U (Sec) is selenocysteine; X (Xaa) is any amino acid; Z (Glx) is glutamine or glutamic acid.

[0100] Table 1. Representative AAV serotypes

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] The contents of each patent, application, and / or publication listed in Table 1 are incorporated herein by reference in their entirety.

[0131] In certain embodiments, the AAV serotype may be or may comprise a sequence as described in International Patent Publication No. WO2015038958 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV9 (SEQ ID NOs: 2 and 11 of WO2015038958 or SEQ ID NOs: 135 and 136 herein), PHP.B (SEQ ID NOs: 8 and 9 of WO2015038958, SEQ ID NOs: 3 and 4 herein), G2B-13 (SEQ ID NO: 12 of WO2015038958, SEQ ID NO: 5 herein), G2B-26 (SEQ ID NO: 13 of WO2015038958 or SEQ ID NO: 3 herein), TH1.1-32 (SEQ ID NO: 14 of WO2015038958 or SEQ ID NO: 6 herein), TH1.1-35 (SEQ ID NO: 15 of WO2015038958 or SEQ ID NO: 7 herein), TH1.1-36 (SEQ ID NO: 16 of WO2015038958 or SEQ ID NO: 8 herein), TH1.1-37 (SEQ ID NO: 17 of WO2015038958 or SEQ ID NO: 9 herein), TH1.1-38 (SEQ ID NO: 18 of WO2015038958 or SEQ ID NO: 19 herein), TH1.1-39 (SEQ ID NO: 20 of WO2015038958 or SEQ ID NO: 21 herein), TH1.1-37 (SEQ ID NO: 21 of WO2015038958 or SEQ ID NO: 22 herein), TH1.1-38 (SEQ ID NO: 15 or SEQ ID NO: 7 herein) or its variants. In addition, any "targeting peptide" or "amino acid insert" described in WO2015038958 (used interchangeably herein to refer to sequences that can be inserted into the AAV capsid sequence to facilitate delivery to CNS tissues) can be inserted into any parent AAV serotype, such as, but not limited to, AAV9 (SEQ ID NO: 135 of the DNA sequence and SEQ ID NO: 136 of the amino acid sequence). In some embodiments, the amino acid insert is inserted between amino acids 586-592 of the parent AAV (e.g., AAV9). In some embodiments, the amino acid insert is inserted between amino acids 588-589 of the parent AAV sequence.The amino acid insert 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 herein), KFPVALT (SEQ ID NO: 3 of WO2015038958; SEQ ID NO: 1261 herein), LAVPFK (SEQ ID NO: 31 of WO2015038958; SEQ ID NO: 1262 herein), AVPFK (SEQ ID NO: 32 of WO2015038958; SEQ ID NO: 1263 herein), VPFK (SEQ ID NO: 33 of WO2015038958; SEQ ID NO: 1264 herein), TLAVPF (SEQ ID NO: 34 of WO2015038958; SEQ ID NO: 1265 herein), TLAVP (SEQ ID NO: 35 of WO2015038958; SEQ ID NO: 1266 herein), NO: 1266), TLAV (SEQ ID NO: 36 of WO2015038958; SEQ ID NO: 1267 herein), SVSKPFL (SEQ ID NO: 28 of WO2015038958; SEQ ID NO: 1268 herein), FTLTTPK (SEQ ID NO: 29 of WO2015038958; SEQ ID NO: 1269 herein), MNATKNV (SEQ ID NO: 30 of WO2015038958; SEQ ID NO: 1270 herein), QSSQTPR (SEQ ID NO: 54 of WO2015038958; SEQ ID NO: 1271 herein), ILGTGTS (SEQ ID NO: 55 of WO2015038958; SEQ ID NO: 1272 herein), TRTNPEA (SEQ ID NO: 56 of WO2015038958; SEQ ID NO: 1273 herein), NO: 1273), NGGTSSS (SEQ ID NO: 58 of WO2015038958; SEQ ID NO: 1274 herein), or YTLSQGW (SEQ ID NO: 60 of WO2015038958; SEQ ID NO: 1275 herein).Non-limiting examples of nucleotide sequences that can encode amino acid inserts include, but are not limited to, the following: AAGTTTCCTGTGGCGTTGACT (SEQ ID NO: 3 of WO2015038958; SEQ ID NO: 1276 herein), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NOs: 24 and 498 of WO201503895; SEQ ID NO: 1277 herein), AGTGTGAGTAAGCCTTTTTTG (SEQ ID NO: 25 of WO2015038958; SEQ ID NO: 1278 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 26 of WO2015038958; SEQ ID NO: 1279 herein), ATGAATGCTACGAAGAATGTG (SEQ ID NO: 27 of WO2015038958; SEQ ID NO: 1280 herein), NO: 1280), CAGTCGTCGCAGACGCCTAGG (SEQ ID NO: 48 of WO2015038958; SEQ ID NO: 1281 herein), ATTCTGGGGACTGGTACTTCG (SEQ ID NOs: 50 and 52 of WO2015038958; SEQ ID NO: 1282 herein), ACGCGGACTAATCCTGAGGCT (SEQ ID NO: 51 of WO2015038958; SEQ ID NO: 1283 herein), AATGGGGGACTAGTAGTTCT (SEQ ID NO: 53 of WO2015038958; SEQ ID NO: 1284 herein), or TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 59 of WO2015038958; SEQ ID NO: 1285 herein).

[0132] In some embodiments, the AAV serotype may be or may comprise a sequence as described in International Patent Publication No. WO2017100671 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV9 K449R (SEQ ID NO: 45 of WO2017100671, SEQ ID NO: 9 herein), PHP.N (SEQ ID NO: 46 of WO2017100671, SEQ ID NO: 2 herein), PHP.S (SEQ ID NO: 47 of WO2017100671, SEQ ID NO: 8 herein), or a variant thereof. In addition, any targeting peptide or amino acid insert described in WO2017100671 may 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 insert is inserted between amino acids 586-592 of the parent AAV (e.g., AAV9). In some embodiments, the amino acid insert is inserted between amino acids 588-589 of the parent AAV sequence. The amino acid insert 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 herein), AQSVSKPFLAQ (SEQ ID NO: 2 of WO2017100671; SEQ ID NO: 1287 herein), AQFTLTTPKAQ (SEQ ID NO: 3 of the WO2017100671 sequence listing; SEQ ID NO: 1288 herein), DGTLAVPFKAQ (SEQ ID NO: 4 of the WO2017100671 sequence listing; SEQ ID NO: 1289 herein), ESTLAVPFKAQ (SEQ ID NO: 5 of WO2017100671; SEQ ID NO: 1290 herein), GGTLAVPFKAQ (SEQ ID NO: 6 of WO2017100671; SEQ ID NO: 1291 herein), NO: 1291), AQTLATPFKAQ (SEQ ID NOs: 7 and 33 of WO2017100671; SEQ ID NO: 1292 herein), ATTLATPFKAQ (SEQ ID NO: 8 of WO2017100671; SEQ ID NO: 1293 herein), DGTLATPFKAQ (SEQ ID NO: 9 of WO2017100671; SEQ ID NO: 1294 herein), GGTLATPFKAQ (SEQ ID NO: 10 of WO2017100671;SEQ ID NO: 1295 herein), SGSLAVPFKAQ (SEQ ID NO: 11 of WO2017100671; SEQ ID NO: 1296 herein), AQTLAQPFKAQ (SEQ ID NO: 12 of WO2017100671; SEQ ID NO: 1297 herein), AQTLQQPFKAQ (SEQ ID NO: 13 of WO2017100671; SEQ ID NO: 1298 herein), AQTLSNPFKAQ (SEQ ID NO: 14 of WO2017100671; SEQ ID NO: 1299 herein), AQTLAVPFSNP (SEQ ID NO: 15 of WO2017100671; SEQ ID NO: 1300 herein), QGTLAVPFKAQ (SEQ ID NO: 16 of WO2017100671; SEQ ID NO: 1707 herein), NO:1301), NQTLAVPFKAQ (SEQ ID NO:17 of WO2017100671; SEQ ID NO:1302 herein), EGSLAVPFKAQ (SEQ ID NO:18 of WO2017100671; SEQ ID NO:1303 herein), SGNLAVPFKAQ (SEQ ID NO:1303 of WO2017100671) 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; SEQ ID NO in this article) NO: 1306), AVTLAVPFKAQ (SEQ ID NO: 22 of WO2017100671; SEQ ID in this article NO: 1307), AQTLSTPFKAQ (SEQ ID NO: 23 of WO2017100671; SEQ ID NO: 1308 herein), AQTLPQPFKAQ (SEQ ID NOs: 24 and 32 of WO2017100671; SEQ ID NO: 1309 herein), AQTLSQPFKAQ (SEQ ID NO: 25 of WO2017100671; SEQ ID NO: 1310 herein), AQTLQLPFKAQ (SEQ ID NO: 26 of WO2017100671;SEQ ID NO: 1311 herein), AQTLTMPFKAQ (SEQ ID NOs: 27 and 34 of WO2017100671 and SEQ ID NO: 35 in the sequence listing of WO2017100671; SEQ ID NO: 1312 herein), AQTLTTPFKAQ (SEQ ID NO: 28 of WO2017100671; SEQ ID NO: 1313 herein), AQYTLSQGWAQ (SEQ ID NO: 29 of WO2017100671; SEQ ID NO: 1314 herein), AQMNATKNVAQ (SEQ ID NO: 30 of WO2017100671; SEQ ID NO: 1315 herein), AQVSGGHHSAQ (SEQ ID NO: 31 of WO2017100671; SEQ ID NO: 1316 herein), NO: 1316), AQTLTAPFKAQ (SEQ ID NO: 35 in Table 1 of WO2017100671; SEQ ID NO: 1317 herein), AQTLSKPFKAQ (SEQ ID NO: 36 in WO2017100671; SEQ ID NO: 1318 herein), QAVRTSL (SEQ ID NO: 37 in WO2017100671; SEQ ID NO: 1319 herein), YTLSQGW (SEQ ID NO: 38 in WO2017100671; SEQ ID NO: 1275 herein), LAKERS (SEQ ID NO: 39 in WO2017100671; SEQ ID NO: 1320 herein), TLAVPFK (SEQ ID NO: 40 in the sequence listing of WO2017100671; SEQ ID NO: 41 herein), NO: 1260), SVSKPFL (SEQ ID NO: 41 of WO2017100671; SEQ ID NO: 1268 herein), FTLTTPK (SEQ ID NO: 42 of WO2017100671; SEQ ID NO: 1269 herein), MNSTKNV (SEQ ID NO: 43 of WO2017100671; SEQ ID NO: 1321 herein), VSGGHHS (SEQ ID NO: 44 of WO2017100671; SEQ ID NO: 1322 herein), SAQTLAVPFKAQAQ (SEQ ID NO: 48 of WO2017100671; SEQ ID NO: 1323 herein), SXXXLAVPFKAQAQ (SEQ ID NO: 49 of WO2017100671, wherein X can be any amino acid;SEQ ID NO: 1324 herein), SAQXXXVPFKAQAQ (SEQ ID NO: 50 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1325 herein), SAQTLXXXFKAQAQ (SEQ ID NO: 51 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1326 herein), SAQTLAVXXXAQAQ (SEQ ID NO: 52 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1327 herein), SAQTLAVPFXXXAQ (SEQ ID NO: 53 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1328 herein), TNHQSAQ (SEQ ID NO: 65 of WO2017100671; SEQ ID NO: 66 herein). NO: 1329), AQAQTGW (SEQ ID NO: 66 of WO2017100671; SEQ ID NO: 1330 herein), DGTLATPFK (SEQ ID NO: 67 of WO2017100671; SEQ ID NO: 1331 herein), DGTLATPFKXX (SEQ ID NO: 68 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1332 herein), LAVPFKAQ (SEQ ID NO: 80 of WO2017100671; SEQ ID NO: 1333 herein), VPFKAQ (SEQ ID NO: 81 of WO2017100671; SEQ ID NO: 1334 herein), FKAQ (SEQ ID NO: 82 of WO2017100671; SEQ ID NO: 1335 herein), NO: 1335), AQTLAV (SEQ ID NO: 83 of WO2017100671; SEQ ID NO: 1336 herein), AQTLAVPF (SEQ ID NO: 84 of WO2017100671; SEQ ID NO: 1337 herein), QAVR (SEQ ID NO: 85 of WO2017100671; SEQ ID NO: 1338 herein), AVRT (SEQ ID NO: 86 of WO2017100671; SEQ ID NO: 1339 herein), VRTS (SEQ ID NO: 87 of WO2017100671; SEQ ID NO: 1340 herein), RTSL (SEQ ID NO: 88 of WO2017100671);SEQ ID NO: 1341 herein), QAVRT (SEQ ID NO: 89 of WO2017100671; SEQ ID NO: 1342 herein), AVRTS (SEQ ID NO: 90 of WO2017100671; SEQ ID NO: 1343 herein), VRTSL (SEQ ID NO: 91 of WO2017100671; SEQ ID NO: 1344 herein), QAVRTS (SEQ ID NO: 92 of WO2017100671; SEQ ID NO: 1345 herein), or AVRTSL (SEQ ID NO: 93 of WO2017100671; SEQ ID NO: 1346 herein). ;

[0133] Non-limiting examples of nucleotide sequences that can encode amino acid insertions include the following: GATGGGACTTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 54 of WO2017100671; SEQ ID NO: 1347 herein),

[0134] GATGGGACGTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 55 of WO2017100671; SEQ ID NO: 1348 herein), CAGGCGGTTAGGACGTCTTTG (SEQ ID NO: 56 of WO2017100671; SEQ ID NO: 1349 herein), CAGGTCTTCACGGACTCAGACTATCAG (SEQ ID NOs: 57 and 78 of WO2017100671; SEQ ID NO: 1350 herein),

[0135] CAAGTAAAACCTCTACAAATGTGGTAAAATCG (SEQ ID NO: 58 of WO2017100671; SEQ ID NO: 1351 herein),

[0136] ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC (SEQ ID NO: 59 of WO2017100671; SEQ ID NO: 1352 herein), GGAAGTATTCCTTGGTTTTGAACCCA (SEQ ID NO: 60 of WO2017100671; SEQ ID NO: 1353 herein), GGTCGCGGTTCTTGTTTGTGGAT (SEQ ID NO: 61 of WO2017100671; SEQ ID NO: 1354 herein), CGACCTTGAAGCGCATGAACTCCT (SEQ ID NO: 62 of WO2017100671; SEQ ID NO: 1355 herein),

[0137] GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNM NNMNNMNNMNNMNNTTGGGCACTCTGGTGGTTTGTC (SEQ ID NO: 63 of WO2017100671, wherein N may be A, C, T, or G; SEQ ID NO: 1356 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCMNNMNNMNNAAAA GGCACCGCCAAAGTTTG (SEQ ID NO: 69 of WO2017100671, wherein N may be A, C, T, or G; SEQ ID NO: 1357 herein),

[0138] GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNCACCGCCAAAGTTTGGGCACT (SEQ ID NO: 70 of WO2017100671, wherein N may be A, C, T, or G; SEQ ID NO: 1358 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAMNNMNNMNNCAAAGTTTGGGCACTCTGGTGG (SEQ ID NO: 71 of WO2017100671, wherein N may be A, C, T, or G; SEQ ID NO: 1359 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAAGG CACMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG (SEQ ID NO: 72 of WO2017100671, wherein N may be A, C, T, or G; SEQ ID NO: 1360 herein), NO: 1360), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NO: 74 of WO2017100671; SEQ ID NO: 1277 herein), AGTGTGAGTAAGCTTTTTTG (SEQ ID NO: 75 of WO2017100671; SEQ ID NO: 1278 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 76 of WO2017100671; SEQ ID NO: 1279 herein), TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 77 of WO2017100671; SEQ ID NO: 1285 herein), or CTTGCGAAGGAGCGGCTTTCG (SEQ ID NO: 79 of WO2017100671; SEQ ID NO: 1361 herein).

