Compositions and methods for treating huntington's disease

Abstract

The present invention relates to compositions and methods for treating Huntington's disease, in particular to adeno-associated virus (AAV) particles encoding siRNA molecules and methods for treating Huntington's disease (HD).

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2018800446517, application date May 4, 2018, and invention name “Compositions and methods for treating Huntington's disease”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 501,786, filed on May 5, 2017, entitled “Compositions and Methods for Treating Huntington’s Disease,” U.S. Provisional Patent Application No. 62 / 507,921, filed on May 18, 2017, entitled “Compositions and Methods for Treating Huntington’s Disease,” and U.S. Provisional Patent Application No. 62 / 520,086, filed on June 15, 2017, the contents of each of which are incorporated herein by reference in their entirety.

[0004] Reference sequence list

[0005] This application is submitted with a sequence listing in electronic format. The title of the provided sequence listing file is 14482_0152_228_SEQ_LISTING.txt, which was created on May 1, 2018 and is 6,780,111 bytes in size. The electronic form of the sequence listing is incorporated herein by reference in its entirety. Field of the Invention

[0006] The present invention relates to compositions, methods and processes for the design, preparation, manufacture, use and / or formulation of AAV particles, which contain regulatory polynucleotides, such as polynucleotides encoding small interfering RNA (siRNA) molecules targeting Huntington protein (HTT) genes (e.g., wild-type or mutant CAG-expanded HTT genes). Targeting mutant HTT genes may interfere with the expression of HTT genes and the resulting HTT protein production. AAV particles containing regulatory polynucleotides encoding siRNA molecules can be inserted into recombinant adeno-associated virus (AAV) vectors. Also disclosed are methods of inhibiting HTT gene expression using AAV particles in subjects with neurodegenerative diseases (e.g., Huntington's disease (HD)). Background of the Invention

[0008] Huntington's disease (HD) is a monogenic fatal neurodegenerative disease characterized by progressive chorea, neuropsychiatric and cognitive dysfunction. Huntington's disease is known to be caused by an autosomal dominant triplet (CAG) repeat expansion of polyglutamine encoding the N-terminus of the Huntington (HTT) protein. This repeat expansion leads to a toxic increase in HTT function and ultimately to striatal neurodegeneration, which progresses to widespread brain atrophy. Symptoms usually appear between the ages of 35-44, and the life expectancy after onset is 10-25 years. Interestingly, the length of the HTT expansion is associated with the age of onset and the rate of disease progression, with longer expansions associated with more severe disease. In a small proportion of the HD population (about 6%), disease onset occurs at 2-20 years of age and akinetic-rigid syndrome occurs. These cases tend to progress faster than later-onset cases and have been classified as juvenile or Westphal variant HD. It is estimated that there are currently about 35,000-70,000 patients with HD in the United States and Europe. Currently, only symptom relief and supportive care are available for the treatment of HD, and no cure has been found. Ultimately, individuals with HD die from other illnesses (e.g., pneumonia, heart failure), suffocation, breathing difficulties, or other complications, such as physical injuries from falls.

[0009] The mechanism by which CAG-expanded HTT causes neurotoxicity is not well understood. Huntingtin is expressed in all cells, although its concentration is highest in the brain. The normal function of HTT is unknown, but in the brain of HD patients, HTT aggregates into abnormal nuclear inclusions. It is now believed that it is this misfolding and aggregation process and the associated protein intermediates (i.e., soluble species and toxic N-terminal fragments) that cause neurotoxicity.

[0010] For example, studies in animal models of HD have shown that phenotype reversal is feasible after gene shut-off in models of regulated expression. In addition, animal models of HTT silencing have been tested, showing promising results, with the treatment both well tolerated and showing potential therapeutic benefit. These findings suggest that HTT silencing could serve as a potential therapeutic target for HD treatment.

[0011] Adeno-associated virus (AAV) is a member of the Parvoviridae family and has emerged as an attractive gene therapy vector, largely because the virus is apparently nonpathogenic; indeed, AAV has not been implicated in any human disease. Further appeal is due to its ability to transduce both dividing and nondividing cells (including efficient neuronal transduction), reduced proinflammatory and immune responses in humans, its inability to replicate autonomously without a helper virus (AAV is a helper-dependent DNA parvovirus, which belongs to the genus Dependovirus), and its long-term gene expression. Although more than 10 recombinant AAV serotypes (rAAV) have been engineered as vectors, rAAV2 is the most commonly used serotype in gene therapy trials. Other rAAV serotypes have been developed and tested in animal models that are more effective at neuronal transduction.

[0012] The present invention develops AAV particles containing regulatory polynucleotides encoding novel double-stranded RNA (dsRNA) constructs and siRNA constructs and design methods thereof to inhibit or prevent the expression of CAG-expanded HTT in HD patients, thereby treating the disease. Summary of the invention

[0013] Described herein are methods, processes, compositions, kits and devices for administering AAV particles comprising regulatory polynucleotides encoding siRNA molecules to treat, prevent, alleviate and / or ameliorate symptoms and conditions associated with Huntington's disease (HD).

[0014] The present invention provides viral genomes comprising regulatory polynucleotides encoding siRNA molecules to target HTT and reduce the expression of HTT in cells and / or subjects.

[0015] In some embodiments, the viral genome comprises a 5' inverted terminal repeat (ITR) sequence region (e.g., but not limited to SEQ ID NOs: 1380 and 1381), an enhancer sequence region (e.g., but not limited to SEQ ID NOs: 1408 and 1409), a promoter sequence region (e.g., but not limited to SEQ ID NOs: 1410-1411 and sequence GTTG), at least one intron sequence region (e.g., but not limited to SEQ ID NOs: 1417-1419), a regulatory polynucleotide sequence region (e.g., but not limited to SEQ ID NOs: 1183-1279 and 1346-1347), a polyadenylation (poly A) signal sequence region (e.g., but not limited to SEQ ID NOs: 1420-1423), and a 3' ITR sequence region (e.g., but not limited to SEQ ID NOs: 1382 and 1383).

[0016] In some embodiments, the viral genome may include at least one additional sequence region.

[0017] In one embodiment, the viral genome comprises at least one multiple cloning site (MCS) sequence region, such as but not limited to SEQ ID NO: 384-1390 and sequence TCGAG. In one embodiment, the viral genome comprises one MCS sequence region. In one embodiment, the viral genome comprises two MCS sequence regions.

[0018] In one embodiment, the viral genome comprises at least one stuffer sequence region, such as but not limited to SEQ ID NO: 1390-1407. In one embodiment, the AAV viral genome comprises one stuffer sequence region. In one embodiment, the AAV viral genome comprises two stuffer sequence regions.

[0019] In one embodiment, the viral genome comprises at least one exon sequence region, such as but not limited to SEQ ID NOs: 1415 and 1416.

[0020] In one embodiment, the viral genome comprises an intron sequence region.

[0021] In one embodiment, the viral genome comprises two intron sequence regions.

[0022] In one embodiment, the viral genome comprises a 5' inverted terminal repeat (ITR) sequence region, an enhancer sequence region, a promoter sequence region, at least one intron sequence region, a regulatory polynucleotide sequence region, a polyadenylation (poly A) signal sequence region, and a 3' ITR sequence region. As a non-limiting example, the AAV viral genome comprises a sequence, such as SEQ ID NO: 1352-1379 or a fragment or variant thereof.

[0023] In one embodiment, the viral genome is, for example but not limited to, any one of SEQ ID NOs: 1352-1379 or a variant thereof having at least 95% identity thereto.

[0024] In one embodiment, the AAV particle may comprise a viral genome such as, but not limited to, any of SEQ ID NOs: 1352-1379 or a variant thereof having at least 95% identity. The AAV particle may comprise a serotype such as, but not limited to, any serotype listed in Table 1.

[0025] Also provided herein are pharmaceutical compositions of AAV particles. The AAV particles may comprise a viral genome, such as but not limited to any of SEQ ID NOs: 1352-1379 or variants thereof having at least 95% identity. The AAV particles may comprise a serotype, such as but not limited to any serotype listed in Table 1.

[0026] In some embodiments, the present invention provides a method for inhibiting / silencing HTT gene expression in a cell. The cell can be a neuron (e.g., medium spiny neurons in the putamen, caudate, or striatum, and cortical neurons in the cerebral cortex), an astrocyte (e.g., astrocytes in the striatum, cortical astrocytes in the cerebral cortex), and / or an oligodendrocyte. As a non-limiting example, inhibition (or reduction) of HTT gene expression in the putamen, caudate, and cortex reduces the impact of Huntington's disease in a subject. As another non-limiting example, inhibition (or reduction) of HTT gene expression in medium spiny neurons in the striatum reduces the impact of Huntington's disease in a subject. As yet another non-limiting example, inhibition (or reduction) of HTT gene expression in astrocytes in the striatum reduces the impact of Huntington's disease in a subject. In some aspects, inhibition of HTT gene expression refers to inhibition or reduction of at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. Thus, the protein product of the targeted gene can be inhibited by at least about 20%, preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0027] In one embodiment, the present invention provides a method for inhibiting / silencing HTT gene expression by at least about 40% in a cell using a viral genome comprising a regulatory polynucleotide encoding an siRNA molecule. The cell can be a neuron (e.g., a medium spiny neuron in the putamen or striatum, and a cortical neuron in the cerebral cortex), an astrocyte (e.g., astrocytes in the striatum, cortical astrocytes in the cerebral cortex) and / or an oligodendrocyte. As a non-limiting example, at least 40% inhibition (or reduction) of HTT gene expression in the putamen and cortex reduces the effect of Huntington's disease in a subject. As another non-limiting example, at least 40% inhibition (or reduction) of HTT gene expression in medium spiny neurons in the striatum reduces the effect of Huntington's disease in a subject. As yet another non-limiting example, at least 40% inhibition (or reduction) of HTT gene expression in astrocytes in the striatum reduces the effect of Huntington's disease in a subject.

[0028] In some embodiments, the present invention provides a method for treating or ameliorating Huntington's disease associated with an HTT gene (e.g., a CAG-expanded HTT gene) and the resulting HTT protein (e.g., a poly-Q protein) in a subject in need of treatment, the method comprising administering to the subject a pharmaceutically effective amount of a pharmaceutical composition comprising the AAV particles described herein, and improving the subject's HD symptoms.

[0029] In some embodiments, an AAV particle comprising a nucleic acid sequence encoding at least one siRNA duplex targeting the HTT gene is administered to a subject in need of treatment and / or amelioration of HD. The AAV vector serotype may be any of the serotypes listed in Table 1.

[0030] In some embodiments, AAV particles can be introduced directly into the central nervous system of a subject, for example, by intracranial injection.