[0139] In some embodiments, the AAV serotype may be or may comprise a sequence as described in U.S. Pat. No. 9,624,274 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, AAV1 (SEQ ID NO: 181 of U.S. Pat. No. 9,624,274), AAV6 (SEQ ID NO: 182 of U.S. Pat. No. 9,624,274), AAV2 (SEQ ID NO: 183 of U.S. Pat. No. 9,624,274), AAV3b (SEQ ID NO: 184 of U.S. Pat. No. 9,624,274), AAV7 (SEQ ID NO: 185 of U.S. Pat. No. 9,624,274), AAV8 (SEQ ID NO: 186 of U.S. Pat. No. 9,624,274), AAV10 (SEQ ID NO: 187 of U.S. Pat. No. 9,624,274), AAV4 (SEQ ID NO: 188 of U.S. Pat. No. 9,624,274), AAV11 (SEQ ID NO: 189 of U.S. Pat. No. 9,624,274), bAAV (SEQ ID NO: 190 of U.S. Pat. No. 9,624,274), or AAV2 (SEQ ID NO: 191 of U.S. Pat. No. 9,624,274). ID NO: 190), AAV5 (SEQ ID NO: 191 of US9624274), GPV (SEQ ID NO: 192 of US9624274; SEQ ID NO: 992 herein), B19 (SEQ ID NO: 193 of US9624274; SEQ ID NO: 993 herein), MVM (SEQ ID NO: 194 of US9624274; SEQ ID NO: 994 herein), FPV (SEQ ID NO: 195 of US9624274; SEQ ID NO: 995 herein), CPV (SEQ ID NO: 196 of US9624274; SEQ ID NO: 996 herein), or variants thereof. In addition, any of the structural protein inserts described in U.S. Pat. No. 9,624,274 can be inserted into, but are not limited to, I-453 and I-587 of any parental AAV serotype, such as, but not limited to, AAV2 (SEQ ID NO: 183 of U.S. Pat. No. 9,624,274). The amino acid insert can be, but is not limited to, any of the following amino acid sequences: VNLTWSRASG (SEQ ID NO: 50 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1362 herein), EFCINHRGYWVCGD (SEQ ID NO: 55 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1363 herein), EDGQVMDVDLS (SEQ ID NO: 85 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1364 herein), EKQRNGTLT (SEQ ID NO: 86 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1367 herein), EFCINHRGYWVCGD (SEQ ID NO: 57 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1368 herein), EDGQVMDVDLS (SEQ ID NO: 87 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1369 herein), EDGQVMDVDLS (SEQ ID NO: 88 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1370 herein), EDGQVMDVDLS (SEQ ID NO: 89 of U.S. Pat. No. 9,624,274; SEQ ID NO: 1371 herein), EDGQVMDVDLSSEQ ID NO: 1365 herein), TYQCRVTHPHLPRALMR (SEQ ID NO: 87 of US9624274; SEQ ID NO: 1366 herein), RHSTTQPRKTKGSG (SEQ ID NO: 88 of US9624274; SEQ ID NO: 1367 herein), DSNPRGVSAYLSR (SEQ ID NO: 89 of US9624274; SEQ ID NO: 1368 herein), TITCLWDLAPSK (SEQ ID NO: 90 of US9624274; SEQ ID NO: 1369 herein), KTKGSGFFVF (SEQ ID NO: 91 of US9624274; SEQ ID NO: 1370 herein), THPHLPRALMRS (SEQ ID NO: 92 of US9624274; SEQ ID NO: 1371 herein), ID NO: 1371), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 93 of US9624274; SEQ ID NO: 1372 herein), LPRALMRS (SEQ ID NO: 94 of US9624274; SEQ ID NO: 1373 herein), INHRGYWV (SEQ ID NO: 95 of US9624274; SEQ ID NO: 1374 herein), CDAGSVRTNAPD (SEQ ID NO: 60 of US9624274; SEQ ID NO: 1375 herein), AKAVSNLTESRSESLQS (SEQ ID NO: 96 of US9624274; SEQ ID NO: 1376 herein), SLTGDEFKKVLET (SEQ ID NO: 97 of US9624274; SEQ ID NO: 1377 herein), REAVAYRFEED (SEQ ID NO: 98 of US9624274; SEQ ID NO: 1378 herein), NO:98; SEQ ID NO:1378 herein), INPEIITLDG (SEQ ID NO:99 of US9624274; SEQ ID NO:1379 herein), DISVTGAPVITATYL (SEQ ID NO:100 of US9624274; SEQ ID NO:1380 herein), DISVTGAPVITA (SEQ ID NO:101 of US9624274; SEQ ID NO:1381 herein), PKTVSNLTESSSESVQS (SEQ ID NO:102 of US9624274;SEQ ID NO: 1382 herein), SLMGDEFKAVLET (SEQ ID NO: 103 of US9624274; SEQ ID NO: 1383 herein), QHSVAYTFEED (SEQ ID NO: 104 of US9624274; SEQ ID NO: 1384 herein), INPEIITRDG (SEQ ID NO: 105 of US9624274; SEQ ID NO: 1385 herein), DISLTGDPVITASYL (SEQ ID NO: 106 of US9624274; SEQ ID NO: 1386 herein), DISLTGDPVITA (SEQ ID NO: 107 of US9624274; SEQ ID NO: 1387 herein), DQSIDFEIDSA (SEQ ID NO: 108 of US9624274; SEQ ID NO: 1389 herein), NO: 1388), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 109 of US9624274; SEQ ID NO: 1389 herein), KNVSEDLPLPT (SEQ ID NO: 110 of US9624274; SEQ ID NO: 1390 herein), CDSGRVRTDAPD (SEQ ID NO: 111 of US9624274; SEQ ID NO: 1391 herein), FPEHLLVDFLQSLS (SEQ ID NO: 112 of US9624274; SEQ ID NO: 1392 herein), DAEFRHDSG (SEQ ID NO: 65 of US9624274; SEQ ID NO: 1393 herein), HYAAAQWDFGNTMCQL (SEQ ID NO: 113 of US9624274; SEQ ID NO: 1394 herein), NO: 1394), YAAQWDFGNTMCQ (SEQ ID NO: 114 of US9624274; SEQ ID NO: 1395 herein), RSQKEGLHYT (SEQ ID NO: 115 of US9624274; SEQ ID NO: 1396 herein), SSRTPSDKPVAHWANPQAE (SEQ ID NO: 116 of US9624274; SEQ ID NO: 1397 herein), SRTPSDKPVAHWANP (SEQ ID NO: 117 of US9624274; SEQ ID NO: 1398 herein), SSRTPSDKP (SEQ ID NO: 118 of US9624274);SEQ ID NO: 1399 herein), NADGNVDYHMNSVP (SEQ ID NO: 119 of US9624274; SEQ ID NO: 1400 herein), DGNVDYHMNSV (SEQ ID NO: 120 of US9624274; SEQ ID NO: 1401 herein), RSFKEFLQSSLRALRQ (SEQ ID NO: 121 of US9624274; SEQ ID NO: 1402 herein); FKEFLQSSLRA (SEQ ID NO: 122 of US9624274; SEQ ID NO: 1403 herein), or QMWAPQWGPD (SEQ ID NO: 123 of US9624274; SEQ ID NO: 1404 herein).

[0140] In some embodiments, the AAV serotype can be or can have a sequence as described in U.S. Pat. No. 9,475,845 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, an AAV capsid protein comprising a modification of one or more amino acids at amino acids 585 to 590 of a native AAV2 capsid protein. In addition, modifications can result in, but are not limited to, amino acid sequences RGNRQA (SEQ ID NO: 3 of US9475845; SEQ ID NO: 1405 herein), SSSTDP (SEQ ID NO: 4 of US9475845; SEQ ID NO: 1406 herein), SSNTAP (SEQ ID NO: 5 of US9475845; SEQ ID NO: 1407 herein), SNSNLP (SEQ ID NO: 6 of US9475845; SEQ ID NO: 1408 herein), SSTTAP (SEQ ID NO: 7 of US9475845; SEQ ID NO: 1409 herein), AANTAA (SEQ ID NO: 8 of US9475845; SEQ ID NO: 1410 herein), QQNTAP (SEQ ID NO: 9 of US9475845; SEQ ID NO: 1411 herein), SAQAQA (SEQ ID NO: 10 of US9475845; SEQ ID NO: 1412 herein), NO: 1412), QANTGP (SEQ ID NO: 11 of US9475845; SEQ ID NO: 1413 herein), NATTAP (SEQ ID NO: 12 of US9475845; SEQ ID NO: 1414 herein), SSTAGP (SEQ ID NOs: 13 and 20 of US9475845; SEQ ID NO: 1415 herein), QQNTAA (SEQ ID NO: 14 of US9475845; SEQ ID NO: 1416 herein), PSTAGP (SEQ ID NO: 15 of US9475845; SEQ ID NO: 1417 herein), NQNTAP (SEQ ID NO: 16 of US9475845; SEQ ID NO: 1418 herein), QAANAP (SEQ ID NO: 17 of US9475845; SEQ ID NO: 1419 herein), SIVGLP (SEQ ID NOs: 13 and 20 of US9475845; SEQ ID NO: 1420 herein), NO: 18; herein SEQ ID NO: 1420), AASTAA (SEQ ID NOs: 19 and 27 of US9475845; herein SEQ ID NO: 1421), SQNTTA (SEQ ID NO: 21 of US9475845;In some embodiments, the amino acid modification is a substitution of amino acid positions 262 to 265 in a native AAV2 capsid protein, or corresponding positions in a capsid protein of another AAV, with a targeting sequence. The targeting sequence can be, but is not limited to, any of the following amino acid sequences: NGRAHA (SEQ ID NO: 38 of US9475845; SEQ ID NO: 1428 herein), QPEHSST (SEQ ID NOs: 39 and 50 of US9475845; SEQ ID NO: 1429 herein), VNTANST (SEQ ID NO: 40 of US9475845; SEQ ID NO: 1430 herein), HGPMQKS (SEQ ID NO: 41 of US9475845; SEQ ID NO: 1431 herein), PHKPPLA (SEQ ID NO: 42 of US9475845; SEQ ID NO: 1432 herein), IKNNEMW (SEQ ID NO: 43 of US9475845; SEQ ID NO: 1433 herein), RNLDTPM (SEQ ID NO: 44 of US9475845; SEQ ID NO: 45 herein), NO: 1434), VDSHRQS (SEQ ID NO: 45 of US9475845; SEQ ID NO: 1435 herein), YDSKTKT (SEQ ID NO: 46 of US9475845; SEQ ID NO: 1436 herein), SQLPHQK (SEQ ID NO: 47 of US9475845; SEQ ID NO: 1437 herein), STMQQNT (SEQ ID NO: 48 of US9475845; SEQ ID NO: 1438 herein), TERYMTQ (SEQ ID NO: 49 of US9475845; SEQ ID NO: 1439 herein), DASLSTS (SEQ ID NO: 51 of US9475845);SEQ ID NO: 1440 herein), DLPNKKT (SEQ ID NO: 52 of US9475845; SEQ ID NO: 1441 herein), DLTAARL (SEQ ID NO: 53 of US9475845; SEQ ID NO: 1442 herein), EPHQFNY (SEQ ID NO: 54 of US9475845; SEQ ID NO: 1443 herein), EPQSNHT (SEQ ID NO: 55 of US9475845; SEQ ID NO: 1444 herein), MSSWPSQ (SEQ ID NO: 56 of US9475845; SEQ ID NO: 1445 herein), NPKHNAT (SEQ ID NO: 57 of US9475845; SEQ ID NO: 1446 herein), PDGMRTT (SEQ ID NO: 58 of US9475845; SEQ ID NO: 1447 herein), NO: 1447), PNNNKTT (SEQ ID NO: 59 of US9475845; SEQ ID NO: 1448 herein), QSTTHDS (SEQ ID NO: 60 of US9475845; SEQ ID NO: 1449 herein), TGSKQKQ (SEQ ID NO: 61 of US9475845; SEQ ID NO: 1450 herein), SLKHQAL (SEQ ID NO: 62 of US9475845; SEQ ID NO: 1451 herein), SPIDGEQ (SEQ ID NO: 63 of US9475845; SEQ ID NO: 1452 herein), WIFPWIQL (SEQ ID NOs: 64 and 112 of US9475845; SEQ ID NO: 1453 herein), CDCRGDCFC (SEQ ID NO: 65 of US9475845; SEQ ID NO: 1454 herein), NO: 1454), CNGRC (SEQ ID NO: 66 of US9475845; SEQ ID NO: 1455 herein), CPRECES (SEQ ID NO: 67 of US9475845; SEQ ID NO: 1456 herein), CTTHWGFTLC (SEQ ID NOs: 68 and 123 of US9475845; SEQ ID NO: 1457 herein), CGRRAGGSC (SEQ ID NO: 69 of US9475845; SEQ ID NO: 1458 herein), CKGGRAKDC (SEQ ID NO: 70 of US9475845);SEQ ID NO: 1459 herein), CVPELGHEC (SEQ ID NOs: 71 and 115 of US9475845; SEQ ID NO: 1460 herein), CRRETAWAK (SEQ ID NO: 72 of US9475845; SEQ ID NO: 1461 herein), VSWFSHRYSPFAVS (SEQ ID NO: 73 of US9475845; SEQ ID NO: 1462 herein), GYRDGYAGPILYN (SEQ ID NO: 74 of US9475845; SEQ ID NO: 1463 herein), XXXYXXX (SEQ ID NO: 75 of US9475845; SEQ ID NO: 1464 herein), YXNW (SEQ ID NO: 76 of US9475845; SEQ ID NO: 1465 herein), RPLPPLP (SEQ ID NO: 77 of US9475845; SEQ ID NO: 1466 herein), NO: 1466), APPLPPR (SEQ ID NO: 78 of US9475845; SEQ ID NO: 1467 herein), DVFYPYPYASGS (SEQ ID NO: 79 of US9475845; SEQ ID NO: 1468 herein), MYWYPY (SEQ ID NO: 80 of US9475845; SEQ ID NO: 1469 herein), DITWDQLWDLMK ​​(SEQ ID NO: 81 of US9475845; SEQ ID NO: 1470 herein), CWDDXWLC (SEQ ID NO: 82 of US9475845; SEQ ID NO: 1471 herein), EWCEYLGGYLRCYA (SEQ ID NO: 83 of US9475845; SEQ ID NO: 1472 herein), YXCXXGPXTWXCXP (SEQ ID NO: 84 of US9475845; SEQ ID NO: 1473 herein), ID NO: 1473), IEGPTLRQWLAARA (SEQ ID NO: 85 of US9475845; SEQ ID NO: 1474 herein), LWXXX (SEQ ID NO: 86 of US9475845; SEQ ID NO: 1475 herein), XFXXYLW (SEQ ID NO: 87 of US9475845; SEQ ID NO: 1476 herein), SSIISHFRWGLCD (SEQ ID NO: 88 of US9475845; SEQ ID NO: 1477 herein), MSRPACPPNDKYE (SEQ ID NO: 89 of US9475845);SEQ ID NO: 1478 herein), CLRSGRGC (SEQ ID NO: 90 of US9475845; SEQ ID NO: 1479 herein), CHWMFSPWC (SEQ ID NO: 91 of US9475845; SEQ ID NO: 1480 herein), WXXF (SEQ ID NO: 92 of US9475845; SEQ ID NO: 1481 herein), CSSRLDAC (SEQ ID NO: 93 of US9475845; SEQ ID NO: 1482 herein), CLPVASC (SEQ ID NO: 94 of US9475845; SEQ ID NO: 1483 herein), CGFECVRQCPERC (SEQ ID NO: 95 of US9475845; SEQ ID NO: 1484 herein), CVALCREACGEGC (SEQ ID NO: 96 of US9475845; SEQ ID NO: 1485 herein), NO: 1485), SWCEPGWCR (SEQ ID NO: 97 of US9475845; SEQ ID NO: 1486 herein), YSGKWGW (SEQ ID NO: 98 of US9475845; SEQ ID NO: 1487 herein), GLSGGRS (SEQ ID NO: 99 of US9475845; SEQ ID NO: 1488 herein), LMLPRAD (SEQ ID NO: 100 of US9475845; SEQ ID NO: 1489 herein), CSCFRDVCC (SEQ ID NO: 101 of US9475845; SEQ ID NO: 1490 herein), CRDVVSVIC (SEQ ID NO: 102 of US9475845; SEQ ID NO: 1491 herein), MARSGL (SEQ ID NO: 103 of US9475845; SEQ ID NO: 104 herein). NO: 1492), MARAKE (SEQ ID NO: 104 of US9475845; SEQ ID NO: 1493 herein), MSRTMS (SEQ ID NO: 105 of US9475845; SEQ ID NO: 1494 herein), KCCYSL (SEQ ID NO: 106 of US9475845; SEQ ID NO: 1495 herein), MYWGDSHWLQYWYE (SEQ ID NO: 107 of US9475845; SEQ ID NO: 1496 herein), MQLPLAT (SEQ ID NO: 108 of US9475845);SEQ ID NO: 1497 herein), EWLS (SEQ ID NO: 109 of US9475845; SEQ ID NO: 1498 herein), SNEW (SEQ ID NO: 110 of US9475845; SEQ ID NO: 1499 herein), TNYL (SEQ ID NO: 111 of US9475845; SEQ ID NO: 1500 herein), WDLAWMFRLPVG (SEQ ID NO: 113 of US9475845; SEQ ID NO: 1501 herein), CTVALPGGYVRVC (SEQ ID NO: 114 of US9475845; SEQ ID NO: 1502 herein), CVAYCIEHHCWTC (SEQ ID NO: 116 of US9475845; SEQ ID NO: 1503 herein), CVFAHNYDYLVC (SEQ ID NO: 117 of US9475845; SEQ ID NO: 118 herein), ID NO: 1504), CVFTSNYAFC (SEQ ID NO: 118 of US9475845; SEQ ID NO: 1505 herein), VHSPNKK (SEQ ID NO: 119 of US9475845; SEQ ID NO: 1506 herein), CRGDGWC (SEQ ID NO: 120 of US9475845; SEQ ID NO: 1507 herein), XRGCDX (SEQ ID NO: 121 of US9475845; SEQ ID NO: 1508 herein), PXXX (SEQ ID NO: 122 of US9475845; SEQ ID NO: 1509 herein), SGKGPRQITAL (SEQ ID NO: 124 of US9475845; SEQ ID NO: 1510 herein), AAAAAAAAAXXXXX (SEQ ID NO: 125 of US9475845; SEQ ID NO: 1511 herein), NO: 1511), VYMSPF (SEQ ID NO: 126 of US9475845; SEQ ID NO: 1512 herein), ATWLPPR (SEQ ID NO: 127 of US9475845; SEQ ID NO: 1513 herein), HTMYYHHYQHHL (SEQ ID NO: 128 of US9475845; SEQ ID NO: 1514 herein), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 129 of US9475845;SEQ ID NO: 1515 herein), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 130 of US9475845; SEQ ID NO: 1516 herein), CKGQCDRFKGLPWEC (SEQ ID NO: 131 of US9475845; SEQ ID NO: 1517 herein), SGRSA (SEQ ID NO: 132 of US9475845; SEQ ID NO: 1518 herein), WGFP (SEQ ID NO: 133 of US9475845; SEQ ID NO: 1519 herein), AEPMPHSLNFSQYLWYT (SEQ ID NO: 134 of US9475845; SEQ ID NO: 1520 herein), WAYXSP (SEQ ID NO: 135 of US9475845; SEQ ID NO: 1521 herein), IELLQAR (SEQ ID NO: 136 of US9475845; SEQ ID NO: 1522 herein), NO: 136 of US9475845; SEQ ID NO: 1522 herein), AYTKCSRQWRTCMTTH (SEQ ID NO: 137 of US9475845; SEQ ID NO: 1523 herein), PQNSKIPGPTFLDPH (SEQ ID NO: 138 of US9475845; SEQ ID NO: 1524 herein), SMEPALPDWWWKMFK (SEQ ID NO: 139 of US9475845; SEQ ID NO: 1525 herein), ANTPCGPYTHDCPVKR (SEQ ID NO: 140 of US9475845; SEQ ID NO: 1526 herein), TACHQHVRMVRP (SEQ ID NO: 141 of US9475845; SEQ ID NO: 1527 herein), VPWMEPAYQRFL (SEQ ID NO: 142 of US9475845; SEQ ID NO: 143 herein), NO: 1528), DPRATPGS (SEQ ID NO: 143 of US9475845; SEQ ID NO: 1529 herein), FRPNRAQDYNTN (SEQ ID NO: 144 of US9475845; SEQ ID NO: 1530 herein), CTKNSYLMC (SEQ ID NO: 145 of US9475845; SEQ ID NO: 1531 herein), CXXTXXXGXGC (SEQ ID NO: 146 of US9475845; SEQ ID NO: 1532 herein), CPIEDRPMC (SEQ ID NO: 147 of US9475845);SEQ ID NO: 1537 herein), KSREHVNNSACPSKRITAAL (SEQ ID NO: 152 of US9475845; SEQ ID NO: 1538 herein), EGFR (SEQ ID NO: 153 of US9475845; SEQ ID NO: 154 herein), SEQ ID NO: 155 of US9475845, SEQ ID NO: 156 herein), SEQ ID NO: 157 of US9475845, SEQ ID NO: 1579 herein), SEQ ID NO: 158 of US9475845, SEQ ID NO: 1590 herein), SEQ ID NO: 1591 of US9475845, SEQ ID NO: 1592 herein), SEQ ID NO: 1593 of US9475845, SEQ ID NO: 1594 herein), SEQ ID NO: 1595 of US9475845, SEQ ID NO: 1596 herein), SEQ ID NO: 1597 of US9475845, SEQ ID NO: 1598 herein), SEQ ID NO: 1599 of US9475845, SEQ ID NO: 1600 herein), SEQ ID NO: 1610 of US9475845, SEQ ID NO: 1611 of US9475845, SEQ ID NO: 1612 herein), SEQ ID NO: 1613 of US9475845, SEQ ID NO: NO: 1539), AGLGVR (SEQ ID NO: 154 of US9475845; SEQ ID NO: 1540 herein), GTRQGHTMRLGVSDG (SEQ ID NO: 155 of US9475845; SEQ ID NO: 1541 herein), IAGLATPGWSHWLAL (SEQ ID NO: 156 of US9475845; SEQ ID NO: 1542 herein), SMSIARL (SEQ ID NO: 157 of US9475845; SEQ ID NO: 1543 herein), HTFEPGV (SEQ ID NO: 158 of US9475845; SEQ ID NO: 1544 herein), NTSLKRISNKRIRRK (SEQ ID NO: 159 of US9475845; SEQ ID NO: 1545 herein), LRIKRKRRKRKKTRK (SEQ ID NO: 150 of US9475845; SEQ ID NO: 1546 herein), NO: 160; herein SEQ ID NO: 1546), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH or GTV. ;

[0141] In some embodiments, the AAV serotype can be or can have a sequence as described in U.S. Patent Application Publication No. US20160369298 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, a site-specific mutated capsid protein of AAV2 (SEQ ID NO: 97 of US20160369298; SEQ ID NO: 1547 herein) or a variant thereof, wherein the specific mutation site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.

[0142] In addition, any mutant sequence described in US20160369298 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; SEQ ID NO: 1548 herein), SPSGASN (SEQ ID NO: 2 of US20160369298; SEQ ID NO: 1549 herein), SHSGASN (SEQ ID NO: 3 of US20160369298; SEQ ID NO: 1550 herein), SRSGASN (SEQ ID NO: 4 of US20160369298; SEQ ID NO: 1551 herein), SKSGASN (SEQ ID NO: 5 of US20160369298; SEQ ID NO: 1552 herein), SNSGASN (SEQ ID NO: 6 of US20160369298; SEQ ID NO: 1553 herein), NO: 1553), SGSGASN (SEQ ID NO: 7 of US20160369298; SEQ ID NO: 1554 herein), SASGASN (SEQ ID NOs: 8, 175, and 221 of US20160369298; SEQ ID NO: 1555 herein), SESGTSN (SEQ ID NO: 9 of US20160369298; SEQ ID NO: 1556 herein), STTGGSN (SEQ ID NO: 10 of US20160369298; SEQ ID NO: 1557 herein), SSAGSTN (SEQ ID NO: 11 of US20160369298; SEQ ID NO: 1558 herein), NNDSQA (SEQ ID NO: 12 of US20160369298; SEQ ID NO: 1559 herein), NO: 1559), NNRNQA (SEQ ID NO: 13 of US20160369298; SEQ ID NO: 1560 herein), NNNKQA (SEQ ID NO: 14 of US20160369298; SEQ ID NO: 1561 herein), NAKRQA (SEQ ID NO: 15 of US20160369298; SEQ ID NO: 1562 herein), NDEHQA (SEQ ID NO: 16 of US20160369298; SEQ ID NO: 1563 herein), NTSQKA (SEQ ID NO: 17 of US20160369298);SEQ ID NO: 1564 herein), YYLSRTNTPSGTDTQSRLVFSQAGA (SEQ ID NO: 18 of US20160369298; SEQ ID NO: 1565 herein), YYLSRTNTDSGTETQSGLDFSQAGA (SEQ ID NO: 19 of US20160369298; SEQ ID NO: 1566 herein), YYLSRTNTESGTPTQSALEFSQAGA (SEQ ID NO: 20 of US20160369298; SEQ ID NO: 1567 herein), YYLSRTNTHSGTHTQSPLHFSQAGA (SEQ ID NO: 21 of US20160369298; SEQ ID NO: 1568 herein), YYLSRTNTSSGTITISHLIFSQAGA (SEQ ID NO: 22 of US20160369298; SEQ ID NO: 1569 herein), NO:1569), YYLSRTNTRSGIMTKSSLMFSQAGA;

[0143] (US20160369298 SEQ ID NO:23; SEQ ID NO:1570 in the text), YYLSRTNTKSGRKTLSNLSFSQAGA(US20160369298 SEQ ID NO:24; SEQ ID NO:1571 in the text), YYLSRTNDGSGPVTPSKLRFSQRGA(US20160369298 SEQ ID NO:25; SEQ ID NO:1572 in the text), YYLSRTNAASGHATHSDLKFSQPGA(SEQ ID NO:26 in the US20160369298; SEQ ID NO:1573 in the text), YYLSRTNGQAGSLTMSELGFSQVGA(SEQ ID NO:27 in the text NO:1574), YYLSRTNSTGGNQTTSQLLFSQLSA(US20160369298's SEQ ID NO:28; SEQ ID NO:1575 in the text), YFLSRTNNNTGLNTNSTLNFSQGRA(US20160369298's SEQ ID NO:29; SEQ ID in the text NO:1576), SKTGADNNNSEYSWTG(US20160369298's SEQ ID NO:30; SEQ ID NO:1577 in the text), SKTDADNNNSEYSWTG(US20160369298's SEQ ID NO:31; SEQ ID in the text NO:1578), SKTEADNNNSEYSWTG(US20160369298 SEQ ID NO:32; SEQ ID in text NO:1579), SKTPADNNNSEYSWTG(US20160369298's SEQ ID NO:33; SEQ ID NO:1580 in the text), SKTHADNNNSEYSWTG(US20160369298's SEQ ID NO:34; SEQ ID in the text NO:1581), SKTQADNNNSEYSWTG(US20160369298's SEQ ID NO:35; SEQ ID NO:1582 in the text), SKTIADNNNSEYSWTG(US20160369298's SEQ ID NO:36; SEQ ID in the text NO:1583), SKTMADNNNSEYSWTG(US20160369298 SEQ ID NO:37;SEQ ID NO: 1584 herein), SKTRADNNNSEYSWTG (SEQ ID NO: 38 of US20160369298; SEQ ID NO: 1585 herein), SKTNADNNNSEYSWTG (SEQ ID NO: 39 of US20160369298; SEQ ID NO: 1586 herein), SKTVGRNNNSEYSWTG (SEQ ID NO: 40 of US20160369298; SEQ ID NO: 1587 herein), SKTADRNNNSEYSWTG (SEQ ID NO: 41 of US20160369298; SEQ ID NO: 1588 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 42 of US20160369298; SEQ ID NO: 1589 herein), SKPTTGNNNSDYSWPG (SEQ ID NO: 43 of US20160369298; SEQ ID NO: 1590 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 44 of US20160369298; SEQ ID NO: 1591 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 45 of US20160369298; SEQ ID NO: 1592 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 46 of US20160369298; SEQ ID NO: 1593 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 47 of US20160369298; SEQ ID NO: 1594 herein), SKKLSQNNNSKYSWQG (SEQ ID NO: 48 of US20160369298; SEQ ID NO: 1595 herein), SKKLSQNNNSKYSWQ NO: 43; SEQ ID NO: 1590 herein), STQKNENNNSNYSWPG (SEQ ID NO: 44 of US20160369298; SEQ ID NO: 1591 herein), HKDDEGKF (SEQ ID NO: 45 of US20160369298; SEQ ID NO: 1592 herein), HKDDNRKF (SEQ ID NO: 46 of US20160369298; SEQ ID NO: 1593 herein), HKDDTNKF (SEQ ID NO: 47 of US20160369298; SEQ ID NO: 1594 herein), HEDSDKNF (SEQ ID NO: 48 of US20160369298; SEQ ID NO: 1595 herein), HRDGADSF (SEQ ID NO: 49 of US20160369298; SEQ ID NO: 1596 herein), NO: 1596), HGDNKSRF (SEQ ID NO: 50 of US20160369298; SEQ ID NO: 1597 herein), KQGSEKTNVDFEEV (SEQ ID NO: 51 of US20160369298; SEQ ID NO: 1598 herein), KQGSEKTNVDSEEV (SEQ ID NO: 52 of US20160369298; SEQ ID NO: 1599 herein), KQGSEKTNVDVEEV (SEQ ID NO: 53 of US20160369298);SEQ ID NO: 1600 herein), KQGSDKTNVDDAGV (SEQ ID NO: 54 of US20160369298; SEQ ID NO: 1601 herein), KQGSSKTNVDPREV (SEQ ID NO: 55 of US20160369298; SEQ ID NO: 1602 herein), KQGSRKTNVDHKQV (SEQ ID NO: 56 of US20160369298; SEQ ID NO: 1603 herein), KQGSKGGNVDTNRV (SEQ ID NO: 57 of US20160369298; SEQ ID NO: 1604 herein), KQGSGEANVDNGDV (SEQ ID NO: 58 of US20160369298; SEQ ID NO: 1605 herein), KQDAAADNIDYDHV (SEQ ID NO: 59 of US20160369298; SEQ ID NO: 1616 herein), KQGSKGGNVDTNRV (SEQ ID NO: 59 of US20160369298; SEQ ID NO: 1617 herein), KQGSGEANVDNGDV (SEQ ID NO: 50 of US20160369298; SEQ ID NO: 1618 herein), KQDAAADNIDYDHV (SEQ ID NO: 51 of US20160369298; NO: 59; SEQ ID NO: 1606 herein), KQSGTRSNAAASSV (SEQ ID NO: 60 of US20160369298; SEQ ID NO: 1607 herein), KENTNTNDTELTNV (SEQ ID NO: 61 of US20160369298; SEQ ID NO: 1608 herein), QRGNNVAATADVNT (SEQ ID NO: 62 of US20160369298; SEQ ID NO: 1609 herein), QRGNNEAATADVNT (SEQ ID NO: 63 of US20160369298; SEQ ID NO: 1610 herein), QRGNNPAATADVNT (SEQ ID NO: 64 of US20160369298; SEQ ID NO: 1611 herein), QRGNNHAATADVNT (SEQ ID NO: 1612 of US20160369298; SEQ ID NO: 1613 herein), NO: 65; SEQ ID NO: 1612 herein), QEENNIAATPGVNT (SEQ ID NO: 66 of US20160369298; SEQ ID NO: 1613 herein), QPPNNMAATHEVNT (SEQ ID NO: 67 of US20160369298; SEQ ID NO: 1614 herein), QHHNNSAATTIVNT (SEQ ID NO: 68 of US20160369298; SEQ ID NO: 1615 herein), QTTNNRAAFNMVET (SEQ ID NO: 69 of US20160369298);SEQ ID NO: 1616 herein), QKKNNNAASKKVAT (SEQ ID NO: 70 of US20160369298; SEQ ID NO: 1617 herein), QGGNNKAADDAVKT (SEQ ID NO: 71 of US20160369298; SEQ ID NO: 1618 herein), QAAKGGAADDAVKT (SEQ ID NO: 72 of US20160369298; SEQ ID NO: 1619 herein), QDDRAAAANESVDT (SEQ ID NO: 73 of US20160369298; SEQ ID NO: 1620 herein), QQQHDDAAYQRVHT (SEQ ID NO: 74 of US20160369298; SEQ ID NO: 1621 herein), QSSSSLAAVSTVQT (SEQ ID NO: 75 of US20160369298; SEQ ID NO: 1622 herein), NO: 75; SEQ ID NO: 1622 herein), QNNQTTAAIRNVTT (SEQ ID NO: 76 of US20160369298; SEQ ID NO: 1623 herein), NYNKKSDNVDFT (SEQ ID NO: 77 of US20160369298; SEQ ID NO: 1624 herein), NYNKKSENVDFT (SEQ ID NO: 78 of US20160369298; SEQ ID NO: 1625 herein), NYNKKSLNVDFT (SEQ ID NO: 79 of US20160369298; SEQ ID NO: 1626 herein), NYNKKSPNVDFT (SEQ ID NO: 80 of US20160369298; SEQ ID NO: 1627 herein), NYSKKSHCVDFT (SEQ ID NO: 81 of US20160369298; SEQ ID NO: 1628 herein), NO: 1628), NYRKTIYVDFT (SEQ ID NO: 82 of US20160369298; SEQ ID NO: 1629 herein), NYKEKKDVHFT (SEQ ID NO: 83 of US20160369298; SEQ ID NO: 1630 herein), NYGHRAIVQFT (SEQ ID NO: 84 of US20160369298; SEQ ID NO: 1631 herein), NYANHQFVVCT (SEQ ID NO: 85 of US20160369298);SEQ ID NO: 1632 herein), NYDDDPTGVLLT (SEQ ID NO: 86 of US20160369298; SEQ ID NO: 1633 herein), NYDDPTGVLLT (SEQ ID NO: 87 of US20160369298; SEQ ID NO: 1634 herein), NFEQQNSVEWT (SEQ ID NO: 88 of US20160369298; SEQ ID NO: 1635 herein), SQSGASN (SEQ ID NO: 89 and SEQ ID NO: 241 of US20160369298; SEQ ID NO: 1636 herein), NNGSQA (SEQ ID NO: 90 of US20160369298; SEQ ID NO: 1637 herein), YYLSRTNTPSGTTTWSRLQFSQAGA (SEQ ID NO: 1638 of US20160369298). ID NO: 91; SEQ ID NO: 1638 herein), SKTSADNNNSEYSWTG (SEQ ID NO: 92 of US20160369298; SEQ ID NO: 1639 herein), HKDDEEKF (SEQ ID NOs: 93, 209, 214, 219, 224, 234, 239, and 244 of US20160369298; SEQ ID NO: 1640 herein), KQGSEKTNVDIEEV (SEQ ID NO: 94 of US20160369298; SEQ ID NO: 1641 herein), QRGNNQAATADVNT (SEQ ID NO: 95 of US20160369298; SEQ ID NO: 1642 herein), NYNKKSVNVDFT (SEQ ID NO: 96 of US20160369298; SEQ ID NO: 1643 herein);

[0144] SQSGASNYNTPSGTTTQSRLQFSTSADNNNSEYSWTGATKYH (SEQ ID NO: 106 of US20160369298; SEQ ID NO: 1644 herein), SASGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 107 of US20160369298; SEQ ID NO: 1645 herein), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSEYSWTGATKYH (SEQ ID NO: 108 of US20160369298; SEQ ID NO: 1646 herein), SASGASNYNTPSGTTTQSRLQFSTSADNNNSEFSWPGATTYH (SEQ ID NO: 109 of US20160369298; SEQ ID NO: 1650 herein). NO:1647), SQSGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO:110 of US20160369298; SEQ ID NO:1648 in this article), SASGASNYNTPSGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO:111 of US20160369298; SEQ ID NO:1648 in this article) 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 NO:1650 in this article) NO: 1651), SGAGASN (SEQ ID NO: 176 of US20160369298; SEQ ID NO: 1652 herein), NSEGGSLTQSSLGFS (SEQ ID NOs: 177, 185, 193 and 202 of US20160369298; SEQ ID NO: 1653 herein), TDGENNNSDFS (SEQ ID NO: 178 of US20160369298; SEQ ID NO: 1654 herein), SEFSWPGATT (SEQ ID NO: 179 of US20160369298);SEQ ID NO: 1657 herein), TSADNNNSDFSWT (SEQ ID NO: 180 of US20160369298; SEQ ID NO: 1658 herein), SQSGASNY (SEQ ID NO: 181, 187, and 198 of US20160369298; SEQ ID NO: 1659 herein), NTPSGTTTQSRLQFS (SEQ ID NO: 182, 188, 191, and 199 of US20160369298; SEQ ID NO: 1660 herein), TSADNNNSEYSWTGATKYH (SEQ ID NO: 183 of US20160369298; SEQ ID NO: 1661 herein), SASGASNF (SEQ ID NO: 184 of US20160369298; SEQ ID NO: 1662 herein), NO: 1660), TDGENNNSDFSWTGATKYH (SEQ ID NOs: 186, 189, 194, 197, and 203 of US20160369298; SEQ ID NO: 1661 herein), SASGASNY (SEQ ID NOs: 190 and 195 of US20160369298; SEQ ID NO: 1662 herein), TSADNNNSEFSWPGATTYH (SEQ ID NO: 192 of US20160369298; SEQ ID NO: 1663 herein), NTPSGSLTQSSLGFS (SEQ ID NO: 196 of US20160369298; SEQ ID NO: 1664 herein), TSADNNNSDFSWTGATKYH (SEQ ID NO: 200 of US20160369298; SEQ ID NO: 201 herein), NO: 1665), SGAGASNF (SEQ ID NO: 201 of US20160369298; SEQ ID NO: 1666 herein);