[0031] In some embodiments, the pharmaceutical composition of the present invention is used as a monotherapy. In other embodiments, the pharmaceutical composition of the present invention is used in combination therapy. Combination therapy can be used in combination with one or more neuroprotectants (e.g., small molecule compounds, growth factors, and hormones) that have been tested and have a neuroprotective effect on neuronal degeneration.

[0032] In some embodiments, the present invention provides methods of treating or ameliorating Huntington's disease by administering to a subject in need thereof a therapeutically effective amount of a plasmid or AAV vector described herein.

[0033] In some embodiments, the present invention provides a method of inhibiting HTT gene expression in a region of the central nervous system of a subject by administering to the subject a composition having at least one AAV particle, the AAV particle comprising a regulatory polynucleotide encoding an siRNA molecule that, when expressed, inhibits or represses the expression of HTT in the subject. The expression may be reduced in a region of the subject, such as, but not limited to, the forebrain or a region of the forebrain of the subject, such as, but not limited to, the putamen. The expression of HTT in the forebrain or a region of the forebrain, such as the putamen, may be reduced by 40-70%, 40-60%, 50-70%, 50-60%, or may be reduced by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%.

[0034] In some embodiments, the present invention provides a method for treating Huntington's disease (HD) in a subject in need of treatment. The method can inhibit the expression of the HTT gene in a region of the central nervous system of the subject, comprising administering to the subject a composition comprising at least one AAV particle, the AAV particle comprising a regulatory polynucleotide encoding an siRNA molecule, the siRNA molecule inhibiting or suppressing the expression of HTT in the subject when expressed. The expression can be reduced in a region of the subject, such as, but not limited to, the forebrain or a region of the forebrain of the subject (such as, but not limited to, the putamen). The expression of HTT in the forebrain or a region of the forebrain (such as the putamen) can be reduced by 40-70%, 40-60%, 50-70%, 50-60%, and may also be reduced by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings. The drawings are not necessarily drawn to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[0037] Figure 1 is a schematic diagram of the viral genome of the present invention.

[0038] Figure 2 is a schematic diagram of the viral genome of the present invention.

[0039] Figure 3 is a schematic diagram of the viral genome of the present invention.

[0040] Figure 4 is a schematic diagram of the viral genome of the present invention.

[0041] Figure 5 is a schematic diagram of the viral genome of the present invention.

[0042] Figure 6 is a schematic diagram of the viral genome of the present invention.

[0043] Figure 7 is a schematic diagram of the viral genome of the present invention.

[0044] Figure 8 is a schematic diagram of the viral genome of the present invention.

[0045] Fig. 9 is a schematic diagram of the viral genome of the present invention.

[0046] The details of one or more embodiments of the present invention are set forth in the following attached description. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred materials and methods are now described. By description, other features, objects and advantages of the present invention will be apparent. In the specification, the singular form also includes the plural form, unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. In the event of a conflict, this specification shall prevail. DETAILED DESCRIPTION OF THE INVENTION

[0048] I. Composition of the Invention

[0049] According to the present invention, compositions for delivering regulatory polynucleotides and / or regulatory polynucleotide-based compositions via adeno-associated viruses (AAVs) are provided. The AAV particles of the present invention can be provided to cells, tissues, organs, or organisms in vivo, ex vivo, or in vitro by any of several routes.

[0050] As used herein, an "AAV particle" is a virus comprising a viral genome having at least one payload region and at least one inverted terminal repeat (ITR) region.

[0051] As used herein, "viral genome" or "vector genome" or "viral vector" refers to the nucleic acid sequence encapsulated in the AAV particle. The viral genome contains at least one payload region encoding a polypeptide or a fragment thereof.

[0052] As used herein, "payload" or "payload region" is any nucleic acid molecule encoding one or more polypeptides of the present invention. The payload region comprises at least a nucleic acid sequence encoding sense and antisense sequences (a siRNA-based composition) or a fragment thereof, but may also optionally comprise one or more functional or regulatory elements to promote transcriptional expression and / or polypeptide translation.

[0053] The nucleic acid sequences and polypeptides disclosed herein can be engineered to include assembled modular elements and / or sequence motifs that achieve expression of the regulatory polynucleotides of the present invention and / or compositions based on regulatory polynucleotides. In some embodiments, the nucleic acid sequence comprising the payload region may include one or more of a promoter region, an intron, a Kozak sequence, an enhancer, or a polyadenylation sequence. The payload region of the present invention typically encodes at least one sense and antisense sequence (a composition based on siRNA) or fragments of the foregoing that are bound to each other or to other polypeptide moieties.

[0054] The payload region of the invention can be delivered to one or more target cells, tissues, organs or organisms within the viral genome of an AAV particle.

[0055] Adeno-associated virus (AAV) and AAV particles

[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 consists of two subfamilies: the Parvovirinae that infects vertebrates and the Densovirinae that infects invertebrates. Due to its relatively simple structure, it is easy to operate using standard molecular biology techniques, so this virus family can be used as a biological tool. The genome of the virus can be modified so that it includes the minimum components for assembling a functional recombinant virus or a viral particle, which is loaded with or engineered to express or deliver the desired payload, which can be delivered to a target cell, tissue, organ or organism.

[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 Edition, 1996), Chapter 69, the contents of which are incorporated by reference in their entirety.

[0058] The Parvoviridae family includes the genus Dependentovirus, which includes adeno-associated viruses (AAV) capable of replicating in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep.

[0059] The AAV viral genome is a linear single-stranded DNA (ssDNA) molecule with a length of about 5,000 nucleotides (nt). The AAV viral genome may include a payload region and at least one inverted terminal repeat (ITR) or ITR region. Traditionally, ITR is located on the flank of the coding nucleotide sequence of non-structural proteins (encoded by the Rep gene) and structural proteins (encoded by the capsid gene or Cap gene). Although not wishing to be bound by theory, the AAV viral genome generally includes two ITR sequences. The AAV viral genome includes a characteristic T-shaped hairpin structure, which is limited by the self-complementary end 145nt of the 5' and 3' ends of the ssDNA that forms an energy-stabilized double-stranded region. The double-stranded hairpin structure includes a variety of functions, including but not limited to being used as a starting point for DNA replication by acting as a primer for the endogenous DNA polymerase complex of the host virus replication cell.

[0060] In addition to the encoded heterologous payload, the AAV vector may also contain, in whole or in part, any naturally occurring and / or recombinant AAV serotype nucleotide sequence or viral genome of a variant. AAV variants may have sequences with significant homology at the nucleic acid (genome or capsid) and amino acid levels (capsid) to produce constructs that are generally physically and functionally equivalent, replicated by similar mechanisms, and assembled by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33 (1997); Srivastava et al., J. Vir. 45: 555-64 (1983); Chiorini et al., J. Vir. 73: 1309-1319 (1999); Rutledge et al., J. Vir. 72: 309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), the contents of each of which are incorporated herein by reference in their entirety.

[0061] In one embodiment, the AAV particles of the invention are replication-defective recombinant AAV vectors that lack sequences encoding functional Rep and Cap proteins in their viral genomes. These defective AAV vectors may lack most or all of the parental coding sequences and essentially carry only one or two AAV ITR sequences and a target nucleic acid for delivery to a cell, tissue, organ or organism.

[0062] In one embodiment, the viral genome of the AAV particles of the present invention comprises at least one control element that provides for replication, transcription and translation of the coding sequence encoded therein. It is not always necessary to have all the control elements present, as long as the coding sequence can be replicated, transcribed and / or translated in an appropriate host cell. Non-limiting examples of expression control elements include sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, effective RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability and / or sequences that enhance protein processing and / or secretion.

[0063] According to the present invention, the AAV particles used for treatment and / or diagnosis contain viruses that have been distilled or reduced to the minimum components required to transduce the target nucleic acid payload or cargo. In this way, the AAV particles are engineered as vectors for specific delivery while lacking the deleterious replication and / or integration features found in wild-type viruses.

[0064] The AAV vectors of the present invention can be recombinantly produced and can be based on a parent or reference sequence of an adeno-associated virus (AAV). As used herein, a "vector" is any molecule or entity that transports, transduces, or otherwise acts as a carrier of a heterologous molecule (e.g., a nucleic acid described herein).

[0065] In addition to single-stranded AAV viral genomes (e.g., ssAAV), the present invention also provides self-complementary AAV (scAAV) viral genomes. The scAAV viral genome comprises DNA strands that anneal together to form double-stranded DNA. By skipping second-strand synthesis, scAAV can be rapidly expressed in cells.

[0066] In one embodiment, the AAV particle of the invention is a scAAV.

[0067] In one embodiment, the AAV particle of the invention is ssAAV.

[0068] Methods for producing and / or modifying AAV particles, such as pseudotyped AAV vectors, are disclosed in the art (PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO 2005005610 and WO 2005072364, the contents of each of which are incorporated herein by reference in their entirety).

[0069] AAV particles can be modified to enhance delivery efficiency. Such modified AAV particles can be effectively packaged and used to successfully infect target cells with high frequency and minimal toxicity. In some embodiments, the capsid of AAV particles is engineered according to the method described in U.S. Publication No. US20130195801, the contents of which are incorporated herein by reference in their entirety.

[0070] In one embodiment, AAV particles comprising a payload region encoding a polypeptide of the invention can be introduced into mammalian cells.

[0071] AAV serotypes

[0072] The AAV particles of the present invention may comprise or be derived from any natural or recombinant AAV serotype. According to the present invention, the AAV particles may utilize or be based on a serotype selected from any of the following: AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3 , AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11 , AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, A AV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5 -22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41 , AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61,AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAV C2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh. .65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVc y.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、AA Vhu.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.4 6、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.t19, 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, caprine AAV, bovine AAV, ovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6,AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV改组100-1, AAV改组100-3, AAV改组100-7, AAV改组10-2, AAV改组10-6, AAV改组10-8, AAV改组100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, 真型AAV(ttAAV), UPENN AAV 10, 日本AAV 10血清型, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAVCBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAVCHt-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, AAVCKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3AAV CKd-B4、AAVCKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAVCKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAVCLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAVCLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAVCLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAVCLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAVCLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAVCLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M1、AAV CLv-M2、AAV CLv-M5、AAVCLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAVCLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAVCSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-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 / HSC 7. AAVF8 / HSC8, AAVF9 / HSC9, AAV-PHP.B(PHP.B), AAV-PHP.A(PHP.A), G2B-26, G2B-13, TH1.1-32, TH 1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP , AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQ P, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, A 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, AAVG2B4 and / or AAVG2B5 and variants thereof.

[0073] In some embodiments, the AAV serotype can be or have modifications described in U.S. Publication No. US20160361439, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof.