[0145] CTCCAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACACAA (SEQ ID NO: 204 of US20160369298; SEQ ID NO: 1667 herein), CTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACA A (SEQ ID NO: 205 of US20160369298; SEQ ID NO: 1668 herein), SAAGASN (SEQ ID NO: 206 of US20160369298; SEQ ID NO: 1669 herein), YFLSRTNTESGSTTQSTLRFSQAG (SEQ ID NO: 207 of US20160369298; SEQ ID NO: 1670 herein), SKTSADNNNSDFS (SEQ ID NOs: 208, 228, and 253 of US20160369298; SEQ ID NO: 1671 herein), NO: 1671), KQGSEKTDVDIDKV (SEQ ID NO: 210 of US20160369298; SEQ ID NO: 1672 herein), STAGASN (SEQ ID NO: 211 of US20160369298; SEQ ID NO: 1673 herein), YFLSRTNTTSGIETQSTLRFSQAG (SEQ ID NO: 212 and SEQ ID NO: 247 of US20160369298; SEQ ID NO: 1674 herein), SKTDGENNNSDFS (SEQ ID NO: 213 and SEQ ID NO: 248 of US20160369298; SEQ ID NO: 1675 herein), KQGAAADDVEIDGV (SEQ ID NO: 215 and SEQ ID NO: 250 of US20160369298; SEQ ID NO: 1676 herein), NO: 1676), SEAGASN (SEQ ID NO: 216 of US20160369298; SEQ ID NO: 1677 herein), YYLSRTNTPSGTTTQSRLQFSQAG (SEQ ID NO: 217, 232, and 242 of US20160369298; SEQ ID NO: 1678 herein), SKTSADNNNSEYS (SEQ ID NO: 218, 233, 238, and 243 of US20160369298; SEQ ID NO: 1679 herein), KQGSEKTNVDIEKV (SEQ ID NO: 220, 225, and 245 of US20160369298);SEQ ID NO: 1680 herein), YFLSRTNDASGSDTKSTLLFSQAG (SEQ ID NO: 222 of US20160369298; SEQ ID NO: 1681 herein), STTPSENNNSEYS (SEQ ID NO: 223 of US20160369298; SEQ ID NO: 1682 herein), SAAGATN (SEQ ID NO: 226 and SEQ ID NO: 251 of US20160369298; SEQ ID NO: 1683 herein), YFLSRTNGEAGSATLSELRFSQAG (SEQ ID NO: 227 of US20160369298; SEQ ID NO: 1684 herein), HGDDADRF (SEQ ID NO: 229 and SEQ ID NO: 254 of US20160369298; SEQ ID NO: 1685 herein). NO: 1685), KQGAEKSDVEVDRV (SEQ ID NO: 230 and SEQ ID NO: 255 of US20160369298; SEQ ID NO: 1686 herein), KQDSGGDNIDIDQV (SEQ ID NO: 235 of US20160369298; SEQ ID NO: 1687 herein), SDAGASN (SEQ ID NO: 236 of US20160369298; SEQ ID NO: 1688 herein), YFLSRTNTEGGHDTQSTLRFSQAG (SEQ ID NO: 237 of US20160369298; SEQ ID NO: 1689 herein), KEDGGGSDVAIDEV (SEQ ID NO: 240 of US20160369298; SEQ ID NO: 1690 herein), SNAGASN (SEQ ID NO: 241 of US20160369298; SEQ ID NO: 1691 herein), ID NO: 246; SEQ ID NO: 1691 herein), and YFLSRTNGEAGSATLSELRFSQPG (SEQ ID NO: 252 of US20160369298; SEQ ID NO: 1692 herein). Non-limiting examples of nucleotide sequences that can encode amino acid mutation sites include the following: AGCVVMDCAGGARSCASCAAC (SEQ ID NO: 97 of US20160369298; SEQ ID NO: 1693 herein), AACRACRRSMRSMAGGCA (SEQ ID NO: 98 of US20160369298;SEQ ID NO: 1694 herein), CACRRGGACRRCRMSRRSARSTTT (SEQ ID NO: 99 of US20160369298; SEQ ID NO: 1695 herein);

[0146] TATTTCTTGAGCAGAACAAACRVCVVSRSCGGAMNCVHSACGMHSTCA VVSCTTVDSTTTTCTCAGSBCRGSGCG (SEQ ID NO: 100 of US20160369298; SEQ ID NO: 1696 herein),

[0147] TCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCGTGGMMAGG A (SEQ ID NO: 101 of US20160369298; SEQ ID NO: 1697 herein), AAGSAARRCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTC (SEQ ID NO: 102 of US20160369298; SEQ ID NO: 1698 herein), CAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACA (SEQ ID NO: 103 of US20160369298; SEQ ID NO: 1699 herein), AACTWCRVSVASMVSVHSDDTGTGSWSTKSACT (SEQ ID NO: 104 of US20160369298; SEQ ID NO: 1691 herein), NO: 1700), TTGTTGAACATCACCACGTGACGCACGTTC (SEQ ID NO: 256 of US20160369298; SEQ ID NO: 1701 herein),

[0148] TCCCCGTGGTTCTACTACATAATGTGGCCG (SEQ ID NO: 257 of US20160369298; SEQ ID NO: 1702 herein),

[0149] TTCCACACTCCGTTTTGGATAATGTTGAAC (SEQ ID NO: 258 of US20160369298; SEQ ID NO: 1703 herein),

[0150] AGGGACATCCCCAGCTCCATGCTGTGGTCG (SEQ ID NO: 259 of US20160369298; SEQ ID NO: 1704 herein),

[0151] AGGGACAACCCCTCCGACTCGCCCTAATCC (SEQ ID NO: 260 of US20160369298; SEQ ID NO: 1705 herein),

[0152] TCCTAGTAGAAGACACCCTCTCACTGCCCG (SEQ ID NO: 261 of US20160369298; SEQ ID NO: 1706 herein),

[0153] AGTACCATGTACACCCACTCTCCCAGTGCC (SEQ ID NO: 262 of US20160369298; SEQ ID NO: 1707 herein),

[0154] ATATGGACGTTCATGCTGATCACCATACCG (SEQ ID NO: 263 of US20160369298; SEQ ID NO: 1708 herein),

[0155] AGCAGGAGCTCCTTGGCCTCAGCGTGCGAG (SEQ ID NO: 264 of US20160369298; SEQ ID NO: 1709 herein),

[0156] ACAAGCAGCTTCACTATGACAACCACTGAC (SEQ ID NO: 265 of US20160369298; SEQ ID NO: 1710 herein),

[0157] CAGCCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGAGAGTC

[0158] TCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCCTGGMMAGG

[0159] AGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCCGG

[0160] ACCAGCTATGGCAAGCCACRRGGACRRCRMSRRSARSTTTTTCCTCAG

[0161] AGCGGGGTTCTCATCTTTGGGAAGSAARRCRSCRVSRVARVCRATRYCG

[0162] MSNHCRVMVRSGTCATGATTACAGACGAAGAGGAGATCTGGAC (SEQ ID NO: 266 of US20160369298; SEQ ID NO: 1711 herein), TGGGACAATGGCGGTCGTCTCTCAGAGTTKTKKT (SEQ ID NO: 267 of US20160369298; SEQ ID NO: 1712 herein),

[0163] AGAGGACCKKTCCTCGATGGTTCATGGTGGAGTTA (SEQ ID NO: 268 of US20160369298; SEQ ID NO: 1713 in this article),

[0164] CCACTTAGGGCCTGGTCGATACCGTTCGGTG (SEQ ID NO: 269 of US20160369298; SEQ ID NO: 1714 herein), and

[0165] TCTCGCCCCAAGAGTAGAAACCCTTCSTTYYG (SEQ ID NO: 270 of US20160369298; SEQ ID NO: 1715 herein).

[0166] In some embodiments, the AAV serotype may comprise an ocular cell targeting peptide as described in International Patent Publication No. WO2016134375 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, SEQ ID NO: 9 and SEQ ID NO: 10 of WO2016134375. Furthermore, any ocular cell targeting peptide or amino acid described in WO2016134375 may be inserted into any parent AAV serotype, such as, but not limited to, AAV2 (SEQ ID NO: 8 of WO2016134375; SEQ ID NO: 1716 herein), or AAV9 (SEQ ID NO: 11 of WO2016134375; SEQ ID NO: 1717 herein). In some embodiments, modifications, such as insertions, are made in the AAV2 protein at P34-A35, T138-A139, A139-P140, G453-T454, N587-R588, and / or R588-Q589. In certain embodiments, insertions are made at D384, G385, I560, T561, N562, E563, E564, E565, N704, and / or Y705 of AAV9. The ocular cell targeting peptide may be, but is not limited to, any of the following amino acid sequences: GSTPPPM (SEQ ID NO: 1 of WO2016134375; SEQ ID NO: 1718 herein) or GETRAPL (SEQ ID NO: 4 of WO2016134375; SEQ ID NO: 1719 herein).

[0167] In some embodiments, the AAV serotypes can be modified, as described in U.S. Patent Application Publication No. US20170145405 (the contents of which are incorporated herein by reference in their entirety). The AAV serotypes can 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).

[0168] In some embodiments, the AAV serotype can be modified as described in International Publication No. WO2017083722 (the contents of which are incorporated herein by reference in their 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).

[0169] In some embodiments, as described in International Patent Publication No. WO2017015102 (the contents of which are incorporated herein by reference in their entirety), an AAV serotype may comprise an engineered epitope comprising amino acids SPAKFA (SEQ ID NO: 24 of WO2017015102; SEQ ID NO: 1720 herein) or NKDKLN (SEQ ID NO: 2 of WO2017015102; SEQ ID NO: 1721 herein). The epitope may be inserted in the region of amino acids 665 to 670 based on VP1 capsid numbering of AAV8 (SEQ ID NO: 3 of WO2017015102) and / or residues 664 to 668 of AAV3B (SEQ ID NO: 3).

[0170] In some embodiments, the AAV serotype may be or may have a sequence as described in International Patent Publication No. WO2017058892 (the contents of which are incorporated herein by reference in their entirety), such as, but not limited to, an AAV variant having a capsid protein comprising amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500, 528-534, 547-552 of AAV1. , 588-597, 709-710, 716-722 (in any combination), or one or more (e.g., 2, 3, 4, 5, 6, or 7) of the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. Amino acid substitutions may be, but are not limited to, any of the amino acid sequences described in WO2017058892.In some embodiments, the AAV may comprise amino acid substitutions at residues 256L, 258K, 259Q, 261S, 263A, 264S, 265T, 266G, 272H, 385S, 386Q, S472R, V473D, N500E 547S, 709A, 710N, 716D, 717N, 718N, 720L, A456T, Q457T, N458Q, K459S, T492S, K493A, S586R, S587G, S588N, T589R and / or 722T (in any combination) of AAV1 (SEQ ID NO: 1 of WO2017058892), AAV5 (SEQ ID NO: 2 708E, 709Y, and / or 710R (in any combination) of AAV5 (SEQ ID NO: 5), amino acid substitutions at 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) of AAV5 (SEQ ID NO: 5), amino acid substitutions at 244N, 246Q, 248R, 249E, 532Q 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D, 706T, 708E, 709Y and / or (in any combination) of AAV6 (SEQ ID NO: 6 WO2017058892), amino acid substitutions at 264S, 266G, 269N, 272H, 457Q, 588S and / or 589I (in any combination) of AAV8 (SEQ ID NO: 6 WO2017058892), amino acid substitutions at 264S, 266G, 269N, 272H, 457Q, 588S and / or 589I (in any combination) of AAV6 (SEQ ID NO: 6 WO2017058892), amino acid substitutions at 264S, 266G, 269N, 272H, 457Q, 588S and / or 589I (in any combination) of AAV8 (SEQ ID NO: 6 WO2017058892), amino acid substitutions at 264S, 266G, 269N, 272H, NO: 8), amino acid substitutions at 457T, 459N, 496G, 499N, 500N, 589Q, 590N and / or 592A (in any combination) of AAV9 (SEQ ID NO: 9WO2017058892), amino acid substitutions at 451I, 452N, 453G, 454S, 455G, 456Q, 457N and / or 458Q (in any combination) of AAV9 (SEQ ID NO: 9WO2017058892).

[0171] In some embodiments, the AAV may include the amino acid sequence at positions 155, 156, and 157 of VP1 or positions 17, 18, 19, and 20 of VP2 as described in International Publication No. WO 2017066764, the contents of which are incorporated herein by reference in their entirety. The amino acid sequence may be, but is not limited to, NSS, SXS, SSY, NXS, NSY, SXY, and NXY, wherein N, X, and Y are, but are not limited to, independently non-serine or non-threonine amino acids, 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 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 can be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

[0172] In some embodiments, the AAV can be a serotype produced by Cre-based AAV targeted evolution (CREATE) as described in Deverman et al. (Nature Biotechnology 34(2):204-209 (2016)), Chan et al., (Nature Neuroscience 20(8):1172-1179 (2017)), and International Patent Application Publication Nos. WO2015038958 and WO2017100671, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the AAV serotype produced in this manner has improved CNS transduction and / or neuronal and astrocyte tropism compared to AAV serotypes not produced in this manner. As non-limiting examples, the AAV serotype can 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, the AAV serotypes can be derivatives of AAV9 (SEQ ID NO: 136) or AAV9K449R (SEQ ID NO: 9) having an amino acid insertion between amino acids 588 and 589. Non-limiting examples of these amino acid insertions include TLAVPFK (PHP.B; SEQ ID NO: 1260), SVSKPFL (PHP.B2; SEQ ID NO: 1268), FTLTTPK (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).

[0173] In some embodiments, the AAV serotype 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.

[0174] 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.

[0175] In some embodiments, the AAV serotype is the K449R AAV9 variant (SEQ ID NO: 9). AAV9 K449R has the same function as wild-type AAV9. In some embodiments, the AAV serotype is AAV9K449R with a peptide insert.

[0176] In some embodiments, the AAV serotype is PHP.B (e.g., as described in WO2015038958). In some embodiments, the AAV serotype is paired with the synapsin promoter to enhance neuronal transduction compared to using a more ubiquitous promoter (i.e., CBA or CMV).

[0177] In some embodiments, the AAV serotype is PHP.N (e.g., as described in WO2017100671).

[0178] In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.N (PHP.N) peptide or a variant thereof.

[0179] In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.B (PHP.B) peptide or a variant thereof.

[0180] In some embodiments, the AAV serotype is a serotype comprising the AAVPHP.A (PHP.A) peptide or a variant thereof.

[0181] In some embodiments, the AAV serotype is a serotype comprising the PHP.S peptide or a variant thereof.

[0182] In some embodiments, the AAV serotype is a serotype comprising the PHP.B2 peptide or a variant thereof.

[0183] In some embodiments, the AAV serotype is a serotype comprising the PHP.B3 peptide or a variant thereof.

[0184] In some embodiments, the AAV serotype is a serotype comprising the G2B4 peptide or a variant thereof.

[0185] In some embodiments, the AAV serotype is a serotype comprising the G2B5 peptide or a variant thereof.

[0186] 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.

[0187] 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.

[0188] In some embodiments, the AAV capsid allows blood-brain barrier penetration after intravenous administration. Non-limiting examples of such AAV capsids include AAV9, AAV9 K449R, VOY101, VOY201, or AAV capsids comprising 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).

[0189] In some embodiments, the AAV serotype can comprise a capsid amino acid sequence that is 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 to any of those described above. In some embodiments, the 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, the 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, the 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, the 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, the 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 sequence of SEQ ID NO: 1, 2, 3, 9, 136, or 1724.

[0190] In some embodiments, an AAV serotype can be encoded by a capsid nucleic acid sequence that is 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 to any of the above-mentioned. In some embodiments, the 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, the 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, the 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, the 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, the 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.

[0191] In some embodiments, the initiation codon for translation of the AAV VP1 capsid protein can be CTG, TTG, or GTG, as described in U.S. Pat. No. 8,163,543, the contents of which are incorporated herein by reference in their entirety.

[0192] The present disclosure relates to structural capsid proteins (including VP1, VP2 and VP3) encoded by capsid (Cap) genes. These capsid proteins form the outer protein structural shell (i.e., capsid) of viral vectors such as AAV. The VP capsid proteins synthesized by Cap polynucleotides typically include methionine as the first amino acid in the peptide sequence (Met1), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, the first methionine (Met1) residue or generally any first amino acid (AA1) is usually cut by a protein processing enzyme such as Met-aminopeptidase after or during polypeptide synthesis. This "Met / AA-clipping" processing is usually associated with the corresponding acetylation of the second amino acid (e.g., alanine, valine, serine, threonine, etc.) in the polypeptide sequence. Met clipping usually occurs in VP1 and VP3 capsid proteins, but may also occur in VP2 capsid protein.

[0193] When Met / AA cleavage is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid may be produced, some of which may include Met1 / AA1 amino acids (Met+ / AA+), and some of which may lack Met1 / AA1 amino acids due to Met / AA cleavage (Met- / AA-). For further discussion of Met / AA cleavage 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. 2010 February 19. 327(5968): 973–977; the contents of each of which are incorporated herein by reference in their entirety.

[0194] According to the present disclosure, reference to a capsid protein is not limited to a cleaved (Met- / AA-) or uncleaved (Met+ / AA+) sequence, and in the context may refer to an individual capsid protein, a viral capsid comprising a mixture of capsid proteins, and / or a polynucleotide sequence (or fragment thereof) encoding, describing, producing or obtaining a capsid protein of the present disclosure. Direct reference to a "capsid protein" or "capsid polypeptide" (such as VP1, VP2 or VP2) may also include: a VP capsid protein comprising Met1 / AA1 amino acids (Met+ / AA+), and a corresponding VP capsid protein lacking Met1 / AA1 amino acids due to Met / AA cleavage (Met- / AA-).

[0195] Further in accordance with the present disclosure, reference to a particular SEQ ID NO: (whether protein or nucleic acid) comprising or encoding one or more capsid proteins including Met1 / AA1 amino acids (Met+ / AA+), respectively, should be understood to teach VP capsid proteins lacking the Met1 / AA1 amino acids, as will be apparent upon inspection of the sequence, any sequence lacking only the first listed amino acid (whether or not methionine).

[0196] As a non-limiting example, a 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 start codon can also be understood as teaching a VP1 polypeptide sequence that is 735 amino acids in length and does not include the "Met1" amino acid (Met-) of the 736th amino acid Met+ sequence. As a second non-limiting example, a 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 can also be understood as teaching a VP1 polypeptide sequence that is 735 amino acids in length and does not include the "AA1" amino acid (AA1-) of the 736th amino acid AA1+ sequence.

[0197] Reference to a viral capsid formed by a VP capsid protein (e.g., reference to a specific AAV capsid serotype) may include a VP capsid protein comprising Met1 / AA1 amino acids (Met+ / AA1+), the corresponding VP capsid protein lacking Met1 / AA1 amino acids due to Met / AA1-cleavage (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0198] As non-limiting examples, 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 similar optional combinations of VP2 (Met+ / AA1) and VP2 (Met- / AA1-).

[0199] expression vector

[0200] In some aspects, the AAV particles of the present disclosure serve as expression vectors encoding FXN. The expression vector is not limited to AAV, and can be an adenovirus, a retrovirus, a lentivirus, a plasmid, a vector, or any variant thereof.

[0201] In some embodiments, the AAV particle expression vector can comprise, from 5' to 3', the ITR, a promoter, an intron, a nucleic acid sequence encoding FXN, a polyA sequence, and the ITR.

[0202] Inverted terminal repeats (ITRs)

[0203] The AAV particles of the present disclosure comprise a viral genome having at least one ITR region and a payload region encoding FXN. As used herein, a "viral genome" or "vector genome" is a polynucleotide comprising at least one inverted terminal ITR and at least one encoded payload. In one embodiment, the viral genome comprises two ITRs. These two ITRs flank the payload region at the 5' and 3' ends. The ITRs function as origins of replication containing replication recognition sites. The ITRs comprise sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the viral genome of the present disclosure can be composed of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0204] The ITRs can be derived from the same serotype as the capsid, selected from any serotype listed in Table 1 or a derivative thereof. The ITRs can be of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In some embodiments, the AAV particle has a viral genome comprising 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 0, 1, or 2 of the ITRs having the same serotype as the capsid. In some embodiments, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0205] In some embodiments, the length of ITR is 140-142 nucleotides. Non-limiting examples of ITR length are 102, 105, 119, 130, 140, 141, 142 or 145 nucleotides in length, and nucleotides with at least 95% identity thereto.