[0074] In some embodiments, the AAV serotype may be or have a mutation described in U.S. Pat. No. US 9546112, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, at least two, but not all, F129L, D418E, K531E, L584F, V598A, and H642N mutations in the sequence of AAV6 (SEQ ID NO: 4 of US 9546112), AAV1 (SEQ ID NO: 6 of US 9546112), AAV2, AAV3, AAV4, AAV5, AAV7, AAV9, AAV10, or AAV11, or derivatives thereof. In yet another embodiment, the AAV serotype may be or have an AAV6 sequence comprising a K53IE mutation (SEQ ID NO: 5 of US 9546112).

[0075] In some embodiments, the AAV serotype may be or have a mutation in the AAV1 sequence described in U.S. Publication No. US20130224836, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, preferably at least one surface-exposed tyrosine residue at positions 252, 273, 445, 701, 705, and 731 of AAV1 (SEQ ID NO: 2 of US20130224836) is substituted with another amino acid, preferably with a phenylalanine residue. In one embodiment, the AAV serotype may be or have a mutation in the AAV9 sequence, such as, but not limited to, preferably at least one surface-exposed tyrosine residue at positions 252, 272, 444, 500, 700, 704, and 730 of AAV2 (SEQ ID NO: 4 of US20130224836) is substituted with another amino acid, preferably with a phenylalanine residue. In one embodiment, the tyrosine residue at position 446 of AAV9 (SEQ ID NO: 6US20130224836) is substituted with a phenylalanine residue.

[0076] In some embodiments, the serotype may be AAV2 or a variant thereof as described in International Publication No. WO2016130589, which is incorporated herein by reference in its entirety. The amino acid sequence of AAV2 may include N587A, E548A, or N708A mutations. In one embodiment, the amino acid sequence of any AAV may include a V708K mutation.

[0077] In some embodiments, the AAV serotype may be or have a sequence as described in U.S. 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: 2-3 of US20030138772), AAV11 (SEQ ID NOs: 2-3 of US20030138772), AAV12 (SEQ ID NOs: 2-3 of US20030138772), AAV13 (SEQ ID NOs: 2-3 of US20030138772), AAV14 (SEQ ID NO: 63 of US20030138772), AAV15 (SEQ ID NO: 114 of US20030138772), AAV16 (SEQ ID NO: 65 of US20030138772), AAV17 (SEQ ID NOs: 2- 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 (US20030138772 SEQ ID NO:18), AAVC1 (US20030138772 SEQ ID NO:19), AAVC3 (US20030138772 SEQ ID NO:20), AAVC5(US20030138772 SEQ ID NO: 21), AAVF1 (US20030138772 SEQ IDNO:22)、AAVF3(US20030138772 SEQ ID NO:23)、AAVF5(US20030138772 SEQ ID NO:24)、AAVH6(US20030138772 SEQ 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(US20030138772 SEQ 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(US20030138772 SEQ 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(US20030138772 SEQ ID NO:40)、AAV43-12(US20030138772 SEQ ID NO:41)、AAV43-20(US20030138772 SEQ ID NO:42)、AAV43-21(US20030138772 SEQ ID NO:43)、AAV43-23(US20030138772 SEQ ID NO:44)、AAV43-25(US20030138772 SEQ ID NO:45)、AAV44.1(US20030138772 SEQ ID NO:46)、AAV44.5(US20030138772 SEQ ID NO:47)、AAV223.1(US20030138772 SEQ ID NO:48)、AAV223.2(US20030138772 SEQ IDNO: 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 thereof.

[0078] In some embodiments, the AAV serotype can be or have a sequence described in U.S. 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), AAV6.1 (SEQ ID NO: 7 of US20150159173), rh80 (SEQ ID NO: 8 of US20150159173), rh91 (SEQ ID NO: 9 of US20150159173), rh10 (SEQ ID NO: 10 of US20150159173), rh110 (SEQ ID NO: 11 of US20150159173), rh120 (SEQ ID NO: 12 of US20150159173), rh131 (SEQ ID NO: 13 of US20150159173), rh140 (SEQ ID NO: 14 of US20150159173), rh150 (SEQ ID NO: 15 of US20150159173), rh160 (SEQ ID NO: 16 of US NO:29), 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 NOs:8 and 24 of US20150159173), rh.10 (SEQ ID NO:43 of US20150159173), NO:9 and 25), 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 33 of US20150159173), hu.37 (SEQ ID NO:18 and 29 of US20150159173), ID NO:18 and 34), 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: 48 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 and hu.48R3. .

[0079] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Pat. No. US 7198951, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV9 (SEQ (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 US7198951), and AAV8 (SEQ ID NO: 7 of US7198951).

[0080] In some embodiments, the AAV serotype can be or have a mutation in the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), incorporated herein by reference in its entirety), such as but not limited to AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

[0081] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Pat. No. 6156303, 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 US 6156303), AAV6 (SEQ ID NOs: 2, 7, and 11 of US 6156303), AAV2 (SEQ ID NOs: 3 and 8 of US 6156303), AAV3A (SEQ ID NOs: 4 and 9 of US 6156303), or derivatives thereof.

[0082] In some embodiments, the AAV serotype can be or have a sequence described in U.S. 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.

[0083] In some embodiments, the serotype can be AAVDJ (or AAV-DJ) 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 include two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772 (the contents of which are incorporated herein by reference in their entirety) can include 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, three mutations may be included: (1) K406R, wherein the lysine (K; Lys) at amino acid 406 is changed to arginine (R; Arg), (2) R587Q, wherein the arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, wherein the arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

[0084] In some embodiments, the AAV serotype may be or have an AAV4 sequence as described in International Publication No. WO 1998011244, the contents of which are incorporated herein by reference in their entirety, such as but not limited to AAV4 (SEQ ID NOs: 1-20 of WO1998011244).

[0085] In some embodiments, the AAV serotype may be or have a mutation in the AAV2 sequence to produce AAV2G9, as described in International Publication No. WO2014144229, which is herein incorporated by reference in its entirety.

[0086] In some embodiments, the AAV serotype may be or have a sequence 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: 7 of WO2005033321), AAV129.1 / hu.50 (SEQ ID NO: 8 of WO2005033321), AAV130.1 / hu.51 (SEQ ID NO: 9 of WO2005033321), AAV131.1 / hu.52 (SEQ ID NO: 10 of WO2005033321), AAV132.1 / hu.53 (SEQ ID NO: 11 of WO2005033321), AAV133.1 / hu.54 (SEQ ID NO: 11 of WO2005033321), AAV134.1 / hu.55 (SEQ ID NO: 11 of WO2005033321), AAV135.1 / hu.56 (SEQ ID NO: 11 of WO2005033321), AAV137.1 / hu.57 (SEQ ID NO: 11 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). 33 and 114 of WO2005033321), AAV2-3 / rh.61 (SEQ ID NO: 21 of WO2005033321), AAV2-4 / rh.50 (SEQ ID NO: 23 and 108 of WO2005033321), AAV2-5 / rh.51 (SEQ ID NO: 211 and 221 of WO2005033321), AAV2-62 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-7 / rh.70 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-8 / rh.81 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-9 / rh.90 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-10 / rh.101 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-11 / rh.102 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-12 / rh.123 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-13 / rh.134 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-14 / rh.145 (SEQ ID NO: 33 and 114 of WO2005033321), AAV2-15 / rh.156 (SEQ ID NO: 33 and 114 of WO200 NO: 104 and 22), AAV3.1 / hu.6 (SEQ ID NO: 5 and 84 of WO2005033321), AAV3.1 / hu.9 of WO2005033321 (SEQ ID NOs: 155 and 58), 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), AAV4-20 / rh.57 (SEQ ID NO: 201 of WO2005033321), AAV4-21 / rh.58 (SEQ ID NO: 203 of WO2005033321), AAV4-22 / hu.59 (SEQ ID NO: 214 of WO2005033321), AAV4-23 / hu.51 (SEQ ID NO: 215 of WO2005033321), AAV4-24 / hu.52 (SEQ ID NO: 216 of WO2005033321), AAV4-25 / hu.53 (SEQ ID NO: 217 of WO2005033321), AAV4-26 / hu.55 (SEQ ID NO: 218 of WO2005033321), 3321), AAV5-22 / rh.58 (SEQ ID No: 27 of WO2005033321), AAV5-3 / rh.57 (SEQ ID No: 106 of WO2005033321), AAV5-4 / 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: 106 of WO2005033321), AAV5-4 / 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: 106 of WO2005033321), AAV5-4 / hu.20 (SEQ ID NO: 63 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 NO: 57 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 WO2005033321), AAVhu.52 (SEQ ID NO:191 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.63 (SEQ ID NO:195 of WO2005033321), AAVhu.64 (SEQ ID NO:196 of WO2005033321), AAVhu.66 (SEQ ID NO:197 of WO2005033321), AAVhu.67 (SEQ ID NO:198 of WO2005033321), AAVhu.7 (SEQ ID NO:150 of WO2005033321), AAVhu.8 (SEQ ID NO:12 of WO2005033321), AAVhu.9 (SEQ ID NO:155 of WO2005033321), AAVLG-10 / rh.40 (SEQ ID No:14 of WO2005033321), AAVLG-4 / rh.38 (SEQ ID NO:86 of WO2005033321), AAVLG-4 / rh.38 (SEQ ID No:7 of WO2005033321), AAVN721-8 / rh.43 (SEQ ID NO:163 of WO2005033321), AAVN721-8 / rh.43 (SEQ ID No:43 of WO2005033321), AAVpi.1 (WO2005033321 SEQ ID NO:28), AAVpi.2 (WO2005033321 SEQ ID NO:30), AAVpi.3 (WO2005033321 SEQ ID NO:29), AAVrh.38 (SEQ ID NO:86 of WO2005033321), AAVrh.40 (SEQ ID NO:92 of WO2005033321), AAVrh.43 (SEQ ID NO:163 of WO2005033321), AAVrh.44 (WO2005033321 SEQ ID NO:34), AAVrh.45 (WO2005033321 SEQ ID NO:41), AAVrh.47 (WO2005033321 SEQ ID NO:38), AAVrh.48 (SEQ ID NO:115 of WO2005033321), AAVrh.49 (SEQ ID NO:103 of WO2005033321), AAVrh.50 (SEQ ID NO:108 of WO2005033321), AAVrh.51 (SEQ ID NO:104 of WO2005033321), AAVrh.52 (SEQ ID NO:96 of WO2005033321), AAVrh.53 (SEQ ID NO:97 of WO2005033321), AAVrh.55 (WO2005033321 SEQ ID NO:37), AAVrh.56 (SEQ ID NO:152 of WO2005033321), AAVrh.57 (SEQ ID NO:105 of WO2005033321), AAVrh.58 (SEQ ID NO:106 of WO2005033321), AAVrh.59 (WO2005033321 SEQ ID NO:42), AAVrh.60 (WO2005033321SEQ ID NO:31), 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 AAVrh.2, AAVrh.3, AAVrh.4, AAVrh.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, and AAVrh14. Non-limiting examples of variants include SEQ ID NOs: 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62, 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219, 224-236 of WO2005033321, the contents of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, the AAV serotype can be or have a sequence described in International Publication No. WO2015168666, the contents of which are incorporated herein 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), AAVrh8RR533A mutant (SEQ ID NO: 11 of WO2015168666), or variants thereof.

[0088] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Pat. No. US9233131, 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 US9233131), AAVhEr1.5 (SEQ ID NO: 45 of US9233131), AAVhER1.14 (SEQ ID NO: 46 of US9233131), AAVhEr1.8 (SEQ ID NO: 47 of US9233131), AAVhEr1.16 (SEQ ID NO: 48 of US9233131), AAVhEr1.18 (SEQ ID NO: 49 of US9233131), AAVhEr1.35 (SEQ ID NO: 50 of US9233131), AAVhEr1.7 (SEQ ID NO: 51 of US9233131), 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), AAVhEr3.2 (SEQ ID NO:55 of US9233131), AAVhEr3.3 (SEQ ID NO:56 of US9233131), AAVhEr3.4 (SEQ ID NO:57 of US9233131), AAVhEr3.5 (SEQ ID NO:58 of US9233131), AAVhEr3.6 (SEQ ID NO:59 of US9233131), AAVhEr3.7 (SEQ ID NO:59 of US9233131), AAVhEr3.8 (SEQ ID NO:51 of US9233131), AAVhEr3.9 (SEQ ID NO:51 of US9233131), AAVhEr3.10 (SEQ ID NO:51 of US9233131), AAVh NO:59), AAV2.5T (SEQ ID NO:42 of US9233131), or variants thereof.

[0089] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Patent 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 NO: 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 US20150376607ID NO: 25), AAV-PAEC11 (SEQ ID NO: 26 of US20150376607), AAV-PAEC12 (SEQ ID NO: 27 of US20150376607), or variants thereof.

[0090] In some embodiments, the AAV serotype may be or have a sequence described in U.S. Pat. No. US9163261, 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).

[0091] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Patent 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.

[0092] In some embodiments, the AAV serotype can be or have a sequence described in U.S. Patent 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: 36 of US20160017295). 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), AAV SM 100-3 (SEQ ID NO:40 of US20160017295), AAV SM 100-10 (SEQ ID NO:41 of US20160017295) or variants thereof.

[0093] In some embodiments, the AAV serotype can be or have a sequence 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.

[0094] In some embodiments, the AAV serotype can be or can have a sequence 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), AAVhu.64 (SEQ ID NO: 165 of US20150315612), AAVhu.65 (SEQ ID NO: 166 of US20150315612), AAVhu.67 (SEQ ID NO: 168 of US20150315612), AAVhu.68 (SEQ ID NO: 169 of US20150315612), AAVhu.71 (SEQ ID NO: 170 of US20150315612), AAVhu.72 (SEQ ID NO: 171 of US20150315612), AAVhu.73 (SEQ ID NO: 174 of US20150315612), AAVhu.74 (SEQ ID NO: 175 of US20150 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. of US20150315612) No: 60), 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. of US20150315612 No:65), AAV54.4R / hu.27 (SEQ ID of US20150315612 No:64), AAV46.2 / hu.28 (SEQ ID No:68 of US20150315612), AAV46.6 / hu.29 (SEQ ID No:69 of US20150315612), AAV128.1 / hu.43 (SEQ ID No:80 of US20150315612) or variants thereof.

[0095] In some embodiments, the AAV serotype can be or have the sequence described in International Publication No. WO2015121501, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), "UPenn AAV10" (SEQ ID NO: 8 of WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501) or variants thereof.

[0096] According to the present invention, the selection or use of AAV capsid serotypes can be from a variety of species. In one embodiment, AAV can be avian AAV (AAAV). The AAAV serotype can be or have a sequence described in U.S. Pat. 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 and 14 of US 9,238,800) or a variant thereof.

[0097] In one embodiment, the AAV may be a bovine AAV (BAAV). The BAAV serotype may be or have a sequence as described in U.S. Pat. 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 variants thereof. The BAAV serotype may be or have a sequence as described in U.S. Pat. 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 variants thereof.

[0098] In one embodiment, the AAV may be a goat AAV. The goat AAV serotype may be or have a sequence described in U.S. Pat. No. US7427396, 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 US7427396) or a variant thereof.

[0099] In other embodiments, the AAV can be engineered as a hybrid AAV 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 the sequence described in U.S. Patent Publication No. US20160017005, the contents of which are incorporated herein by reference in their entirety.

[0100] In one embodiment, the AAV can be a serotype having mutations in amino acids 390-627 (VP1 numbering) generated from an AAV9 capsid library 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 the corresponding nucleotide and amino acid substitutions can 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; 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; A AV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A, C 1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9.4 4(A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W5 09R, 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) and AAV9.95 (T1605A; F535L).

[0101] In some embodiments, the AAV serotype can be or have a sequence 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: 8 and 10 of WO2016049230), AAVF8 / HSC8 (SEQ ID NOs: 9 and 11 of WO2016049230), AAVF9 / HSC9 (SEQ ID NOs: 10 and 11 of WO2016049230), AAVF10 / HSC11 (SEQ ID NOs: 11 and 11 of WO2016049230), AAVF11 / HSC12 (SEQ ID NOs: 12 and 13 of WO2016049230), AAVF12 / HSC13 (SEQ ID NOs: 13 and 13 of WO2016049230), AAVF13 / HSC14 (SEQ ID NOs: 14 and 15 of WO2016049 49230), AAVF14 / HSC14 (SEQ ID NOs: 15 and 32 of WO2016049230), AAVF15 / HSC15 (SEQ ID NOs: 16 and 17 of WO2016049230), AAVF16 / HSC16 (SEQ ID NOs: 17 and 18 of WO2016049230), AAVF17 / HSC18 (SEQ ID NOs: 19 and 20 of WO2016049230), AAVF18 / HSC19 (SEQ ID NOs: 21 and 22 of WO2016049230), AAVF19 / HSC20 (SEQ ID NOs: 23 and 24 of WO2016049230), AAVF20 / HSC21 (SEQ ID NOs: 24 and 25 of WO2016049230), AAVF21 / HSC22 (SEQ ID NOs: 25 and 26 of WO2016049230), AAVF22 / HSC23 (SEQ ID NOs: 26 and 27 of WO2016049230), AAVF2 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.

[0102] In some embodiments, the AAV serotype can be or have a sequence 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 94 of U.S. Pat. No. 8,734,809), 809), AAV CBr-E8 (SEQ ID NOs: 21 and 95 of US8734809), AAV CLv-D1 (SEQ ID NOs: 22 and 96 of US8734809), AAV CLv-D2 (SEQ ID NOs: 23 and 97 of US8734809), AAV CLv-D3 (SEQ ID NOs: 24 and 98 of US8734809), AAV CLv-D4 (SEQ ID NOs: 25 and 99 of US8734809), AAV CLv-D5 (SEQ ID NOs: 26 and 100 of US8734809), AAV CLv-D6 (SEQ ID NOs: 27 and 101 of US8734809), AAV CLv-D7 (SEQ ID NOs: 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), AAV CLv-R8 (SEQ ID NOs: 37 and 111 of US8734809), AAV CLv-R9 (SEQ ID NOs: 38 and 112 of US8734809), AAV CLg-F1 (SEQ ID NOs: 39 and 113 of US8734809), AAV CLg-F2 (SEQ ID NOs: 40 and 114 of US8734809), AAV CLg-F3 (SEQ ID NOs: 41 and 115 of US8734809), AAV CLg-F4 (SEQ ID NOs: 42 and 116 of US8734809), AAV CLg-F5 (SEQ ID NOs: 43 and 117 of US8734809), AAV CLg-F6 (SEQ ID NOs: 43 and 117 of US8734809), AAV CLg-F7 (SEQ ID NOs: 44 and 118 of US8734809), AAV CLg-F8 (SEQ ID NOs: 43 and 117 of US8734809), AAV CSp-1 (SEQ ID NOs: 45 and 119 of US8734809), AAV CSp-10 (SEQ ID NOs: 46 and 120 of US8734809), AAV CSp-11 (SEQ ID NOs: 47 and 121 of US8734809), AAV CSp-2 (SEQ ID NOs: 48 and 122 of US8734809), AAV CSp-3 (SEQ ID NOs: 49 and 123 of US8734809), AAV CSp-4 (SEQ ID NOs: 49 and 124 of US8734809), AAV CSp-5 (SEQ ID NOs: 41 and 125 of US8734809), AAV CSp-6 (SEQ ID NOs: 43 and 117 of US8734809), AAV CSp-7 (SEQ ID NOs: 44 and 118 of US8734809), AAV CSp-8 (SEQ ID NOs: 45 and 119 of US8734809), AAV CSp-9 (SEQ ID NOs: 46 and 120 of US8734809), AAV CSp-10 (SEQ ID NOs: 46 and 120 of US8734809), AAV CSp-11 (SEQ ID NOs: 47 and 121 of US8734809), AAV CSp- 809), 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 CKd-2 (SEQ ID NOs: 59 and 140 of US8734809), AAV CKd-3 (SEQ ID NOs: 51 and 141 of US8734809), AAV CKd-4 (SEQ ID NOs: 51 and 142 of US8734809), AAV CKd-5 (SEQ ID NOs: 52 and 143 of US8734809), AAV CKd-6 (SEQ ID NOs: 51 and 125 of US8734809), AAV CKd-7 (SEQ ID NOs: 52 and 126 of US8734809), AAV CKd-8 (SEQ ID NOs: 53 and 127 of US8734809), AAV CKd-9 (SEQ ID NOs: 54 and 128 of US8734809), NO:59 and 133), 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: 60 and 134 of US8734809), AAV CLv-4 (SEQ ID NOs: 61 and 135 of US8734809), AAV CLv-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-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), AAVCLv1-1 (SEQ ID NO: 171 of US8734809), AAV CLv1-2 (SEQ ID NO: 172 of US8734809), AAVCLv1-3 (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.

[0103] In some embodiments, the AAV serotype can be or have a sequence 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: 6 and 57 of WO2016065001), AAV CBr-7.5 (SEQ ID NOs: 7 and 8 of WO2016065001), AAV CBr-7.6 (SEQ ID NOs: 8 and 8 of WO2016065001), AAV CBr-7.7 (SEQ ID NOs: 9 and 10 of WO2016065001), AAV CBr-7.8 (SEQ ID NOs: 10 and 11 of WO2016065001), AAV CBr-7.9 (SEQ ID NOs: 11 and 12 of WO2016065001), AAV CBr-7.10 (SEQ ID NOs: 11 and 12 of WO2016065001), AAV CBr-7. 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), AAV CLv-M8 (SEQ ID NOs: 27 and 28 of WO2016065001), AAV CLv-M9 (SEQ ID NOs: 30 and 31 of WO2016065001), AAV CLv- 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). 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. .