[0206] In some embodiments, 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, 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. In some embodiments, the viral genome comprises 5' and 3' ITRs as described above, and a payload region encoding a frataxin, e.g., encoding SEQ ID NO: 1725 or a variant thereof having at least 90% sequence identity. In some embodiments, the viral genome comprises 5' and 3' ITRs as described above, and a payload region encoding a frataxin, e.g., comprising SEQ ID NO: 1824 or a variant thereof having at least 90% sequence identity, e.g., a variant that retains one or more functional properties of a wild-type frataxin.

[0207] promoter

[0208] 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).

[0209] In some embodiments, delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) comprises a composition wherein the AAV genome further comprises a cell-specific promoter region. In some embodiments, delivery comprises a composition wherein the AAV genome further comprises a ubiquitous promoter region.

[0210] In some embodiments, the promoter is effective to drive expression of a payload or transgene. In some embodiments, the promoter is effective to drive expression of FXN.

[0211] 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 expression.

[0212] In some embodiments, the promoter is a weak promoter that expresses the payload (e.g., FXN) for a sustained period of time in a target tissue, such as, but not limited to, nervous system tissue (e.g., CNS tissue). Expression can continue for 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, 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, 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. Expression can continue for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years. In some embodiments, the promoter is a weak promoter for sustained expression of the payload in neural tissue.

[0213] In some embodiments, the promoter may be a promoter that is less than 1 kb in size. The promoter may have a length of 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, 480 700, 780, 790, 800 or more nucleotides. The promoter may have a length of between 50-100, 100-150, 150-200, 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800 nucleotides.

[0214] In some embodiments, the promoter can be a combination of two or more components, such as, but not limited to, CMV and CBA. Each component can have a length of 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, 701, 710, 720, 730, 740, 750, 760, 770, 780, 790, 801, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 700, 780, 790, 800 or more nucleotides. In some embodiments, the promoter is a combination of a CMV-enhancer sequence of 382 nucleotides and a CBA-promoter sequence of 260 nucleotides. In some embodiments, the promoter is a combination of a 380-nucleotide CMV-enhancer sequence and a 260-nucleotide CBA-promoter sequence.

[0215] In some embodiments, the vector genome comprises at least one element that enhances the 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 herein by reference in their entirety). Non-limiting examples of elements that enhance expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, upstream enhancers (USEs), CMV enhancers, and / or introns. In certain embodiments, elements for enhancing the target specificity and / or expression of FXN are referred to as "enhancers" or "enhancer sequences." In some embodiments, the promoter may comprise an enhancer sequence. In some embodiments, the enhancer may be a separate component in the viral genome that is distinct from the promoter. In some embodiments, the enhancer may be at the 5' end of the promoter sequence in the viral genome. In some embodiments, the enhancer may be at the 3' end of the promoter sequence in the viral genome. In some embodiments, the enhancer comprises or consists of SEQ ID NO: 1777.

[0216] As used herein, "intron" or "intron sequence" encompasses full-length introns or fragments thereof. As used herein, "exon" or "exon sequence" encompasses full-length exons or fragments thereof. In some embodiments, the enhancer may comprise at least one intron or exon sequence. In some embodiments, the enhancer may comprise at least one intron sequence. In some embodiments, the enhancer may comprise at least one exon sequence. In some embodiments, the enhancer comprises one intron sequence and one exon sequence. In some embodiments, the enhancer sequence comprises two intron sequences. In some embodiments, the enhancer sequence comprises two exon sequences. In some embodiments, the enhancer sequence comprises two intron sequences and two exon sequences. In some embodiments, the enhancer comprises SEQ ID NO: 1818. In some embodiments, the enhancer may comprise two intron sequences and two exon sequences. In some embodiments, the enhancer may comprise an ie1 exon (e.g., exon 1), an ie1 intron (e.g., intron 1), a human β-globin intron (e.g., intron 2), and a human β-globin exon (e.g., exon 3). In some embodiments, the enhancer may comprise SEQ ID NOs: 1817, 1819, 1820, 1821 from 5' to 3'. In some embodiments, the enhancer may comprise SEQ ID NO: 1816.

[0217] Promoters that promote expression in most tissues include, but are not limited to, human elongation factor 1α subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (βglucuronidase) (GUSB) or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types, such as, but not limited to, nervous system promoters, which can be used to restrict expression to neurons, astrocytes or oligodendrocytes. Non-limiting examples of neuronal tissue-specific expression elements 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 oligodendrocytes include myelin basic protein (MBP) promoter.

[0218] In some embodiments, the viral genome comprises a ubiquitous promoter. 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 contents of which are incorporated herein by reference in their entirety) used lentiviral vectors to evaluate the expression of eGFP under CAG, EFIα, PGK, and UBC promoters in rat DRG cells and primary DRG cells and found that UBC showed weaker expression than the other three promoters, and only 10-12% glial expression was seen for all promoters. Soderblom et al. (E. Neuro 2015; the contents of which are incorporated herein by reference in their entirety) studied the expression of eGFP in AAV8 with CMV and UBC promoters and in AAV2 with CMV promoter after injection in the motor cortex. Intranasal administration of plasmids containing the UBC or EFIα promoters showed sustained airway expression greater than that with the CMV promoter (see, e.g., Gillet et al., Gene Therapy 2001, Vol. 8, 1539-1546; the contents of which are incorporated herein by reference in their entirety). Husain et al. (Gene Therapy 2009; the contents of which are incorporated herein by reference in their entirety) evaluated HβH constructs with the hGUSB promoter, the HSV-1 LAT promoter, and the NSE promoter and found that the HβH constructs showed weaker expression than NSE in the mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, the contents of which are incorporated herein by reference in their entirety) evaluated the long-term effects of HβH vectors after intraventricular injection in neonatal mice and found that expression persisted for at least 1 year. Xu et al. (Gene Therapy 2001, 8, 1323-1332; the contents of which are incorporated herein by reference in their entirety) found low expression in all brain regions when using the NF-L and NF-H promoters compared to 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). Xu et al. found that the descending order of promoter activity was 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. Both are absent in the liver, but NFH is abundant in sensory proprioceptive neurons, brain, and spinal cord, and 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 seen in hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus, and hypothalamus (see, e.g., Drews et al. 2007 and Raymond et al. 2004; the contents of each of which are herein incorporated by reference in their entirety).

[0219] In some embodiments, the vector genome comprises a UBC promoter. The UBC promoter can have a size of 300-350 nucleotides. In some embodiments, the UBC promoter is 332 nucleotides in length.

[0220] In some embodiments, the vector genome comprises a GUSB promoter. The GUSB promoter can have a size of 350-400 nucleotides. In some embodiments, the GUSB promoter is 378 nucleotides in length. In some embodiments, the construct can be AAV-promoter-CMV / globin intron-FXN-RBG, wherein the AAV can be self-complementary and the AAV can be AAV6, AAVrh10 or AAVDJ serotype.

[0221] In some embodiments, the vector genome comprises an NFL promoter. The NFL promoter can have a size of 600-700 nucleotides. In some embodiments, the NFL promoter is 650 nucleotides in length.

[0222] In some embodiments, the vector genome comprises a NFH promoter. The NFH promoter may be 900-950 nucleotides in size. In some embodiments, the NFH promoter is 920 nucleotides in length.

[0223] In some embodiments, the vector genome comprises a scn8a promoter. The scn8a promoter can have a size of 450-500 nucleotides. In some embodiments, the scn8a promoter is 470 nucleotides in length.

[0224] In some embodiments, the vector genome comprises a FXN promoter.

[0225] In some embodiments, the vector genome comprises a PGK promoter.

[0226] In some embodiments, the vector genome comprises a CBA promoter.

[0227] In some embodiments, the vector genome comprises a CMV promoter.

[0228] In some embodiments, the vector genome comprises an H1 promoter.

[0229] In some embodiments, the vector genome comprises a U6 promoter.

[0230] In some embodiments, the vector genome comprises 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, and C5-12.

[0231] In some embodiments, the AAV vector comprises an enhancer element, a promoter, and / or a 5'UTR intron. The enhancer may be, but is not limited to, a CMV enhancer; the promoter may be, but is not limited to, a CMV, CBA, FXN, UBC, GUSB, NSE, synapsin, MeCP2, or GFAP promoter; and the 5'UTR / intron may be, but is 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, SV 40 5'UTR intron; (3) CMV enhancer, CBA promoter, CBA-MVM 5'UTR intron; (4) UBC promoter; (5) GUSB promoter; (6) NSE promoter; (7) synapsin promoter; (8) MeCP2 promoter; (9) GFAP promoter; (10) H1 promoter; and / or (11) U6 promoter.

[0232] In some embodiments, the AAV vector has an engineered promoter.

[0233] In some embodiments, the AAV vector comprises a promoter comprising a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1734-1777. In some embodiments, the promoter is a CMV promoter or is derived from a CMV promoter and comprises a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1743-1751, 1767, 1772-1774, and 1777. In some embodiments, the promoter is a CBA promoter or is derived from a CBA promoter and comprises a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1734-1742, 1760-1766, 1768, and 1775-1776. In some embodiments, the promoter is or is derived from the FXN promoter and comprises a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1752-1759 and 1769-1770.

[0234] In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1738 and a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1738 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0235] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1740 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0236] In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1742 and a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1742 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0237] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1750 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0238] In some embodiments, the viral genome comprises an enhancer, such as the immediate early "ie" enhancer or the CMV / globin enhancer. In some embodiments, the enhancer comprises ie1 exon 1 and ie1 intron 1, or a fragment thereof. In some embodiments, the enhancer comprises ie1 exon 1, ie1 intron 1, or a fragment thereof, human β-globin intron 2, and human β-globin 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 any one of the sequences set forth in 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 comprises an enhancer as described above and a payload region encoding a fraternal protein, e.g., encoding SEQ ID NO: 1725 or a variant thereof having at least 90% sequence identity, or comprises the nucleic acid sequence SEQ ID NO: 1824 or a variant thereof having at least 90% sequence identity.

[0239] introns

[0240] In some embodiments, the vector genome comprises at least one intron or a fragment or derivative thereof. In some embodiments, 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). Non-limiting examples of introns include MVM (67-97 bp), F.IX truncated intron 1 (300 bp), β-globulin 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).

[0241] In some embodiments, the intron can be 100-500 nucleotides in length. Introns can have a length of 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. Introns can have a length of between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500 nucleotides.

[0242] In some embodiments, the AAV vector may comprise an SV40 intron or a fragment or variant thereof. In some embodiments, the promoter may be CMV. In some embodiments, the promoter may be CBA. In some embodiments, the promoter may be H1.

[0243] In some embodiments, the AAV vector may include one or more β-globin introns or fragments or variants thereof. In some embodiments, the intron includes one or more human β-globin sequences (e.g., including fragments / variants thereof). In some embodiments, the intron includes a sequence that is at least 90%, at least 95%, at least 99% or 100% identical to a sequence given as any one of SEQ ID NOs: 1815-1821. In some embodiments, the viral genome includes an intron as described above and a payload region encoding a comitatin, for example, encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity, or comprising a nucleic acid sequence SEQ ID NO: 1824 or a variant thereof with at least 90% sequence identity. In some embodiments, the promoter may be CMV. In some embodiments, the promoter may be CBA. In some embodiments, the promoter may be H1.

[0244] In some embodiments, the encoded FXN can be located downstream of an intron in the expression vector, such as, but not limited to, an SV40 intron or a beta globin intron or other introns known in the art. In addition, the encoded FXN can also be located upstream of a polyadenylation sequence in the expression vector. In some embodiments, the encoded FXN can be located within 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 more 30 nucleotides downstream of a promoter having an intron and / or upstream of a polyadenylation sequence in the expression vector. In some embodiments, the encoded FXN may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream of an intron and / or upstream of a polyadenylation sequence in an expression vector. In some embodiments, the encoded FXN may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of the nucleotides downstream of an intron and / or upstream of a polyadenylation sequence in an expression vector. In some embodiments, the encoded FXN may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% of the sequence downstream of the intron and / or upstream of the polyadenylation sequence in the expression vector.

[0245] In certain embodiments, the intron sequence is not an enhancer sequence.

[0246] In certain embodiments, the intron sequence is not a subcomponent of the promoter sequence.

[0247] Untranslated region (UTR)

[0248] By definition, the wild-type untranslated region (UTR) of a gene is transcribed but not translated. Typically, the 5'UTR begins at the transcription start site and ends at the start codon, while the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0249] Features commonly found in genes that are abundantly expressed in specific target organs can be engineered into UTRs to enhance stability and protein production. As non-limiting examples, 5'UTRs from mRNAs normally expressed in the liver (e.g., albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can be used in the viral genome of the AAV particles of the present disclosure to enhance expression in hepatocyte cell lines or the liver.

[0250] While not wishing to be bound by theory, the wild-type 5' untranslated region (UTR) includes features that play a role in translation initiation. A Kozak sequence, known to be involved in the process by which ribosomes initiate translation of many genes, is often included in the 5'UTR. The Kozak sequence has a consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), followed by another 'G'.

[0251] In some embodiments, the 5'UTR in the viral genome includes a Kozak sequence.

[0252] In some embodiments, the 5'UTR in the viral genome does not include a Kozak sequence.

[0253] While not wishing to be bound by theory, wild-type 3'UTRs are known to have segments of adenosine and uridine embedded within them. These 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 classes (Chen et al., 1995, the contents of which are incorporated herein by reference in their entirety): Class I AREs, such as but not limited to c-Myc and MyoD, contain several dispersed copies of the AUUUA motif within a U-rich region. Class II AREs, such as but not limited to GM-CSF and TNF-α, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III AREs are less well defined, such as but not limited to c-Jun and myogenin. These U-rich regions do not contain the AUUUA motif. Most proteins that bind to AREs are known to destabilize the messenger, but members of the ELAV family (most notably HuR) have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thus stabilization of the message in vivo.

[0254] The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, such as the payload region of a viral genome, one or more copies of AREs can be introduced to reduce the stability of the polynucleotide, thereby reducing translation and reducing the yield of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and yield of the resulting protein.

[0255] In some embodiments, the 3'UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

[0256] Any UTR from any gene known in the art can be incorporated into the viral genome of the AAV particle. These UTRs or portions thereof can be placed in the same orientation as in the gene from which they are selected, or the orientation or position can be changed. In some embodiments, the UTR used in the viral genome of the AAV particle can be reversed, shortened, extended, or prepared with one or more other 5'UTRs or 3'UTRs known in the art. As used herein, the term "altered" when it relates to a UTR means that the UTR has been changed in some way relative to a reference sequence. For example, a 3' or 5'UTR can be changed relative to a wild-type or native UTR by a change in orientation or position as taught above, or can be changed by including additional nucleotides, deletions of nucleotides, exchanges of nucleotides, or transposition.

[0257] In some embodiments, the viral genome of the AAV particle comprises at least one artificial UTR that is not a variant of a wild-type UTR.

[0258] In some embodiments, the viral genome of the AAV particle comprises UTRs that have been selected from a family of transcripts whose proteins share a common function, structure, characteristic, or property.

[0259] miRNA target sites

[0260] In some embodiments, the viral genome can include at least one miRNA binding site. MicroRNA (or miRNA or miR) is a non-coding RNA of 19-25 nucleotides that binds to a site of a nucleic acid target and downregulates gene expression by reducing nucleic acid molecule stability 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.

[0261] In some embodiments, the viral genome comprises at least one sequence encoding a miRNA target site to reduce transgenic expression in a specific tissue. MiRNAs and their target tissues are well known in the art. In some embodiments, a miR-122 miRNA target site (miR-122TS) or a tandem copy of the same can be encoded in the viral genome to reduce expression of the viral genome in the liver, where miR-122 is abundantly expressed.

[0262] In some embodiments, the viral genome comprises 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, for example, three copies of SEQ ID NO: 1827 or a variant thereof with at least 90% sequence identity. In some embodiments, the miR122 binding site series 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 comprises one, two or three miR122 binding sites as described above and a payload region encoding a symtaxin, for example, encoding SEQ ID NO: 1725 or a variant thereof with at least 90% sequence identity, or comprising the nucleic acid sequence SEQ ID NO: 1824 or a variant thereof with at least 90% sequence identity. In some embodiments, the viral genome comprises three miR122 binding sites as described above and a payload region encoding a fraternal protein, e.g., encoding SEQ ID NO: 1725 or a variant thereof having at least 90% sequence identity, or comprises the nucleic acid sequence SEQ ID NO: 1824 or a variant thereof having at least 90% sequence identity.

[0263] Main Chain

[0264] In certain embodiments, cis-elements such as a vector backbone are incorporated into viral particles encoding FXN. The backbone sequence can regulate transcription during viral production. The backbone sequence can contribute to the stability of FXN expression. The backbone sequence can contribute to the expression level of FXN and can be cloned into the pAAVsc or pcDNA3.1 vector backbone.

[0265] polyadenylation sequence

[0266] In certain embodiments, the viral genome of the AAV particles of the present disclosure comprises at least one polyadenylation sequence.The viral genome of the AAV particles may comprise a polyadenylation sequence between the 3' end of the payload encoding sequence and the 5' end of the 3' ITR.

[0267] In some embodiments, the polyadenylation sequence or "poly A sequence" can range in length from absent 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、2 202, 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.

[0268] In some embodiments, the polyadenylation sequence is 50-100 nucleotides in length.

[0269] In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length.

[0270] In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length.

[0271] In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length.

[0272] In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length.

[0273] In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length.

[0274] In some embodiments, the polyadenylation sequence is 60-160 nucleotides in length.

[0275] In some embodiments, the polyadenylation sequence is 60-200 nucleotides in length.

[0276] In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length.

[0277] In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length.

[0278] In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length.

[0279] In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length.

[0280] In some embodiments, the polyadenylation sequence is 80-100 nucleotides in length.

[0281] In some embodiments, the polyadenylation sequence is 80-150 nucleotides in length.

[0282] In some embodiments, the polyadenylation sequence is 80-160 nucleotides in length.

[0283] In some embodiments, the polyadenylation sequence is 80-200 nucleotides in length.

[0284] In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length.

[0285] In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length.

[0286] In some embodiments, the polyadenylation sequence is 90-160 nucleotides in length.

[0287] In some embodiments, the polyadenylation sequence is 90-200 nucleotides in length.