[0104] In one embodiment, the AAV can be a serotype selected from any of the serotypes present in Table 1.

[0105] In one embodiment, the AAV may comprise a sequence, fragment or variant of the sequences in Table 1.

[0106] In one embodiment, the AAV may be encoded by a sequence, fragment or variant described in Table 1.

[0107] Table 1. AAV serotypes

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] Each patent, application, and / or publication listed in Table 1 is incorporated herein by reference in its entirety.

[0160] In one embodiment, the AAV serotype may be or may have 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: 127 and 126, respectively, herein), PHP.B (SEQ ID NOs: 8 and 9 of WO2015038958, SEQ ID NOs: 868 and 869, herein), G2B-13 (SEQ ID NO: 12 of WO2015038958, SEQ ID NO: 870, herein), G2B-26 (SEQ ID NO: 13 of WO2015038958, SEQ ID NOs: 868 and 869, herein), TH1.1-32 (SEQ ID NO: 14 of WO2015038958, SEQ ID NO: 15 of WO2015038958, SEQ ID NO: 16 of WO2015038958, SEQ ID NO: 170, herein), TH1.1-33 (SEQ ID NO: 15 of WO2015038958, SEQ ID NO: 171, herein), TH1.1-34 (SEQ ID NO: 16 of WO2015038958, SEQ ID NO: 162, herein), TH1.1-35 (SEQ ID NO: 16 of WO2015038958, SEQ ID NO: 163, herein), TH1.1-36 (SEQ ID NO: 16 of WO2015038958, SEQ ID NO: 164, herein), TH1.1-37 (SEQ ID NO: NO:871), TH1.1-35 (SEQ ID NO:15 of WO2015038958, SEQ ID NO:872 herein) or variants thereof. In addition, any targeting peptide or amino acid insert described in WO2015038958 can be inserted into any parent AAV serotype, such as but not limited to AAV9 (DNA sequence is SEQ ID NO:126, amino acid sequence is SEQ ID NO:127). In one embodiment, the amino acid insert is inserted between amino acids 586-592 of the parent AAV (e.g., AAV9). In another embodiment, 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: 873 herein), KFPVALT (SEQ ID NO: 3 of WO2015038958; SEQ ID NO: 874 herein), LAVPFK (SEQ ID NO: 31 of WO2015038958; SEQ ID NO: 875 herein), AVPFK (SEQ ID NO: 32 of WO2015038958; SEQ ID NO: 876 herein), VPFK (SEQ ID NO: 33 of WO2015038958; SEQ ID NO: 877 herein), TLAVPF (SEQ ID NO: 34 of WO2015038958; SEQ ID NO: 878 herein), TLAVP (SEQ ID NO: 35 of WO2015038958; SEQ ID NO: 879 herein), NO: 879), TLAV (SEQ ID NO: 36 of WO2015038958; SEQ ID NO: 880 herein), SVSKPFL (SEQ ID NO: 28 of WO2015038958; SEQ ID NO: 881 herein), FTLTTPK (SEQ ID NO: 29 of WO2015038958; SEQ ID NO: 882 herein), MNATKNV (SEQ ID NO: 30 of WO2015038958; SEQ ID NO: 883 herein), QSSQTPR (SEQ ID NO: 54 of WO2015038958; SEQ ID NO: 884 herein), ILGTGTS (SEQ ID NO: 55 of WO2015038958; SEQ ID NO: 885 herein), TRTNPEA (SEQ ID NO: 56 of WO2015038958; SEQ ID NO: 886 herein), ID NO: 886), NGGTSSS (SEQ ID NO: 58 of WO2015038958; SEQ ID NO: 887 herein) or YTLSQGW (SEQ ID NO: 60 of WO2015038958; SEQ ID NO: 888 herein).Non-limiting examples of nucleotide sequences that can encode the amino acid insert include the following: AAGTTTCCTGTGGCGTTGACT (SEQ ID NO: 3 for WO2015038958; SEQ ID NO: 889 herein), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NOs: 24 and 49 of WO2015038958; SEQ ID NO: 890 herein), AGTGTGAGTAAGCCTTTTTTG (SEQ ID NO: 25 of WO2015038958; SEQ ID NO: 891 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 26 of WO2015038958; SEQ ID NO: 892 herein), ATGAATGCTACGAAGAATGTG (SEQ ID NO: 27 of WO2015038958; SEQ ID NO: 893 herein), NO: 893), CAGTCGTCGCAGACGCCTAGG (SEQ ID NO: 48 of WO2015038958; SEQ ID NO: 894 herein), ATTCTGGGGACTGGTACTTCG (SEQ ID NOs: 50 and 52 of WO2015038958; SEQ ID NO: 895 herein), ACGCGGACTAATCCTGAGGCT (SEQ ID NO: 51 of WO2015038958; SEQ ID NO: 896 herein), AATGGGGGGACTAGTAGTTCT (SEQ ID NO: 53 of WO2015038958; SEQ ID NO: 897 herein) or TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 59 of WO2015038958; SEQ ID NO: 898 herein).

[0161] In one embodiment, the AAV serotype can be engineered to contain at least one AAV capsid CD8+ T cell epitope for AAV2, such as, but not limited to, SADNNNSEY (SEQ ID NO:899), LIDQYLYYL (SEQ ID NO:900), VPQYGYLTL (SEQ ID NO:901), TTSTRTWAL (SEQ ID NO:902), YHLNGRDSL (SEQ ID NO:903), SQAVGRSSF (SEQ ID NO:904), VPANPSTTF (SEQ ID NO:905), FPQSGVLIF (SEQ ID NO:906), YFDFNRFHCHFSPRD (SEQ ID NO:907), VGNSSGNWHCDSTWM (SEQ ID NO:908), QFSQAGASDIRDQSR (SEQ ID NO:909), GASDIRQSRNWLP (SEQ ID NO:910), and GNRQAATADVNTQGV (SEQ ID NO:911).

[0162] In one embodiment, the AAV serotype can be engineered to contain at least one AAV capsid CD8+ T cell epitope for AAV1, such as, but not limited to, LDRLMNPLI (SEQ ID NO:912), TTSTRTWAL (SEQ ID NO:902), and QPAKKRLNF (SEQ ID NO:913).

[0163] In one embodiment, peptides contained in AAV serotypes can be identified using the method described by Hui et al. (Molecular Therapy-Methods & Clinical Development (2015) 2, 15029 doi: 10.1038 / mtm.2015.29; the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, the method includes isolating human splenocytes, restimulating splenocytes in vitro using individual peptides spanning the amino acid sequence of the AAV capsid protein, using IFN-γ ELISpots of the individual peptides used for in vitro restimulation, bioinformatics analysis for determining the HLA restriction of the 15-mer recognized by the IFN-γ ELISpot, identification of candidate reactive 9-mer epitopes for a given HLA allele, synthesis of candidate 9-mers, performing a second IFN-γ ELISpot screening of splenocytes from subjects carrying the HLA allele to which the AAV epitope expected to be recognized binds, determining AAV capsid reactive CD8+ T cell epitopes, and determining the frequency of responses of subjects to a given AAV epitope.

[0164] In one embodiment, the AAV can be a serotype produced by Cre-recombination-based AAV targeted evolution (CREATE) described by Deverman et al. (Nature Biotechnology 342): 204-209 (2016), the contents of which are incorporated herein by reference in their entirety. In one embodiment, the AAV serotype produced in this manner has improved CNS transduction and / or neuronal and astrocyte tropism compared to other AAV serotypes. As non-limiting examples, the AAV serotypes can be PHP.B, PHP.B2, PHP.B3, PHP.A, G2A12, G2A15. In one embodiment, these AAV serotypes can be AAV9 (SEQ ID NOs: 126 and 127) derivatives with a 7 amino acid insert between amino acids 588-589. Non-limiting examples of these 7 amino acid insertions include LAVPFK (SEQ ID NO:873), SVSKPFL (SEQ ID NO: 1249), FTLTTPK (SEQ ID NO:882), YTLSQGW (SEQ ID NO:888), QAVRTSL (SEQ ID NO: 1176), and / or LAKERLS (SEQ ID NO: 1177).

[0165] In one embodiment, the AAV serotype may be or may have a sequence described in International Patent Publication WO2017100671, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV9 (SEQ ID NO: 45 of WO2017100671, SEQ ID NO: 1441 herein), PHP.N (SEQ ID NO: 46 of WO2017100671, SEQ ID NO: 1439 herein), PHP.S (SEQ ID NO: 47 of WO2017100671, SEQ ID NO: 1440 herein) or variants 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: 122 or SEQ ID NO: 1441). In one embodiment, the amino acid insert is inserted between amino acids 586-592 of the parent AAV (e.g., AAV9). In another embodiment, 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: 1442 herein), AQSVSKPFLAQ (SEQ ID NO: 2 of WO2017100671; SEQ ID NO: 1443 herein), AQFTLTTPKAQ (SEQ ID NO: 3 in the sequence listing of WO2017100671; SEQ ID NO: 1444 herein), DGTLAVPFKAQ (SEQ ID NO: 4 in the sequence listing of WO2017100671; SEQ ID NO: 1445 herein), ESTLAVPFKAQ (SEQ ID NO: 5 of WO2017100671; SEQ ID NO: 1446 herein), GGTLAVPFKAQ (SEQ ID NO: 6 of WO2017100671; SEQ ID NO: 1447 herein), NO: 1447), AQTLATPFKAQ (SEQ ID NOs: 7 and 33 of WO2017100671; SEQ ID NO: 1448 herein), ATTLATPFKAQ (SEQ ID NO: 8 of WO2017100671; SEQ ID NO: 1449 herein), DGTLATPFKAQ (SEQ ID NO: 9 of WO2017100671; SEQ ID NO: 1450 herein), GGTLATPFKAQ (SEQ ID NO: 10 of WO2017100671;: SEQ ID NO: 1451 herein), SGSLAVPFKAQ (SEQ ID NO: 11 of WO2017100671; SEQ ID NO: 1452 herein), AQTLAQPFKAQ (SEQ ID NO: 12 of WO2017100671; SEQ ID NO: 1453 herein), AQTLQQPFKAQ (SEQ ID NO: 13 of WO2017100671; SEQ ID NO: 1454 herein), AQTLSNPFKAQ (SEQ ID NO: 14 of WO2017100671; SEQ ID NO: 1455 herein), AQTLAVPFSNP (SEQ ID NO: 15 of WO2017100671; SEQ ID NO: 1456 herein), QGTLAVPFKAQ (SEQ ID NO: 16 of WO2017100671; SEQ ID NO: 1457 herein), NO: 1457), NQTLAVPFKAQ (SEQ ID NO: 17 of WO2017100671; SEQ ID NO: 1458 herein), EGSLAVPFKAQ (SEQ ID NO: 18 of WO2017100671; SEQ ID NO: 1459 herein), SGNLAVPFKAQ (SEQ ID NO: 19 of WO2017100671; SEQ ID NO: 1460 herein), EGTLAVPFKAQ (SEQ ID NO: 20 of WO2017100671; SEQ ID NO: 1461 herein), DSTLAVPFKAQ (SEQ ID NO: 21 in Table 1 of WO2017100671; SEQ ID NO: 1462 herein), AVTLAVPFKAQ (SEQ ID NO: 22 of WO2017100671; SEQ ID NO: 1463 herein), NO: 1463), AQTLSTPFKAQ (SEQ ID NO: 23 of WO2017100671; SEQ ID NO: 1464 herein), AQTLPQPFKAQ (SEQ ID NO: 24 and 32 of WO2017100671; SEQ ID NO: 1465 herein), AQTLSQPFKAQ (SEQ ID NO: 25 of WO2017100671; SEQ ID NO: 1466 herein), AQTLQLPFKAQ (SEQ ID NO: 26 of WO2017100671;: SEQ ID NO: 1467 herein), AQTLTMPFKAQ (SEQ ID NOs: 27 and 34 of WO2017100671 and SEQ ID NO: 35 in the sequence listing of WO2017100671; SEQ ID NO: 1468 herein), AQTLTTPFKAQ (SEQ ID NO: 28 of WO2017100671; SEQ ID NO: 1469 herein), AQYTLSQGWAQ (SEQ ID NO: 29 of WO2017100671; SEQ ID NO: 1470 herein), AQMNATKNVAQ (SEQ ID NO: 30 of WO2017100671; SEQ ID NO: 1471 herein), AQVSGGHHSAQ (SEQ ID NO: 31 of WO2017100671; SEQ ID NO: 1472 herein), NO: 1472), AQTLTAPFKAQ (SEQ ID NO: 35 in Table 1 of WO2017100671; SEQ ID NO: 1473 herein), AQTLSKPFKAQ (SEQ ID NO: 36 of WO2017100671; SEQ ID NO: 1474 herein), QAVRTSL (SEQ ID NO: 37 of WO2017100671; SEQ ID NO: 1475 herein), YTLSQGW (SEQ ID NO: 38 of WO2017100671; SEQ ID NO: 888 herein), LAKERLS (SEQ ID NO: 39 of WO2017100671; SEQ ID NO: 1476 herein), TLAVPFK (SEQ ID NO: 40 in the sequence listing of WO2017100671; SEQ ID NO: 873 herein), SVSKPFL (SEQ ID NO: 41 of WO2017100671; SEQ ID NO: 889 herein), NO:41; SEQ ID NO:881 herein), FTLTTPK (SEQ ID NO:42 of WO2017100671; SEQ ID NO:882 herein), MNSTKNV (SEQ ID NO:43 of WO2017100671; SEQ ID NO:1477 herein), VSGGHHS (SEQ ID NO:44 of WO2017100671; SEQ ID NO:1478 herein), SAQTLAVPFKAQAQ (SEQ ID NO:48 of WO2017100671; SEQ ID NO:1479 herein), SXXXLAVPFKAQAQ (SEQ ID NO:49 of WO2017100671, wherein X can be any amino acid;100671, wherein X can be any amino acid; SEQ ID NO: 1484 herein), SAQXXXVPFKAQAQ (SEQ ID NO: 50 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1481 herein), SAQTLXXXFKAQAQ (SEQ ID NO: 51 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1482 herein), SAQTLAVXXXAQAQ (SEQ ID NO: 52 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1483 herein), SAQTLAVPFXXXAQ (SEQ ID NO: 53 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1484 herein), TNHQSAQ (SEQ ID NO: 65 of WO2017100671; SEQ ID NO: 66 herein NO: 1485), AQAQTGW (SEQ ID NO: 66 of WO2017100671; SEQ ID NO: 1486 herein), DGTLATPFK (SEQ ID NO: 67 of WO2017100671; SEQ ID NO: 1487 herein), DGTLATPFKXX (SEQ ID NO: 68 of WO2017100671, wherein X can be any amino acid; SEQ ID NO: 1488 herein), LAVPFKAQ (SEQ ID NO: 80 of WO2017100671; SEQ ID NO: 1489 herein), VPFKAQ (SEQ ID NO: 81 of WO2017100671; SEQ ID NO: 1490 herein), FKAQ (SEQ ID NO: 82 of WO2017100671; SEQ ID NO: 83 herein), NO: 1491), AQTLAV (SEQ ID NO: 83 of WO2017100671; SEQ ID NO: 1492 herein), AQTLAVPF (SEQ ID NO: 84 of WO2017100671; SEQ ID NO: 1493 herein), QAVR (SEQ ID NO: 85 of WO2017100671; SEQ ID NO: 1494 herein), AVRT (SEQ ID NO: 86 of WO2017100671; SEQ ID NO: 1495 herein), VRTS (SEQ ID NO: 87 of WO2017100671; SEQ ID NO: 1496 herein), RTSL (SEQ ID NO: 88 of WO2017100671);SEQ ID NO: 1497 herein), QAVRT (SEQ ID NO: 89 of WO2017100671; SEQ ID NO: 1498 herein), AVRTS (SEQ ID NO: 90 of WO2017100671; SEQ ID NO: 1499 herein), VRTSL (SEQ ID NO: 91 of WO2017100671; SEQ ID NO: 1500 herein), QAVRTS (SEQ ID NO: 92 of WO2017100671; SEQ ID NO: 1501 herein) or AVRTSL (SEQ ID NO: 93 of WO2017100671; SEQ ID NO: 1502 herein). ;

[0166] Non-limiting examples of nucleotide sequences that can encode the amino acid insert include the following GATGGGACTTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 54 of WO2017100671; SEQ ID NO: 1503 herein), GATGGGACGTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID NO: 55 of WO2017100671; SEQ ID NO: 1504 herein), CAGGCGGTTAGGACGTCTTTG (SEQ ID NO: 56 of WO2017100671; SEQ ID NO: 1505 herein), CAGGTCTTCACGGACTCAGACTATCAG (SEQ ID NOs: 57 and 78 of WO2017100671; SEQ ID NO: 1506 herein), CAAGTAAAACCTCTACAAATGTGGTAAAATCG (SEQ ID NO: 58 of WO2017100671; SEQ ID NO: 1507 herein). NO: 1507), ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC (SEQ ID NO: 59 of WO2017100671; SEQ ID NO: 1508 herein), GGAAGTATTCCTTGGTTTTGAACCCA (SEQ ID NO: 60 of WO2017100671; SEQ ID NO: 1509 herein), GGTCGCGGTTCTTGTTTGTGGAT (SEQ ID NO: 61 of WO2017100671; SEQ ID NO: 1510 herein), CGACCTTGAAGCGCATGAACTCCT (SEQ ID NO: 62 of WO2017100671; SEQ ID NO: 1511 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNMNNMNNMNNMNNTTGGGCACTCTGGTGGTTTGTC (SEQ ID NO: 63 of WO2017100671; SEQ ID NO: 1512 herein), ID NO: 63, wherein N may be A, C, T or G; SEQ ID NO: 1512 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCMNNMNNMNNAAAAGGCACCGCCAAAGTTTG (SEQ ID NO: 69 of WO2017100671, wherein N may be A, C, T or G;1514), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCMNNMNNMNNCACCGCC AAAGTTTGGGCACT (SEQ ID NO: 70 of WO2017100671, wherein N may be A, C, T or G; SEQ ID NO: 1515 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAMNNMNNMNNC AAAGTTTGGGCACTCTGGTGG (SEQ ID NO: 71 of WO2017100671, wherein N may be A, C, T or G; SEQ ID NO: 1516 herein), GTATTCCTTGGTTTTGAACCCAACCGGTCTGCGCCTGTGCCTTAAAAGGCACMNNMNNMNNTTGGGCACTCTGGTGGTTTGTG (SEQ ID NO: 72 of WO2017100671, wherein N may be A, C, T or G; SEQ ID NO: 1517 herein), NO: 72, wherein N may be A, C, T or G; SEQ ID NO: 1516 herein), ACTTTGGCGGTGCCTTTTAAG (SEQ ID NO: 74 of WO2017100671; SEQ ID NO: 890 herein), AGTGTGAGTAAGCTTTTTTG (SEQ ID NO: 75 of WO2017100671; SEQ ID NO: 891 herein), TTTACGTTGACGACGCCTAAG (SEQ ID NO: 76 of WO2017100671; SEQ ID NO: 892 herein), TATACTTTGTCGCAGGGTTGG (SEQ ID NO: 77 of WO2017100671; SEQ ID NO: 898 herein), or CTTGCGAAGGAGCGGCTTTCG (SEQ ID NO: 79 of WO2017100671; SEQ ID NO: 1517 herein). ;