[0288] In some embodiments, the encoded FXN can be located upstream of a polyadenylation sequence in an expression vector. In addition, the encoded FXN can be located downstream of a promoter in an expression vector or a fragment thereof (e.g., one disclosed herein), such as, but not limited to, a CMV, U6, CBA, or CBA promoter with an SV40 intron. In some embodiments, the encoded FXN can be located within 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 more than 30 nucleotides downstream of a promoter and / or upstream of a polyadenylation sequence in an expression vector. In some embodiments, the encoded FXN may be located within 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 5-10, 5-15, 5-20, 5-25, 5-30, 10-15, 10-20, 10-25, 10-30, 15-20, 15-25, 15-30, 20-25, 20-30, or 25-30 nucleotides downstream of the intron and / or upstream of the polyadenylation sequence in the expression vector. In some embodiments, the encoded FXN may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of the nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector. In some embodiments, the encoded FXN may be located within the first 1-5%, 1-10%, 1-15%, 1-20%, 1-25%, 5-10%, 5-15%, 5-20%, 5-25%, 10-15%, 10-20%, 10-25%, 15-20%, 15-25%, or 20-25% downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector.

[0289] In some embodiments, the viral genome comprises a human growth hormone (hGH) polyA sequence. 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 an hGH polyA sequence as described above and a payload region encoding a frataxin, e.g., encoding SEQ ID NO: 1725 or a variant thereof having at least 90% sequence identity, or comprises the nucleic acid sequence SEQ ID NO: 1824 or a variant thereof having at least 90% sequence identity.

[0290] Filler sequence

[0291] In some embodiments, the viral genome comprises one or more filler 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 filler sequence is a derivative of human albumin.

[0292] In some embodiments, the viral genome comprises one or more filler sequences to bring the length of the viral genome to an optimal size for packaging. In some embodiments, the viral genome comprises at least one filler sequence to bring the length of the viral genome to about 2.3 kb. In some embodiments, the viral genome comprises at least one filler sequence to bring the length of the viral genome to about 4.6 kb.

[0293] In some embodiments, the viral genome is a single-stranded (ss) viral genome and comprises one or more stuffer sequences, independently or collectively having a length between about 0.1 kb and 3.8 kb, for example, 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. In some embodiments, the full-length filler sequence in the vector genome is 3.1 kb. In some embodiments, the full-length filler sequence in the vector genome is 2.7 kb. In some embodiments, the full-length filler sequence in the vector genome is 0.8 kb. In some embodiments, the full-length filler sequence in the vector genome is 0.4 kb. In some embodiments, the length of each filler sequence in the vector genome is 0.8 kb. In some embodiments, each stuffer sequence in the vector genome is 0.4 kb in length.

[0294] In some embodiments, the viral genome is a self-complementary (sc) viral genome and comprises one or more filler sequences, independently or collectively having a length between about 0.1 kb and 1.5 kb, 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 filler sequence in the vector genome is 0.8 kb. In some embodiments, the full-length filler sequence in the vector genome is 0.4 kb. In some embodiments, the length of each filler sequence in the vector genome is 0.8 kb. In some embodiments, the length of each filler sequence in the vector genome is 0.4 kb.

[0295] In some embodiments, the viral genome comprises any portion of the filler sequence. The viral genome may comprise 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%, or 99% of the filler sequence.

[0296] In some embodiments, the viral genome is a single-stranded (ss) viral genome and comprises one or more filler sequences to bring the length of the viral genome to approximately 4.6 kb. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 3' to the 5' ITR sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 5' to the promoter sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 5' to the 3' ITR sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located between two intron sequences. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located within an intron sequence. In some embodiments, the viral genome comprises two filler sequences, and the first filler sequence is located 3' to the 5' ITR sequence, and the second filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located 5' of the promoter sequence, and the second stuffer sequence is located 3' of the polyadenylation signal sequence. In some embodiments, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located 3' of the 5' ITR sequence, and the second stuffer sequence is located 5' of the 5' ITR sequence.

[0297] In some embodiments, the viral genome is a self-complementary (sc) viral genome and comprises one or more filler sequences to bring the length of the viral genome to approximately 2.3 kb. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 3' to the 5' ITR sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 5' to the promoter sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located 5' to the 3' ITR sequence. In some embodiments, the viral genome comprises at least one filler sequence, and the filler sequence is located between two intron sequences. As a non-limiting example, the viral genome comprises at least one filler sequence, and the filler sequence is located within an intron sequence. In some embodiments, the viral genome comprises two filler sequences, and the first filler sequence is located 3' to the 5' ITR sequence, and the second filler sequence is located 3' to the polyadenylation signal sequence. In some embodiments, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located 5' of the promoter sequence, and the second stuffer sequence is located 3' of the polyadenylation signal sequence. In some embodiments, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located 3' of the 5' ITR sequence, and the second stuffer sequence is located 5' of the 5' ITR sequence.

[0298] In some embodiments, the viral genome may comprise one or more filler sequences between one of the plurality of regions of the viral genome. In some embodiments, the filler region may be located before regions such as, but not limited to, the payload region, inverted terminal repeats (ITRs), the promoter region, the intron region, the enhancer region, the polyadenylation signal sequence region, and / or the exon region. In some embodiments, the filler region may be located after regions such as, but not limited to, the payload region, inverted terminal repeats (ITRs), the promoter region, the intron region, the enhancer region, the polyadenylation signal sequence region, and / or the exon region. In some embodiments, the filler region may be located before and after regions such as, but not limited to, the payload region, inverted terminal repeats (ITRs), the promoter region, the intron region, the enhancer region, the polyadenylation signal sequence region, and / or the exon region.

[0299] In some embodiments, the viral genome may include one or more filler 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%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the region 5' of the filler sequence region. In some embodiments, the filler sequence may bifurcate at least one region such that 10% of the region is located 5' of the filler sequence and 90% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may bifurcate at least one region such that 20% of the region is located 5' of the filler sequence and 80% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 30% of the region is located 5' of the filler sequence and 70% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 40% of the region is located 5' of the filler sequence and 60% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 50% of the region is located 5' of the filler sequence and 50% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 60% of the region is located 5' of the filler sequence and 40% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 70% of the region is located 5' of the filler sequence and 30% of the region is located 3' of the filler sequence. In some embodiments, the filler sequence may fork at least one region such that 80% of the region is located 5' of the filler sequence and 20% of the region is located 3' of the filler sequence. In some embodiments, the stuffer sequence may bifurcate at least one region such that 90% of the region is located 5' to the stuffer sequence and 10% of the region is located 3' to the stuffer sequence.

[0300] In some embodiments, the viral genome comprises a stuffer sequence after the 5' ITR.

[0301] In some embodiments, the viral genome comprises a filler sequence after the promoter region. In some embodiments, the viral genome comprises a filler sequence after the payload region. In some embodiments, the viral genome comprises a filler sequence after the intron region. In some embodiments, the viral genome comprises a filler sequence after the enhancer region. In some embodiments, the viral genome comprises a filler sequence after the polyadenylation signal sequence region. In some embodiments, the viral genome comprises a filler sequence after the exon region.

[0302] In some embodiments, the viral genome comprises a filler sequence before the promoter region. In some embodiments, the viral genome comprises a filler sequence before the payload region. In some embodiments, the viral genome comprises a filler sequence before the intron region. In some embodiments, the viral genome comprises a filler sequence before the enhancer region. In some embodiments, the viral genome comprises a filler sequence before the polyadenylation signal sequence region. In some embodiments, the viral genome comprises a filler sequence before the exon region.

[0303] In some embodiments, the viral genome comprises a stuffer sequence preceding the 3' ITR.

[0304] In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a 5' ITR and a promoter region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a 5' ITR and a payload region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a 5' ITR and an intron region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a 5' ITR and an enhancer region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a 5' ITR and a polyadenylation signal sequence region.

[0305] In some embodiments, a stuffer sequence may be located between two regions, such as, but not limited to, a 5' ITR and an exonic region.

[0306] In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a promoter region and a payload region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a promoter region and an intron region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a promoter region and an enhancer region. In some embodiments, a filler 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 filler sequence may be located between two regions, such as, but not limited to, a promoter region and an exon region. In some embodiments, a filler sequence may be located between two regions, such as, but not limited to, a promoter region and a 3' ITR.

[0307] In some embodiments, a filler 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 an enhancer region. In some embodiments, a filler 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 filler sequence may be located between two regions, such as, but not limited to, a payload region and an exon region.

[0308] In some embodiments, a stuffer sequence may be located between two regions, such as, but not limited to, the payload region and the 3' ITR.

[0309] Self-complementary and single-stranded vectors

[0310] In some embodiments, the AAV vector used in the present disclosure is a single-stranded vector (ssAAV).

[0311] In some embodiments, the AAV vector can be a self-complementary AAV vector (scAAV). See, for example, U.S. Patent No. 7,465,583. The scAAV vector contains two DNA strands that anneal together to form double-stranded DNA. By skipping second-strand synthesis, scAAV allows rapid expression in cells.

[0312] In some embodiments, the AAV vector used in the present disclosure is scAAV.

[0313] Methods for producing and / or modifying AAV vectors are disclosed in the art, such as pseudotyped AAV vectors (International PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO 2005005610 and WO2005072364, the contents of each of which are herein incorporated by reference in their entirety).

[0314] Genome size

[0315] In some embodiments, the viral genome of the AAV particles of the present disclosure can be single-stranded or double-stranded. The size of the vector genome can be small, medium, large, or maximum size.

[0316] In some embodiments, the nucleic acid sequence encoding FXN described herein may 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.

[0317] In some embodiments, the vector genome comprising the nucleic acid sequence encoding FXN described herein can be a small double-stranded vector genome. The size of the small double-stranded vector genome can be about 1.3 to about 1.7 kb, for example, about 1.3, about 1.4, about 1.5, about 1.6, or about 1.7 kb. In some embodiments, the size of the small double-stranded vector genome can be 1.6 kb.

[0318] In some embodiments, the vector genome comprising the nucleic acid sequence encoding FXN described herein can be a medium single-stranded vector genome. The size of the medium single-stranded vector genome can be about 3.6 to about 4.3 kb, for example, 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, the size of the medium single-stranded vector genome can be 4.0 kb.

[0319] In some embodiments, the vector genome comprising the nucleic acid sequence encoding FXN described herein can be a medium single-stranded or double-stranded vector genome. The medium single-stranded or double-stranded vector genome size can be about 1.8 to about 2.1 kb, for example, about 1.8, about 1.9, about 2.0, or about 2.1 kb. In some embodiments, the medium single-stranded or double-stranded vector genome size can be 2.0 kb. In addition, the vector genome can include a promoter and a polyA tail. In one embodiment, the vector genome comprising the nucleic acid sequence encoding FXN described herein can be a large single-stranded vector genome. The large single-stranded vector genome size can be 4.4 to 6.0 kb, for example, about 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, the large single-stranded vector genome size can be 4.8 kb. As another non-limiting example, the size of a large single-stranded vector genome can be 6.0 kb.

[0320] In one embodiment, the vector genome comprising the nucleic acid sequence encoding FXN described herein can be a large double-stranded vector genome. The size of the large double-stranded vector genome can be 2.2 to 3.0 kb, for example, about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0 kb. As a non-limiting example, the size of the large double-stranded vector genome can be 2.4 kb.

[0321] Payload

[0322] In some embodiments, the present disclosure provides constructs that allow for improved expression of FXN delivered by gene therapy vectors.

[0323] In some aspects, the present disclosure relates to compositions containing or comprising nucleic acid sequences encoding frataxin (FXN) or functional fragments thereof and methods of administering these compositions in vitro or in vivo in humans and / or animal models of disease.

[0324] The AAV particles of the present disclosure may include 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 the viral genome, or the expression products of such polynucleotides or polynucleotide regions, such as a transgene, a polynucleotide encoding a polypeptide, or a polynucleotide encoding a poly-polypeptide, such as FXN or a variant thereof. The payload may include any nucleic acid known in the art that can be used to express FXN in target cells transduced or contacted with the AAV particles carrying the payload (by supplementing the protein product or using a regulatory nucleic acid in place of the gene).

[0325] Payload constructs may include a combination of coding and non-coding nucleic acid sequences.

[0326] The viral genome and any segments, fragments or all thereof, and the payload construct may be codon-optimized.

[0327] In some embodiments, the nucleic acid sequence of the AAV particle can be a payload construct that includes at least a portion encoding FXN.

[0328] In some embodiments, the payload construct encodes more than one payload. As a non-limiting example, a payload construct encoding more than one payload can be replicated and packaged into viral particles. Target cells transduced with viral particles containing more than one payload can express each payload in a single cell.

[0329] In some embodiments, the payload construct may encode a coding or non-coding RNA.In certain embodiments, the adeno-associated viral vector particle further comprises at least one cis-element selected from the group consisting of a Kozak sequence, a backbone sequence, and an intron sequence.

[0330] In some embodiments, the payload is a polypeptide that can be a peptide or protein. The protein encoded by the payload construct can include secreted proteins, intracellular proteins, extracellular proteins, and / or membrane proteins. The encoded protein can be structural or functional. The protein encoded by the payload construct includes, but is 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 useful in the fields of human disease, veterinary applications, and various in vivo and in vitro settings.

[0331] In some embodiments, the payload can include polypeptides used as marker proteins to assess cell transformation and expression, fusion proteins, polypeptides with desired biological activity, gene products that can complement genetic defects, RNA molecules, transcription factors, and other gene products related to regulation and / or expression. In some embodiments, the payload can include nucleotide sequences that provide desired effects or regulatory functions (e.g., transposons, transcription factors).

[0332] The encoded payload may include a gene therapy product. Gene therapy products may include, but are not limited to, polypeptides, RNA molecules, or other gene products that, when expressed in target cells, provide a desired therapeutic effect. In some embodiments, gene therapy products may include replacements for nonfunctional genes or genes that are absent, underexpressed, or mutated. In some embodiments, gene therapy products may include replacements for nonfunctional proteins or polypeptides or proteins or polypeptides that are absent, underexpressed, misfolded, degraded excessively, or mutated. For example, a gene therapy product may include a FXN polypeptide or a polynucleotide encoding a FXN polypeptide for treating FXN deficiency or FA.

[0333] 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 can be translated into the encoded polypeptide produced in vitro, in vivo, in situ, or ex vivo. Certain embodiments provide mRNA encoding FXN or a variant thereof.

[0334] Components of mRNA include, but are not limited to, the 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 can be codon-optimized.

[0335] In some embodiments, the length of 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 this context, "X amino acid length" refers to X amino acid residues). In some embodiments, the length of the encoded protein or polypeptide is 50-2000 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-1000 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-1500 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-1000 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-800 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-600 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-400 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-200 amino acids. In some embodiments, the length of the encoded protein or polypeptide is 50-100 amino acids.

[0336] The payload construct encoding the payload can include or encode a selective marker. The selective marker can include a gene sequence or a protein or polypeptide encoded by a gene sequence expressed in a host cell, allowing identification, selection and / or purification of the host cell from a cell population that may or may not express the selective marker. In some embodiments, the selective marker provides resistance to survive the selection process, otherwise the host cell will be killed, for example, by antibiotic treatment. In some embodiments, the antibiotic selective marker can include one or more antibiotic resistance factors, including but not limited to neomycin resistance (e.g., Neo), hygromycin resistance, kanamycin resistance and / or puromycin resistance.

[0337] In some embodiments, any nucleic acid sequence encoding a protein or polypeptide can be used as a selectable marker including one recognized by a specific antibody.

[0338] In some embodiments, the payload construct encoding the payload may include a selectable marker, including but not limited to β-lactamase, luciferase, β-galactosidase, or any other reporter gene as that term is understood in the art, including 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. USA (1995); or Heim et al., Science 373:663-664 (1995); for β-lactamase, see WO 96 / 30540); the contents of each of which are incorporated herein by reference in their entirety.

[0339] In some embodiments, the payload construct encoding the selective marker may comprise a fluorescent protein. Fluorescent proteins as described herein may comprise any fluorescent marker, including but not limited to green, yellow and / or red fluorescent proteins (GFP, YFP and / or RFP). In some embodiments, the payload construct encoding the selective marker may comprise a human influenza hemagglutinin (HA) tag.

[0340] In certain embodiments, a nucleic acid for expression of a payload in a target cell is incorporated into the viral genome and positioned between two ITR sequences.

[0341] Payload: Fraxin

[0342] In some embodiments, the payload is a frataxin. As used herein, the terms "frataxin" or "FXN protein" are used interchangeably with "frataxin polypeptide" or "FXN polypeptide" and encompass wild-type FXN as well as functional variants thereof. A functional variant is a variant that retains some or all of the activity of its wild-type counterpart to achieve a desired therapeutic effect. For example, in some embodiments, a functional variant can be effectively used in gene therapy to treat a disorder or condition, such as FXN deficiency or FA. Unless otherwise indicated, variants of FXN as described herein (e.g., constructs, vectors, genomes, methods, kits, compositions, etc. in the context of the present disclosure) are functional variants.

[0343] Friedreich's ataxia (FA) is an autosomal recessive disorder that occurs when the frataxin (FXN) gene contains expanded intrinsic GAA repeats (an example of a trinucleotide repeat expansion). See Parkinson et al., Journal of Neurochemistry, 2013, 126 (Suppl. 1), 103-117, the contents of which are incorporated herein by reference in their entirety. GAA repeat expansion within the gene leads to reduced FXN protein levels. FXN is an iron-binding protein responsible for the formation of iron-sulfur clusters. One consequence of FXN protein deficiency is mitochondrial iron overload, which can cause damage to many proteins. See Nageshwaran and Festenstein, Frontiers in Neurology, Vol. 6, Art. 262 (2015), the contents of which are incorporated herein by reference in their entirety. The FXN gene is located on chromosome 9. See Sandi et al., Frontiers in Genetics, Vol. 5, Art. 165 (June 2014), the contents of which are incorporated herein by reference in their entirety.

[0344] The mutant gene contains an expanded GAA triplet repeat in the first intron, and in several cases, point mutations have been detected. Because the defect is located in the intron (removed from the mRNA transcript between transcription and translation), the mutation does not result in abnormal FXN protein production. See Nageshwaran and Festenstein, Frontiers in Neurology, Vol.6, Art.262 (2015). Instead, the mutation causes gene silencing (i.e., the mutation reduces gene transcription) in a manner similar to position-effect variegation through the induction of heterologous chromatin structure. In addition to reducing the expression of FXN protein, in in vivo yeast studies, long bundles of GAA repeats induce chromosome breakage.