[0167] In one embodiment, the AAV serotype may be or may have 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 US9,624,274), AAV6 (SEQ ID NO: 182 of US9,624,274), AAV2 (SEQ ID NO: 183 of US9,624,274), AAV3b (SEQ ID NO: 184 of US9,624,274), AAV7 (SEQ ID NO: 185 of US9,624,274), AAV8 (SEQ ID NO: 186 of US9,624,274), AAV10 (SEQ ID NO: 187 of US9,624,274), AAV4 (SEQ ID NO: 188 of US9,624,274), AAV11 (SEQ ID NO: 189 of US9,624,274), bAAV (SEQ ID NO: 190 of US9,624,274), AAV2 (SEQ ID NO: 191 of US9,624,274), AAV3b (SEQ ID NO: 192 of US9,624,274), AAV7 (SEQ ID NO: 193 of US9,624,274), AAV8 (SEQ ID NO: 194 of US9,624,274), AAV10 (SEQ ID NO: 195 of US9,624,274), AAV11 (SEQ ID NO: 196 of US9,624,274), AAV12 (SEQ ID NO: 117 of US9,624,274), AAV13 (SEQ ID NO: 138 of US9,624,274), AAV14 (SEQ ID NO: NO: 190), AAV5 (SEQ ID NO: 191 of US9624274), GPV (SEQ ID NO: 192 of US9624274; SEQ ID NO: 1518 herein), B19 (SEQ ID NO: 193 of US9624274; SEQ ID NO: 1519 herein), MVM (SEQ ID NO: 194 of US9624274; SEQ ID NO: 1520 herein), FPV (SEQ ID NO: 195 of US9624274; SEQ ID NO: 1522 herein), CPV (SEQ ID NO: 196 of US9624274; SEQ ID NO: 1866 herein), or variants thereof. In addition, any structural protein insert described in US 9624274 can be inserted into, but not limited to, I-453 and I-587 of any parent AAV serotype, such as, but not limited to, AAV2 (SEQ ID NO: 183 of US9624274). The amino acid insert can be, but not limited to, any of the following amino acid sequences: VNLTWSRASG (SEQ ID NO: 50 of US9624274; SEQ ID NO: 1901 herein), EFCINHRGYWVCGD (SEQ ID NO: 55 of US9624274; SEQ ID NO: 1902 herein), EDGQVMDVDLS (SEQ ID NO: 85 of US9624274; SEQ ID NO: 1903 herein), EKQRNGTLT (SEQ ID NO: 86 of US9624274; SEQ ID NO: 1904 herein),: 1904 herein), TYQCRVTHPHLPRALMR (SEQ ID NO: 87 of US9624274; SEQ ID NO: 1905 herein), RHSTTQPRKTKGSG (SEQ ID NO: 88 of US9624274; SEQ ID NO: 1906 herein), DSNPRGVSAYLSR (SEQ ID NO: 89 of US9624274; SEQ ID NO: 1907 herein), TITCLWDLAPSK (SEQ ID NO: 90 of US9624274; SEQ ID NO: 1908 herein), KTKGSGFFVF (SEQ ID NO: 91 of US9624274; SEQ ID NO: 1909 herein), THPHLPRALMRS (SEQ ID NO: 92 of US9624274; SEQ ID NO: 1930 herein). NO: 1910), GETYQCRVTHPHLPRALMRSTTK (SEQ ID NO: 93 of US9624274; SEQ ID NO: 1911 herein), LPRALMRS (SEQ ID NO: 94 of US9624274; SEQ ID NO: 1912 herein), INHRGYWV (SEQ ID NO: 95 of US9624274; SEQ ID NO: 1913 herein), CDAGSVRTNAPD (SEQ ID NO: 60 of US9624274; SEQ ID NO: 1914 herein), AKAVSNLTESRSESLQS (SEQ ID NO: 96 of US9624274; SEQ ID NO: 1915 herein), SLTGDEFKKVLET (SEQ ID NO: 97 of US9624274; SEQ ID NO: 1916 herein), REAVAYRFEED (SEQ ID NO: 98 of US9624274; SEQ ID NO: 1917 herein), NO:98; SEQ ID NO:1917 herein), INPEIITLDG (SEQ ID NO:99 of US9624274; SEQ ID NO:1918 herein), DISVTGAPVITATYL (SEQ ID NO:100 of US9624274; SEQ ID NO:1919 herein), DISVTGAPVITA (SEQ ID NO:101 of US9624274; SEQ ID NO:1920 herein), PKTVSNLTESSSESVQS (SEQ ID NO:102 of US9624274;: SEQ ID NO: 1921 herein), SLMGDEFKAVLET (SEQ ID NO: 103 of US9624274; SEQ ID NO: 1922 herein), QHSVAYTFEED (SEQ ID NO: 104 of US9624274; SEQ ID NO: 1923 herein), INPEIITRDG (SEQ ID NO: 105 of US9624274; SEQ ID NO: 1924 herein), DISLTGDPVITASYL (SEQ ID NO: 106 of US9624274; SEQ ID NO: 1925 herein), DISLTGDPVITA (SEQ ID NO: 107 of US9624274; SEQ ID NO: 1926 herein), DQSIDFEIDSA (SEQ ID NO: 108 of US9624274; SEQ ID NO: 1927 herein), NO: 1927), KNVSEDLPLPTFSPTLLGDS (SEQ ID NO: 109 of US9624274; SEQ ID NO: 1928 herein), KNVSEDLPLPT (SEQ ID NO: 110 of US9624274; SEQ ID NO: 1929 herein), CDSGRVRTDAPD (SEQ ID NO: 111 of US9624274; SEQ ID NO: 1930 herein), FPEHLLVDFLQSLS (SEQ ID NO: 112 of US9624274; SEQ ID NO: 1931 herein), DAEFRHDSG (SEQ ID NO: 65 of US9624274; SEQ ID NO: 1932 herein), HYAAAQWDFGNTMCQL (SEQ ID NO: 113 of US9624274; SEQ ID NO: 1934 herein). NO: 1933), YAAQWDFGNTMCQ (SEQ ID NO: 114 of US9624274; SEQ ID NO: 1934 herein), RSQKEGLHYT (SEQ ID NO: 115 of US9624274; SEQ ID NO: 1935 herein), SSRTPSDKPVAHWANPQAE (SEQ ID NO: 116 of US9624274; SEQ ID NO: 1936 herein), SRTPSDKPVAHWANP (SEQ ID NO: 117 of US9624274; SEQ ID NO: 1937 herein), SSRTPSDKP (SEQ ID NO: 118 of US9624274;SEQ ID NO: 1938 herein), NADGNVDYHMNSVP (SEQ ID NO: 119 of US9624274; SEQ ID NO: 1939 herein), DGNVDYHMNSV (SEQ ID NO: 120 of US9624274; SEQ ID NO: 1940 herein), RSFKEFLQSSLRALRQ (SEQ ID NO: 121 of US9624274; SEQ ID NO: 1941 herein); FKEFLQSSLRA (SEQ ID NO: 122 of US9624274; SEQ ID NO: 1942 herein), or QMWAPQWGPD (SEQ ID NO: 123 of US9624274; SEQ ID NO: 1943 herein). ;