[0345] Low levels of FXN protein lead to 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, Vol. 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, occurring in all eukaryotes and some prokaryotes. The FXN protein has various known functions; most notably, it aids in the synthesis of iron-sulfur proteins in the electron transport chain to ultimately produce adenosine triphosphate (ATP), which is the energy flow required for metabolic functions in the cell. The FXN protein also regulates iron transfer in the mitochondria to provide the appropriate amount of reactive oxygen species (ROS) to maintain normal processes. Without the FXN protein, energy in the mitochondria is lost, and excess iron causes additional ROS production, leading to further cell damage.

[0346] It may eventually be discovered that other disorders of the central nervous system are associated with abnormal expression or deficiency of the amount or function of 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, spinal muscular atrophy, or other neurological or neuromuscular diseases, disorders, or conditions described herein.

[0347] As used herein, "associated with decreased frataxin levels" or "associated with decreased expression" refers to one or more symptoms caused by lower than normal frataxin levels in a target tissue or in a biological fluid such as blood. A disease or condition associated with decreased frataxin levels or expression may be a disorder of the central nervous system. Such a disease or condition may be a neuromuscular or neurological disorder or condition. For example, a disease associated with decreased frataxin levels may be FA, or may be another neurological or neuromuscular disorder described herein.

[0348] The present disclosure addresses the need for new technologies by providing FXN-related therapeutics delivered by AAV-based compositions and complexes for the treatment of FA.

[0349] Delivery is exemplified in the context of AAV, but other viral vectors, non-viral vectors, nanoparticles, or liposomes can similarly be used to deliver therapeutic FXN, and include, but are not limited to, vector genomes of any AAV serotype, or other viral delivery vehicles or lentiviruses, etc. The observations and teachings extend to any macromolecular structure, including modified cells, introduced into the CNS in the manner described herein.

[0350] Table 2 shows sequence identifiers for representative polynucleotide and polypeptide sequences of fraxins that can be used in the viral genomes disclosed herein and that can constitute fraxin payloads. Functional variants can also be used, for example, those that retain at least about 90% or at least 95% sequence identity with the sequences shown in Table 2. Other variants that are codon-optimized and encode the same or substantially the same FXN amino acid sequence (e.g., those having at least about 90% amino acid sequence identity) can also be used.

[0351] Table 2. Representative fraxin sequences

[0352] 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 encodes NP_000135.2 1729 DNA Homo sapiens NM_181425.2 encodes NP_852090.1 1730 DNA Homo sapiens NM_001161706.1 encodes NP_001155178.1 1731 PRT cynomolgus macaque A0A2K5VX49(UniProt) 1732 PRT cynomolgus macaque NP_001271967.1 1733 PRT Rhesus monkey NP_001247670.1

[0353] In some embodiments, the viral genome comprises a payload region encoding a symtaxin. The encoded symtaxin can be derived from any species, such as, but not limited to, humans, non-human primates, or rodents.

[0354] In some embodiments, the viral genome comprises a payload region encoding human (Homo sapiens) frataxin or a variant thereof.

[0355] Various embodiments of the present disclosure provide adeno-associated virus (AAV) particles comprising a viral genome comprising at least one inverted terminal repeat region and a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to the human frataxin (hFXN) sequence SEQ ID NO: 1725, 1726 and / or 7271, or a variant thereof.

[0356] 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 comprises 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 comprises at least one inverted terminal repeat region and a nucleic acid sequence encoding SEQ ID NO: 1725.

[0357] 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 to 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 to 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 to 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 to 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 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.

[0358] 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 to 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 to 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 to 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 to 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 of SEQ ID NO: 1823 or a fragment thereof. In some embodiments, the nucleic acid sequence further comprises a stop codon.

[0359] 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 to 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 to 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 to 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 to 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 of SEQ ID NO: 1824 or a fragment thereof.

[0360] In some embodiments, the FXN polypeptide is derived from a non-human primate FXN sequence, such as a macaque or cynomolgus monkey (cynoFXN). Certain embodiments provide FXN polypeptides as humanized versions of the cynomolgus monkey (HcynoFXN) sequence. In some embodiments, the FXN polypeptide sequence has at least about 90% sequence identity with the art-accepted canonical human FXN amino acid sequence SEQ ID NO: 1725, which can be 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 with the art-accepted canonical human FXN amino acid sequence SEQ ID NO: 1726, which is 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 with the art-accepted canonical human FXN amino acid sequence SEQ ID NO: 1727, which is encoded by the nucleic acid sequence of SEQ ID NO: 1730.

[0361] In some embodiments, the viral genome comprises a payload region encoding a cynomolgus or cynomolgus (long-tailed) macaque (Macaca fascicularis) frataxin or a variant thereof.

[0362] In some embodiments, the viral genome comprises a payload region encoding a rhesus macaque (Macaca mulatta) frataxin or a variant thereof.

[0363] 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 to SEQ ID NO: 1822 or a fragment thereof.

[0364] 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 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 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 99% 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 of SEQ ID NO: 1822 or a fragment thereof.

[0365] In some embodiments, the frataxin polypeptide may comprise an amino acid sequence that is 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 to any of the above.

[0366] In some embodiments, the frataxin polypeptide may be encoded by a nucleic acid sequence that is 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 to any of the above.

[0367] Viral genome: promoter

[0368] In some embodiments, the payload region of the viral genome comprises elements that enhance or regulate payload expression, 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 comprises at least one promoter. In some embodiments, the viral genome comprises more than one promoter.

[0369] In some embodiments, the promoter is a wild-type frataxin promoter, or a derivative (e.g., truncated or variant) thereof. Suitable derivatives of the wild-type frataxin promoter are those that are functional, e.g., effective in expressing a payload at at least a slightly detectable level.

[0370] In some embodiments, the promoter is an engineered comitatin promoter. Shorter variants of the comitatin promoter are included herein. The comitatin promoter can be 200-1400 nt in length or any length therebetween. In some embodiments, the comitatin promoter variant can be 223, 363, 534, 747, 906, 1060, 1226, or 1353 nucleotides in length. 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 comitatin promoter variant may be shorter than the wild-type comitatin promoter sequence.

[0371] In some embodiments, promoter is any promoter described herein in one or more combinations. In some embodiments, promoter is used together with enhancer sequence. In some embodiments, enhancer sequence can be derived from cytomegalovirus immediate early gene (CMVie). In some embodiments, enhancer can be located at promoter upstream (5 '). In some embodiments, enhancer comprises SEQ ID NO:1777.

[0372] In some embodiments, the promoter is a CBA promoter, or a derivative (eg, a truncated or variant) thereof. It will be understood that suitable derivatives of the CBA promoter are functional, eg, effective in expressing a payload.

[0373] In some embodiments, the CBA promoter comprises a CMV enhancer, a backbone sequence, and a CB promoter sequence, when recited 5' to 3'. Each of the three components (CMV enhancer, backbone, and CB sequence) can be a different length in the variant.

[0374] In some embodiments, a CBA promoter comprises a backbone sequence and a CB promoter sequence when recited 5' to 3'.

[0375] In some embodiments, the CBA promoter comprises a CB promoter sequence.

[0376] In some embodiments, the length of the CBA promoter can be 100-700 nt or any length therebetween. In some embodiments, the CBA promoter variant can be 100, 180, 260, 270, 332, 412, 492, or 572 nucleotides in length. A CBA promoter variant may be shorter than the wild-type CBA promoter sequence due to deletions in any region of the enhancer, backbone, or 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.

[0377] In some embodiments, the promoter is a CMV promoter, or a derivative thereof (e.g., truncated or variant). It should be understood that suitable derivatives of the CMV promoter are functional, e.g., it is effective to express a payload. The CMV promoter may comprise a CMV enhancer and a CMV promoter sequence, or may comprise only a CMV promoter sequence. The CMV enhancer and CMV promoter sequence may be different lengths between promoter variants.

[0378] In some embodiments, the length of the CMV promoter can be 50-700nt or any length therebetween. In some embodiments, the CMV promoter variant can be 55, 109, 163, 217, 289, 361, 433 or 505 nucleotides in length. Due to deletions in any region of the enhancer or 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 variant may be shorter than the wild-type CMV promoter sequence.

[0379] In some embodiments, the promoter is a deletion variant of a parent promoter sequence, wherein one or more nucleotides have been removed from the parent sequence.

[0380] In some embodiments, the promoter is an insertional variant of a parent promoter sequence, wherein one or more nucleotides are added to the parent sequence.

[0381] In some embodiments, the promoter comprises one or more mutations compared to a parent promoter sequence.

[0382] In some embodiments, a promoter is modified in one or more ways (eg, deletion, mutation, and / or insertion) to generate a promoter variant.

[0383] In some embodiments, the promoter may comprise a sequence, fragment, or variant thereof of any of the sequences in Table 3. For example, the promoter may comprise a sequence having 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, e.g., a sequence having a specified percentage identity and providing the same function as part or all of a sequence selected from the group consisting of SEQ ID NOs: 1734-1777. In some embodiments, the promoter is a CMV promoter or is derived from a CMV promoter and comprises a sequence having 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: 1743-1751, 1767, 1772-1772, and 1777. In some embodiments, the promoter is a CBA promoter or is derived from a CBA promoter and comprises a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1734-1742, 1760-1766, 1768, and 1775-1776. In some embodiments, the promoter is a FXN promoter or is derived from a FXN promoter and comprises a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1752-1759 and 1769-1770.

[0384] In some embodiments, the promoter may comprise a combination of more than one sequence of any of those listed in Table 3. In some embodiments, the promoter sequence may further comprise at least one intron / exon sequence as given in Table 6.

[0385] In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1738 and a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1738 and a payload region encoding a fraternin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824). In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0386] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1740 and a payload region encoding a frataxin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824). In some embodiments, the frataxin 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, the frataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0387] In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1742 and a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1742 and a payload region encoding a fraternin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824). In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0388] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1750 and a payload region encoding a fraternin polypeptide having the amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824). In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0389] In some embodiments, the promoter used in the viral genome disclosed herein comprises any one of the promoter sequences in Table 3. In Table 3, CMV stands for "cytomegalovirus." CBA stands for "chicken β-actin," which may have a CMVIE ("immediate early") enhancer region and promoter region; CAG stands for CMV enhancer, CBA promoter, and rabbit β-globin splice acceptor site; FXN stands for "fraxin," and mCBA stands for a variant of the CBA promoter generated using PCR.

[0390] Table 3. Representative promoters

[0391] Promoter name Start promoter Promoter length SEQ ID NO of the promoter 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

[0392] In some embodiments, the promoter is used to regulate the expression of a fraxin in a target cell. In certain embodiments, the promoter can be used to increase the expression of a fraxin in a target cell to a level greater than normal endogenous fraxin expression. In certain embodiments, the promoter can be used to induce the expression of a fraxin in a target cell to a level close to or equivalent to normal endogenous fraxin expression.

[0393] In some embodiments, the junction sequence can be used in combination with the promoter described herein, such as, but not limited to, those listed in Table 3. In certain embodiments, the junction sequence can be located at the 5' promoter in the viral genome. In certain embodiments, the junction sequence can be located at the 3' promoter in the viral genome. In certain embodiments, the viral genome may include more than one junction sequence. As a non-limiting example, the viral genome may include a junction sequence at the 5' end and at the 3' end of the promoter. The junction sequence can be the same sequence, two different sequences, or a sequence separated (split) on either side of the promoter sequence. In certain embodiments, the junction sequence comprises SEQ SEQ ID NO: 1813. In certain embodiments, the junction sequence comprises SEQ SEQ ID NO: 1814.

[0394] In some embodiments, promoters are used to enhance comitatin expression in target cells (e.g., nervous system or cardiac tissue). For the target cells, comitatin expression can increase endogenous comitatin expression by 0.01 to 100 (0.01-100x) times. In some embodiments, promoters are used in target cells to maintain comitatin expression at 0.5-3x (e.g., 0.5-1x, 1-1.5x, 1.5-2x, 2-2.5x, 2.5-3x) of endogenous comitatin (i.e., normal human levels or about 5.5-32.8 ng / mg protein).

[0395] In some embodiments, a promoter, such as a promoter in Table 3, is used in an AAV vector genome that further comprises a sequence encoding a frataxin polypeptide sequence (e.g., a human frataxin polypeptide sequence). In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1742 and a payload region encoding a frataxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 1725 (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 a payload region encoding a fraternin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1742 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11 or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0396] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1750 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0397] In some embodiments, the promoter comprises a sequence having 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 comprises a promoter sequence having at least 90% sequence identity to SEQ ID NO: 1738 and a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1738 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0398] In some embodiments, the promoter comprises a sequence having 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 a payload region encoding a frataxin polypeptide having at least 90% identical amino acid sequence to SEQ ID NO: 1725 (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 a payload region encoding a frataxin polypeptide having at least 95% identical amino acid sequence to SEQ ID NO: 1725 (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 comprises a promoter sequence of SEQ ID NO: 1740 and a payload region encoding a fraternin polypeptide having an amino acid sequence of SEQ ID NO: 1725 (e.g., a payload region comprising SEQ ID NO: 1824) and / or further comprises one or more sequences as provided in Tables 5-11, or 95% identical variants thereof. In some embodiments, the fraternin 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, the fraternin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824.

[0399] In various embodiments, any promoter disclosed herein (e.g., a promoter from Table 3 or a promoter with 90% or greater homology thereto) can be paired with one or more components disclosed in Tables 5-11 or a component with 90% or greater homology thereto, alone or in combination with other sequences (e.g., a stuffer sequence) in the AAV viral vector genome. In some embodiments, the AAV vector genome may comprise multiple copies (e.g., two, three or more copies) of one or more viral genome components described herein. In some embodiments, the viral genome comprises two miR binding sites (e.g., two miR122 binding sites). In some embodiments, the viral genome comprises three miR binding sites (e.g., three miR122 binding sites). In some embodiments, the viral genome comprises any promoter disclosed herein (e.g., a promoter from Table 3 or a promoter with 90% or greater homology thereto), and one or more components provided in any one of Tables 5-11 or otherwise described herein, in 5' to 3' order as shown in any one of Tables 4, 12, 13, 14, 15, 16 or 17. In some embodiments, the viral genome comprises a promoter provided in Table 3 and one or more components provided in any one of Tables 5-11 or otherwise described herein, in a 5' to 3' order as shown in any one of Tables 4, 12, 13, 14, 15, 16, or 17. In some embodiments, the viral genome comprises all of the components shown in any one of Tables 4, 12, 13, 14, 15, 16, or 17, in a 5' to 3' order.

[0400] For example, a promoter comprising SEQ ID NO: 1742, or a promoter having 90% or greater homology thereto, can be paired with any component in Tables 5-11 (or a component having 90% or greater homology thereto) in the AAV vector genome, for example, the promoter is located between the 5'ITR sequence and ie1 exon 1 (for example, directly contacting two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome comprises three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a comitatin.

[0401] In another example, a promoter comprising SEQ ID NO: 1750, or a promoter having 90% or greater homology thereto, can be paired with any component in Tables 5-11 (or a component having 90% or greater homology thereto) in the AAV vector genome, for example, the promoter is located between the 5'ITR sequence and ie1 exon 1 (for example, directly contacting the two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome comprises three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a comitatin.

[0402] For example, a promoter comprising SEQ ID NO: 1738, or a promoter having 90% or greater homology thereto, can be paired with any component in Tables 5-11 (or a component having 90% or greater homology thereto) in the AAV vector genome, for example, the promoter is located between the 5'ITR sequence and ie1 exon 1 (for example, directly contacting two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome comprises three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a comitatin.

[0403] In another example, a promoter comprising SEQ ID NO: 1740, or a promoter having 90% or greater homology thereto, can be paired with any component in Tables 5-11 (or a component having 90% or greater homology thereto) in the AAV vector genome, for example, the promoter is located between the 5'ITR sequence and ie1 exon 1 (for example, directly contacting two other components or separated by one or more non-coding sequences). In some embodiments, the viral genome comprises three miR122 binding sites. In some embodiments, the viral genome further comprises a payload region, for example, a payload region encoding a comitatin.

[0404] In some embodiments, the promoter is a CBA promoter or is derived from a CBA promoter. The CBA promoter can drive the payload to be expressed in various tissues of the subject. As a non-limiting example, Example 4 of co-owned International Patent Application No. PCT / US2019 / 032387 (the contents of which are incorporated herein by reference in their entirety), including Tables 16-28, shows the expression of FXN using a CBA promoter (promoter provided in SEQ ID NO: 1776, ITR to ITR provided in SEQ ID NO: 1778). Table 16 of co-owned International Patent Application No. PCT / US2019 / 032387 shows the expression of FXN in mice after IV injection, wherein expression is seen in the cortex, lumbar spinal cord, lumbar dorsal root ganglion, trigeminal ganglion, heart, and liver with VOY101 particles harboring the CBA promoter. Table 18 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows expression of FXN in NHPs after IV injection, where expression was seen in the brainstem, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar / sacral DRG, ventricle, atrium, liver, soleus, and jejunum with VOY101 particles carrying the CBA promoter. Table 19 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows expression of FXN in NHPs after IV injection with VOY101 particles carrying the CBA promoter. 11 VG / kg, 2×10 12 VG / kg or 2×10 13 FXN expression in NHPs following IV injection of VOY201 particles with the CBA promoter was observed in the brainstem, brainstem, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar / sacral DRG, ventricle, atrium, liver, kidney, lung, soleus muscle, and / or spleen. The expression of FXN in NHPs following IV injection of VOY201 particles with the CBA promoter was observed in the brainstem, brainstem, cervical spinal cord, thoracic spinal cord, lumbar / sacral DRG, ventricle, atrium, liver, kidney, lung, soleus muscle, and / or spleen. Table 20 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows the expression of FXN in NHPs after IV injection of VOY201 particles with the CBA promoter. 12 VG / kg or 4.89×10 13Expression of FXN in NHPs following IV injection of VOY101 (400 mg / kg) with expression seen in the brainstem, cerebellum, cortex, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar / sacral DRG, ventricles, atria, liver, kidneys, soleus muscle, sympathetic thoracic chain ganglia, and / or adrenal glands with VOY101 particles harboring the CBA promoter. Distribution of vector genomes following IV injection in mice is shown in Table 17 of commonly owned International Patent Application No. PCT / US2019 / 032387, with distribution seen in the cortex, lumbar spinal cord, thoracic dorsal root ganglia, trigeminal ganglia, heart, and liver with VOY101 and AAV9 particles harboring the CBA promoter. Table 18 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows the distribution of vector genomes in NHPs after IV injection, where distribution was seen in the frontal cortex, striatum, brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, cervical dorsal root ganglia, thoracic dorsal root ganglia, lumbar / sacral dorsal root ganglia, ventricles, atria, liver, kidneys, lungs, soleus muscle, jejunum, and spleen with VOY101 particles carrying the CBA promoter. Table 19 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows the distribution of vector genomes in NHPs after IV injection, where distribution was seen in the frontal cortex, striatum, brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, cervical dorsal root ganglia, thoracic dorsal root ganglia, lumbar / sacral dorsal root ganglia, ventricles, atria, liver, kidneys, lungs, soleus muscle, jejunum, and spleen with VOY101 particles carrying the CBA promoter. 11 VG / kg, 2×10 12 VG / kg or 2×10 13 The distribution of vector genomes in NHPs after IV injection of VOY201 particles with the CBA promoter was observed in the frontal cortex, striatum, brainstem, cerebellum, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical DRG, thoracic DRG, lumbar / sacral DRG, ventricle, atrium, liver, kidney, lung, soleus muscle, jejunum, and / or spleen. ... Table 20 of commonly owned International Patent Application No. PCT / US2019 / 032387 shows the distribution of vector genomes in NHPs after IV injection of VOY201 particles with the CBA promoter. 12 VG / kg or 4.89×10 13 Distribution of vector genomes in NHPs following IV injection of VOY101 particles harboring the CBA promoter was seen in the motor cortex, sensorimotor cortex, striatum, brainstem, cerebellar cortex, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, thoracic spinal cord, cervical DRG, thoracic DRG, lumbar / sacral DRG, ventricles, atria, liver, kidney, soleus muscle, jejunum, spleen, sympathetic thoracic chain ganglia, and / or adrenal glands.

[0405] In some embodiments, the promoter is a promoter including a CMVie enhancer, CBA, CMV, ataxin promoter, a truncated CBA and / or a truncated CMV promoter or a promoter derived therefrom. The promoter can drive the payload to be expressed in various tissues of the subject. As a non-limiting example, as shown in Example 5 of co-owned International Patent Application No. PCT / US2019 / 032387 (the contents of which are incorporated herein by reference in their entirety), the mouse model of Friedreich's ataxia can be used to assess the in vivo distribution, expression and efficacy of the IV administration of VOY101 particles with FXN. In certain embodiments, as outlined in Example 5 of co-owned International Patent Application No. PCT / US2019 / 032387, the expression of FXN in mice driven by promoters such as, but not limited to, Table 3 can be assessed. As another non-limiting example, as shown in Example 5 of co-owned International Patent Application No. PCT / US2019 / 032387 (the contents of which are incorporated herein by reference in their entirety), a Friedreich's ataxia NHP model was used to evaluate the in vivo distribution and expression of IV administration of VOY101 particles with FXN. In certain embodiments, as outlined in Example 5 of co-owned International Patent Application No. PCT / US2019 / 032387, promoters such as, but not limited to, those in Table 3 can be evaluated to drive the expression of FXN in NHPs.

[0406] In some embodiments, the promoter is or is derived from a CBA promoter, which can drive expression of a payload in various tissues in a subject. As a non-limiting example, Example 14 of co-owned International Patent Application No. PCT / US2019 / 032387 (the contents of which are incorporated herein by reference in their entirety), including Tables 33-34, shows expression of FXN using a CBA promoter (promoter provided in SEQ ID NO: 1776, ITR to ITR provided in SEQ ID NO: 1778). Table 33 of co-owned International Patent Application No. PCT / US2019 / 032387 shows expression of FXN in mice after IV injection, wherein expression is seen in the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, thoracic DRG, heart, and / or liver of VOY101, VOY801, and / or VOY1101 particles harboring the CBA promoter. Table 34 of co-owned International Patent Application No. PCT / US2019 / 032387 shows the distribution of vector genomes after IV injection in mice, where distribution was seen in the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, heart, and liver of VOY101, VOY801, and / or VOY1101 particles harboring the CBA promoter. As another non-limiting example, Example 14 of co-owned Provisional Patent Application No. 62 / 839,889 (the contents of which are incorporated herein by reference in their entirety), including Tables 35-36, shows expression of FXN in VOY701 and VOY101 capsids using the CBA promoter (promoter provided as SEQ ID NO: 1776, ITR to ITR provided as SEQ ID NO: 1778). Table 35 of commonly owned provisional patent application No. 62 / 839,889 shows expression of FXN following IV injection in mice, where expression was seen in the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, and / or liver of mice harboring VOY701 and / or VOY101 particles with a CBA promoter. Table 36 of commonly owned provisional patent application No. 62 / 839,889 shows distribution of vector genomes following IV injection in mice, where distribution was seen in the cortex, striatum, hippocampus, brainstem, thoracic spinal cord, and / or liver of mice harboring VOY701 and / or VOY101 particles with a CBA promoter.

[0407] In some embodiments, the AAV particles described herein comprise a viral genome having a payload region encoding a frataxin. The viral genome can be engineered to optimize frataxin expression in target cells.

[0408] Viral genome: ITR-to-ITR sequence including the fraxin payload

[0409] Any of the components described herein can be used to design and optimize the ITR sequence of the viral genome for desired facitin expression. The viral genome can contain any number of components, such as, but not limited to, one or more ITRs, enhancers, promoters, introns, UTRs, payload regions, tags or selectable markers, miR binding sites or target sites, backbone regions, polyA sequences, and / or filler sequences. In a given viral genome, each of these components can be present zero, one, two, or more times.

[0410] Each of the ITRs, enhancers, promoters, introns, exons, payloads, tags, miR binding sites, PolyA, and / or stuffer components can be selected independently or in any combination from the sequences provided in Tables 3 and 5-11.

[0411] In some embodiments, the AAV viral genome comprises 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 a 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 comprises ie1 exon 1 and ie1 intron 1, or a fragment 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 NOs: 1817 and / or 1819. In some embodiments, the enhancer comprises one or more human β-globin sequences, for example, a sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs: 1816, 1820, and / or 1821. In some embodiments, the enhancer comprises SEQ ID NOs: 1817, 1819, 1820, and 1821. In some embodiments, the enhancer comprises SEQ ID NO: 1816.

[0412] In some embodiments, the payload region comprises a nucleic acid sequence encoding a polypeptide having at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1725, 1726, 1727, 1731, 1732, or 1733, for example, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO: 1725. In some embodiments, the payload region comprises a nucleic acid sequence having 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, a fragment of SEQ ID NO: 1728 comprises nucleotides 221-853 of SEQ ID NO: 1728. In some embodiments, the frataxin polypeptide is encoded by a nucleic acid sequence comprising SEQ ID NO: 1822, 1823, or 1824. In some embodiments, the tag is absent. In some embodiments, the tag is present and is a human influenza hemagglutinin HA tag. In some embodiments, the HA tag comprises SEQ ID NO: 1825. In some embodiments, the miR binding site is absent. In some embodiments, at least one miR binding site is present and comprises 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 the miR122 binding site, for example, three copies of SEQ ID NO: 1827 or a variant thereof with at least 90% sequence identity. In some embodiments, the set of miR binding sites comprising three copies of the miR122 binding site comprises SEQ ID NO: 1826. In some embodiments, the viral genome comprises a human growth hormone polyA sequence. 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 comprises a filler sequence, e.g., an albumin filler sequence. In some embodiments, the filler sequence comprises any one of those given by 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.

[0413] In certain embodiments, the AAV particle comprises at least one cis-element, including but not limited to a Kozak sequence, a backbone sequence, and / or an intron sequence. Certain embodiments provide that the AAV particle further comprises a promoter region. For example, the promoter may comprise any one of the CBA, CMV, FXN, and / or SV40 genes, or variants thereof. Non-limiting examples of ITR to ITR sequences of AAV particles comprising a viral genome having a payload region encoding a frataxin are described in Table 4.

[0414] In Table 4, cFXN denotes macaque (cynomolgus monkey) frataxin, hFXN denotes human (Homo sapiens) frataxin, hβ-globin denotes human β-globin, HA denotes human influenza hemagglutinin HA tag, and hGH denotes human growth hormone. Alb denotes albumin. The number following alb denotes the length of the albumin filler. miR-122BS denotes the miR-122 binding site. A “–” sign indicates that the construct does not contain a component or sequence. A “+” sign indicates that the construct contains a component or sequence.

[0415] Table 4. Representative ITR to ITR sequences

[0416]

[0417]

[0418] In some embodiments, the AAV particle comprises a viral genome comprising a sequence having a percent identity to any one of SEQ ID NOs: 1778-1810. The viral genome can 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% identical to any one of SEQ ID NOs: 1778-1810. The viral genome can be 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 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% identical to any one of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1778-1810.

[0419] In some embodiments, the viral genome comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 1778-1810. In some embodiments, the viral genome comprises a sequence that is at least 99% identical to any one of SEQ ID NOs: 1778-1810.

[0420] In some embodiments, the viral genome comprises a sequence having 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 having 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 having at least 95% sequence identity to SEQ ID NO: 1808. In some embodiments, the viral genome comprises a sequence having 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 present disclosure may comprise any combination of the sequence regions described in Tables 2-11, or otherwise described herein, encapsulated in any capsid listed in Table 1 or described herein.

[0421] In some embodiments, the AAV particle viral genome may comprise at least one sequence region as described in Tables 2-11. This region may be located before or after any other sequence region described herein. The viral genome may further comprise more than one copy of one or more sequence regions as described in Tables 2-11.

[0422] Viral genome: inverted terminal repeats (ITRs)

[0423] In some embodiments, the AAV particle viral genome may comprise at least one inverted terminal repeat (ITR) region. The ITR region can independently have a length of, for example, 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 6, 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, 1 170, 165-170, 165-175, and 170-175 nucleotides. As a non-limiting example, the viral genome comprises a 5' ITR that is about 141 nucleotides in length. As a non-limiting example, the viral genome comprises a 5' ITR that is approximately 141 nucleotides in length.As a non-limiting example, the viral genome comprises a 5' ITR that is approximately 130 nucleotides in length.As a non-limiting example, the viral genome comprises a 5' ITR having a length of approximately 119 nucleotides. As a non-limiting example, the viral genome comprises a 3' ITR having a length of approximately 141 nucleotides. As a non-limiting example, the viral genome comprises a 3' ITR having a length of approximately 130 nucleotides. As a non-limiting example, the viral genome comprises a 3' ITR having a length of approximately 119 nucleotides. As a non-limiting example, the 5' ITR and the 3' ITR can comprise the same length and / or the same sequence. As another non-limiting example, the 5' ITR and the 3' ITR are different in length and / or sequence.

[0424] In some embodiments, the AAV particle viral genome comprises at least one inverted terminal repeat sequence region. Non-limiting examples of ITR sequence regions are described in Table 5.

[0425] Table 5. Representative inverted terminal repeat (ITR) sequence regions

[0426]

[0427] In some embodiments, the AAV particle viral genome may have an ITR comprising ITR1. In some embodiments, the AAV particle viral genome may have an ITR comprising 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.

[0428] Viral genome: intron and exon sequences of the payload region

[0429] In some embodiments, the AAV particle viral genome comprises at least one intron and / or exon sequence region. Non-limiting examples of intron and exon sequence regions are described in Table 6.

[0430] Table 6. Representative intron and exon sequence regions

[0431] Sequence region name Sequence length SEQ ID NO introns 1016 1815 hB globin intron / exon 566 1816 ie1 exon 1 134 1817 CMV / globin intron 379 1818 ie1 intron 1 (partial) 32 1819 hB globin intron 2 347 1820 hB globin exon 3 53 1821

[0432] In some embodiments, the AAV particle viral genome may comprise at least one intron sequence region.

[0433] 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 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, 1 83, 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 4, 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, 315 347, 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, 1 75-200, 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 comprises an intron sequence region with a length of about 32 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 53 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 134 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 347 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 379 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 566 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of about 1016 nucleotides. As a non-limiting example, the viral genome comprises an intron sequence region with a length of more than about 1016 nucleotides. In some embodiments, the AAV particle viral genome comprises two intron sequence regions. In some embodiments, the AAV particle viral genome comprises three intron sequence regions. In some embodiments, the AAV particle viral genome comprises more than three intron sequence regions.

[0434] In some embodiments, the AAV particle viral genome can comprise at least one exon sequence region. The exon sequence regions can independently have a 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 3, 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 8, 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, 311 344, 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 comprises an exon region that is about 32 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 53 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 134 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 347 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 379 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 566 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is about 1016 nucleotides in length. As a non-limiting example, the viral genome comprises an exon sequence region that is more than about 1016 nucleotides in length.

[0435] In some embodiments, the AAV particle viral genome comprises two exon sequence regions. In some embodiments, the AAV particle viral genome comprises three exon sequence regions. In some embodiments, the AAV particle viral genome comprises more than three exon sequence regions.

[0436] In some embodiments, the AAV particle viral genome comprises a hybrid intron / exon sequence region comprising at least one intron and at least one exon. In some embodiments, the hybrid intron / exon sequence region comprises one intron and one exon. In some embodiments, the hybrid intron / exon sequence region comprises two introns and two exons. In some embodiments, the intron or exon sequence may comprise a full-length intron or exon. In some embodiments, the intron or exon sequence may comprise a fragment or variant of an intron or exon sequence.

[0437] The hybrid intron / exon sequence region can independently have, for example, 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 comprises a hybrid intron / exon sequence region having a length of about 379 nucleotides. As a non-limiting example, the viral genome comprises a hybrid intron / exon sequence region having a length of about 566 nucleotides. As a non-limiting example, the viral genome comprises a hybrid intron / exon region having a length of about 379 nucleotides.

[0438] In some embodiments, the intron / exon sequence region is an enhancer sequence. In some embodiments, the intron / exon sequence region is not an enhancer sequence.

[0439] In some embodiments, the intron / exon sequence region is a component of the promoter sequence.In certain embodiments, the intron / exon sequence region is not a component of the promoter sequence.

[0440] Viral genome: frataxin payload

[0441] In some embodiments, the payload may contain any of the sequences given in Table 7.

[0442] Table 7. Representative fraxin payload sequences

[0443] Sequence region name Sequence length SEQ ID NO FXN 630 1822 hFXN 630 1823 hFXN+ termination 633 1824

[0444] In some embodiments, the payload sequence encodes a symtaxin derived from macaque (cynomolgus monkey) or a variant thereof. In some embodiments, the payload sequence encodes a symtaxin derived from macaque (cynomolgus monkey) but differs from at least one amino acid. In some embodiments, the payload sequence encodes a symtaxin derived from macaque (cynomolgus monkey) but differs from the wild type by at least one amino acid. In some embodiments, the payload sequence encodes a symtaxin derived from macaque (cynomolgus monkey) but differs from the wild type by at least two amino acids.

[0445] In some embodiments, the payload sequence encodes a frataxin derived from human (Homo sapiens) or a variant thereof. In some embodiments, the payload sequence comprises a stop codon.

[0446] Viral genome: tag sequence

[0447] In some embodiments, the AAV particle viral genome may include at least one tag sequence region. As used herein, the term "tag" refers to a polynucleotide sequence attached to a payload, which can be used to identify the expressed payload once expressed. Alternatively, the term "tag" can refer to a polynucleotide sequence attached to a payload, the signal of which is used to retain the expressed payload in a specific region of the cell (e.g., endoplasmic reticulum). The tag sequence region can independently have a 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 or more than 30 nucleotides. The length of the tag sequence region of 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 includes a tag sequence region having a length of about 27 nucleotides.

[0448] In some embodiments, the AAV particle viral genome comprises at least one tag sequence region. Non-limiting examples of tag sequence regions are shown in Table 8.

[0449] Table 8. Representative tag sequence regions

[0450] Sequence region name Sequence length SEQ ID NO HA 27 1825

[0451] In some embodiments, the AAV particle viral genome comprises a tag sequence region. In some embodiments, the tag sequence region is a human influenza hemagglutinin (HA) tag.

[0452] In some embodiments, the AAV particle viral genome comprises more than one tag sequence region. In one embodiment, the AAV particle viral genome comprises two tag sequence regions. In one embodiment, the AAV particle viral genome comprises three tag sequence regions. In one embodiment, the AAV particle viral genome comprises more than three tag sequence regions.

[0453] Viral genome: microRNA (miR) binding sites

[0454] 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.

[0455] Table 9. Representative miR binding site sequence regions

[0456]

[0457] In some embodiments, the AAV particle viral genome comprises a single miR binding site sequence. As a non-limiting example, the miR binding site sequence can be a miR-122 binding site.

[0458] In some embodiments, the viral genome may comprise more than one miR binding site sequence. As non-limiting examples, the viral genome may comprise two, three, four, or five miR binding site sequences.

[0459] In some embodiments, the viral genome may comprise a miR binding site array (SEQ ID NO: 1826) comprising three unique miR binding site sequences (SEQ ID NO: 1827).

[0460] 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, 110 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 nucleotides.

[0461] Viral genome: polyA signal

[0462] In some embodiments, the AAV particle viral genome can comprise at least one polyadenylation (PolyA) sequence region. The polyadenylation sequence region can independently have a length of, for example, 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 ,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,1 43, 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 7, 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 about 477 nucleotides in length.

[0463] In some embodiments, the AAV particle viral genome comprises at least one polyA sequence region. Non-limiting examples of polyA sequence regions are described in Table 10.

[0464] Table 10. Representative PolyA sequence regions

[0465] Sequence region name Sequence length SEQ ID NO hGHpA 477 1828

[0466] In some embodiments, the AAV particle viral genome comprises a polyA sequence region. As a non-limiting example, the polyA sequence comprises a human growth hormone polyadenylation sequence.

[0467] In one embodiment, the AAV particle viral genome comprises more than one polyA sequence region.

[0468] Viral genome: filler (or stuffer) sequence

[0469] In one embodiment, the AAV particle viral genome may comprise at least one or more stuffer sequence regions. The filler zones can 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 7, 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, 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Claims

1. An adeno-associated virus (AAV) genome comprising the nucleotide sequence of SEQ ID NO: 1797.

2. The AAV genome of claim 1 , wherein the AAV genome consists of the nucleotide sequence of SEQ ID NO: 1797.

3. AAV particles comprising the AAV genome and AAV capsid according to claim 1 or 2.

4. The AAV particle of claim 3, wherein the AAV capsid is an AAV5 capsid or a variant thereof, or an AAV9 capsid or a variant thereof.

5. A pharmaceutical composition comprising the AAV particles of claim 3 or 4 and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is formulated for intravenous administration.

7. Use of the AAV particle of claim 3 or 4 in the preparation of a medicament for treating Friedreich's ataxia in a subject.

8. The use according to claim 7, wherein the medicament is formulated for intravenous administration.

9. Use of the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for treating Friedreich's ataxia in a subject.

10. The use according to claim 9, wherein the medicament is formulated for intravenous administration.

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