[0168] In one embodiment, the AAV serotype may be or may have a sequence 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 one or more amino acid modifications at amino acid positions 585 to 590 of a native AAV2 capsid protein. Further modifications may result in, but are not limited to, the amino acid sequences RGNRQA (SEQ ID NO:3 of US9475845; SEQ ID NO:1944 herein), SSSTDP (SEQ ID NO:4 of US9475845; SEQ ID NO:1945 herein), SSNTAP (SEQ ID NO:5 of US9475845; SEQ ID NO:1946 herein), SNSNLP (SEQ ID NO:6 of US9475845; SEQ ID NO:1947 herein), SSTTAP (SEQ ID NO:7 of US9475845; SEQ ID NO:1948 herein), AANTAA (SEQ ID NO:8 of US9475845; SEQ ID NO:1949 herein), QQNTAP (SEQ ID NO:9 of US9475845; SEQ ID NO:1950 herein), SAQAQA (SEQ ID NO:10 of US9475845; SEQ ID NO:11 herein), NO: 1951), QANTGP (SEQ ID NO: 11 of US9475845; SEQ ID NO: 1952 herein), NATTAP (SEQ ID NO: 12 of US9475845; SEQ ID NO: 1953 herein), SSTAGP (SEQ ID NOs: 13 and 20 of US9475845; SEQ ID NO: 1954 herein), QQNTAA (SEQ ID NO: 14 of US9475845; SEQ ID NO: 1955 herein), PSTAGP (SEQ ID NO: 15 of US9475845; SEQ ID NO: 1956 herein), NQNTAP (SEQ ID NO: 16 of US9475845; SEQ ID NO: 1957 herein), QAANAP (SEQ ID NO: 17 of US9475845; SEQ ID NO: 1958 herein), SIVGLP (SEQ ID NOs: 18 and 20 of US9475845; SEQ ID NO: 1959 herein), ID NO: 18; SEQ ID NO: 1959 herein), AASTAA (SEQ ID NO: 19 and 27 of US9475845; SEQ ID NO: 1960 herein), SQNTTA (SEQ ID NO: 21 of US9475845;1961), QQDTAP (SEQ ID NO: 22 of US9475845; SEQ ID NO: 1962 herein), QTNTGP (SEQ ID NO: 23 of US9475845; SEQ ID NO: 1963 herein), QTNGAP (SEQ ID NO: 24 of US9475845; SEQ ID NO: 1964 herein), QQNAAP (SEQ ID NO: 25 of US9475845; SEQ ID NO: 1965 herein), or AANTQA (SEQ ID NO: 26 of US9475845; SEQ ID NO: 1966 herein). In one embodiment, the amino acid modification is substitution with a targeting sequence at amino acid positions 262 to 265 in a native AAV2 capsid protein or corresponding positions in a capsid protein of another AAV. 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: 1967 herein), QPEHSST (SEQ ID NO: 39 and 50 of US9475845; SEQ ID NO: 1968 herein), VNTANST (SEQ ID NO: 40 of US9475845; SEQ ID NO: 1969 herein), HGPMQKS (SEQ ID NO: 41 of US9475845; SEQ ID NO: 1970 herein), PHKPPLA (SEQ ID NO: 42 of US9475845; SEQ ID NO: 1971 herein), IKNNEMW (SEQ ID NO: 43 of US9475845; SEQ ID NO: 1972 herein), RNLDTPM (SEQ ID NO: 44 of US9475845; SEQ ID NO: 45 herein). NO: 1973), VDSHRQS (SEQ ID NO: 45 of US9475845; SEQ ID NO: 1974 herein), YDSKTKT (SEQ ID NO: 46 of US9475845; SEQ ID NO: 1975 herein), SQLPHQK (SEQ ID NO: 47 of US9475845; SEQ ID NO: 1976 herein), STMQQNT (SEQ ID NO: 48 of US9475845; SEQ ID NO: 1977 herein), TERYMTQ (SEQ ID NO: 49 of US9475845; SEQ ID NO: 1978 herein), DASLSTS (SEQ ID NO: 51 of US9475845; SEQ ID NO: 52 of US9475845);: 1979 herein), DLPNKKT (SEQ ID NO: 52 of US9475845; SEQ ID NO: 1980 herein), DLTAARL (SEQ ID NO: 53 of US9475845; SEQ ID NO: 1981 herein), EPHQFNY (SEQ ID NO: 54 of US9475845; SEQ ID NO: 1982 herein), EPQSNHT (SEQ ID NO: 55 of US9475845; SEQ ID NO: 1983 herein), MSSWPSQ (SEQ ID NO: 56 of US9475845; SEQ ID NO: 1984 herein), NPKHNAT (SEQ ID NO: 57 of US9475845; SEQ ID NO: 1985 herein), PDGMRTT (SEQ ID NO: 58 of US9475845; SEQ ID NO: 1986 herein). NO: 1986), PNNNKTT (SEQ ID NO: 59 of US9475845; SEQ ID NO: 1987 herein), QSTTHDS (SEQ ID NO: 60 of US9475845; SEQ ID NO: 1988 herein), TGSKQKQ (SEQ ID NO: 61 of US9475845; SEQ ID NO: 1989 herein), SLKHQAL (SEQ ID NO: 62 of US9475845; SEQ ID NO: 1990 herein), SPIDGEQ (SEQ ID NO: 63 of US9475845; SEQ ID NO: 1991 herein), WIFPWIQL (SEQ ID NOs: 64 and 112 of US9475845; SEQ ID NO: 1992 herein), CDCRGDCFC (SEQ ID NO: 65 of US9475845; SEQ ID NO: 1993 herein). NO: 1993), CNGRC (SEQ ID NO: 66 of US9475845; SEQ ID NO: 1994 herein), CPRECES (SEQ ID NO: 67 of US9475845; SEQ ID NO: 1995 herein), CTTHWGFTLC (SEQ ID NO: 68 and 123 of US9475845; SEQ ID NO: 1996 herein), CGRRAGGSC (SEQ ID NO: 69 of US9475845; SEQ ID NO: 1997 herein), CKGGRAKDC (SEQ ID NO: 70 of US9475845;: SEQ ID NO: 1998 herein), CVPELGHEC (SEQ ID NOs: 71 and 115 of US9475845; SEQ ID NO: 1999 herein), CRRETAWAK (SEQ ID NO: 72 of US9475845; SEQ ID NO: 2000 herein), VSWFSHRYSPFAVS (SEQ ID NO: 73 of US9475845; SEQ ID NO: 2001 herein), GYRDGYAGPILYN (SEQ ID NO: 74 of US9475845; SEQ ID NO: 2002 herein), XXXYXXX (SEQ ID NO: 75 of US9475845; SEQ ID NO: 2003 herein), YXNW (SEQ ID NO: 76 of US9475845; SEQ ID NO: 2004 herein), RPLPPLP (SEQ ID NO: 77 of US9475845; SEQ ID NO: 2005 herein), NO: 2005), APPLPPR (SEQ ID NO: 78 of US9475845; SEQ ID NO: 2006 herein), DVFYPYPYASGS (SEQ ID NO: 79 of US9475845; SEQ ID NO: 2007 herein), MYWYPY (SEQ ID NO: 80 of US9475845; SEQ ID NO: 2008 herein), DITWDQLWDLMK ​​(SEQ ID NO: 81 of US9475845; SEQ ID NO: 2009 herein), CWDDXWLC (SEQ ID NO: 82 of US9475845; SEQ ID NO: 2010 herein), EWCEYLGGYLRCYA (SEQ ID NO: 83 of US9475845; SEQ ID NO: 2011 herein), YXCXXGPXTWXCXP (SEQ ID NO: 84 of US9475845; SEQ ID NO: 2012 herein), ID NO: 2012), IEGPTLRQWLAARA (SEQ ID NO: 85 of US9475845; SEQ ID NO: 2013 herein), LWXXX (SEQ ID NO: 86 of US9475845; SEQ ID NO: 2014 herein), XFXXYLW (SEQ ID NO: 87 of US9475845; SEQ ID NO: 2015 herein), SSIISHFRWGLCD (SEQ ID NO: 88 of US9475845;: SEQ ID NO: 2016 herein), MSRPACPPNDKYE (SEQ ID NO: 89 of US9475845; SEQ ID NO: 2017 herein), CLRSGRGC (SEQ ID NO: 90 of US9475845; SEQ ID NO: 2018 herein), CHWMFSPWC (SEQ ID NO: 91 of US9475845; SEQ ID NO: 2019 herein), WXXF (SEQ ID NO: 92 of US9475845; SEQ ID NO: 2020 herein), CSSRLDAC (SEQ ID NO: 93 of US9475845; SEQ ID NO: 2021 herein), CLPVASC (SEQ ID NO: 94 of US9475845; SEQ ID NO: 2022 herein), CGFECVRQCPERC (SEQ ID NO: 95 of US9475845; SEQ ID NO: 2023 herein), NO: 2023), CVALCREACGEGC (SEQ ID NO: 96 of US9475845; SEQ ID NO: 2024 herein), SWCEPGWCR (SEQ ID NO: 97 of US9475845; SEQ ID NO: 2025 herein), YSGKWGW (SEQ ID NO: 98 of US9475845; SEQ ID NO: 2026 herein), GLSGGRS (SEQ ID NO: 99 of US9475845; SEQ ID NO: 2027 herein), LMLPRAD (SEQ ID NO: 100 of US9475845; SEQ ID NO: 2028 herein), CSCFRDVCC (SEQ ID NO: 101 of US9475845; SEQ ID NO: 2029 herein), CRDVVSVIC (SEQ ID NO: 102 of US9475845; SEQ ID NO: 2030 herein), NO: 2030), MARSGL (SEQ ID NO: 103 of US9475845; SEQ ID NO: 2031 herein), MARAKE (SEQ ID NO: 104 of US9475845; SEQ ID NO: 2032 herein), MSRTMS (SEQ ID NO: 105 of US9475845; SEQ ID NO: 2033 herein), KCCYSL (SEQ ID NO: 106 of US9475845; SEQ ID NO: 2034 herein), MYWGDSHWLQYWYE (SEQ ID NO: 107 of US9475845;: SEQ ID NO: 2035 herein), MQLPLAT (SEQ ID NO: 108 of US9475845; SEQ ID NO: 2036 herein), EWLS (SEQ ID NO: 109 of US9475845; SEQ ID NO: 2037 herein), SNEW (SEQ ID NO: 110 of US9475845; SEQ ID NO: 2038 herein), TNYL (SEQ ID NO: 111 of US9475845; SEQ ID NO: 2039 herein), WDLAWMFRLPVG (SEQ ID NO: 113 of US9475845; SEQ ID NO: 2040 herein), CTVALPGGYVRVC (SEQ ID NO: 114 of US9475845; SEQ ID NO: 2041 herein), CVAYCIEHHCWTC (SEQ ID NO: 116 of US9475845; SEQ ID NO: 2047 herein). NO: 2042), CVFAHNYDYLVC (SEQ ID NO: 117 of US9475845; SEQ ID NO: 2043 herein), CVFTSNYAFC (SEQ ID NO: 118 of US9475845; SEQ ID NO: 2044 herein), VHSPNKK (SEQ ID NO: 119 of US9475845; SEQ ID NO: 2045 herein), CRGDGWC (SEQ ID NO: 120 of US9475845; SEQ ID NO: 2046 herein), XRGCDX (SEQ ID NO: 121 of US9475845; SEQ ID NO: 2047 herein), PXXX (SEQ ID NO: 122 of US9475845; SEQ ID NO: 2048 herein), SGKGPRQITAL (SEQ ID NO: 124 of US9475845; SEQ ID NO: 2049 herein). NO:2049), AAAAAAAAAXXXXX (SEQ ID NO:125 of US9475845; SEQ ID NO:2050 herein), VYMSPF (SEQ ID NO:126 of US9475845; SEQ ID NO:2051 herein), ATWLPPR (SEQ ID NO:127 of US9475845; SEQ ID NO:2052 herein), HTMYYHHYQHHL (SEQ ID NO:128 of US9475845;: SEQ ID NO: 2053 herein), SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 129 of US9475845; SEQ ID NO: 2054 herein), CGLLPVGRPDRNVWRWLC (SEQ ID NO: 130 of US9475845; SEQ ID NO: 2055 herein), CKGQCDRFKGLPWEC (SEQ ID NO: 131 of US9475845; SEQ ID NO: 2056 herein), SGRSA (SEQ ID NO: 132 of US9475845; SEQ ID NO: 2057 herein), WGFP (SEQ ID NO: 133 of US9475845; SEQ ID NO: 2058 herein), AEPMPHSLNFSQYLWYT (SEQ ID NO: 134 of US9475845; SEQ ID NO: 2059 herein). NO: 2059), WAYXSP (SEQ ID NO: 135 of US9475845; SEQ ID NO: 2060 herein), IELLQAR (SEQ ID NO: 136 of US9475845; SEQ ID NO: 2061 herein), AYTKCSRQWRTCMTTH (SEQ ID NO: 137 of US9475845; SEQ ID NO: 2062 herein), PQNSKIPGPTFLDPH (SEQ ID NO: 138 of US9475845; SEQ ID NO: 2063 herein), SMEPALPDWWWKMFK (SEQ ID NO: 139 of US9475845; SEQ ID NO: 2064 herein), ANTPCGPYTHDCPVKR (SEQ ID NO: 140 of US9475845; SEQ ID NO: 2065 herein), TACHQHVRMVRP (SEQ ID NO: 141 of US9475845; SEQ ID NO: 2066 herein), NO: 141; SEQ ID NO: 2066 herein), VPWMEPAYQRFL (SEQ ID NO: 142 of US9475845; SEQ ID NO: 2067 herein), DPRATPGS (SEQ ID NO: 143 of US9475845; SEQ ID NO: 2068 herein), FRPNRAQDYNTN (SEQ ID NO: 144 of US9475845; SEQ ID NO: 2069 herein), CTKNSYLMC (SEQ ID NO: 145 of US9475845;: SEQ ID NO: 2070 herein), CXXTXXXGXGC (SEQ ID NO: 146 of US9475845; SEQ ID NO: 2071 herein), CPIEDRPMC (SEQ ID NO: 147 of US9475845; SEQ ID NO: 2072 herein), HEWSYLAPYPWF (SEQ ID NO: 148 of US9475845; SEQ ID NO: 2073 herein), MCPKHPLGC (SEQ ID NO: 149 of US9475845; SEQ ID NO: 2074 herein), RMWPSSTVNLSAGRR (SEQ ID NO: 150 of US9475845; SEQ ID NO: 2075 herein), SAKTAVSQRVWLPSHRGGEP (SEQ ID NO: 151 of US9475845; SEQ ID NO: 2076 herein). NO: 2076), KSREHVNNSACPSKRITAAL (SEQ ID NO: 152 of US9475845; SEQ ID NO: 2077 herein), EGFR (SEQ ID NO: 153 of US9475845; SEQ ID NO: 2078 herein), AGLGVR (SEQ ID NO: 154 of US9475845; SEQ ID NO: 2079 herein), GTRQGHTMRLGVSDG (SEQ ID NO: 155 of US9475845; SEQ ID NO: 2080 herein), IAGLATPGWSHWLAL (SEQ ID NO: 156 of US9475845; SEQ ID NO: 2081 herein), SMSIARL (SEQ ID NO: 157 of US9475845; SEQ ID NO: 2082 herein), HTFEPGV (SEQ ID NO: 158 of US9475845; SEQ ID NO: 2083 herein), NO:2083), NTSLKRISNKRIRRK (SEQ ID NO:159 of US9475845; SEQ ID NO:2084 herein), LRIKRKRRKRKKTRK (SEQ ID NO:160 of US9475845; SEQ ID NO:2085 herein), GGG, GFS, LWS, EGG, LLV, LSP, LBS, AGG, GRR, GGH, and GTV. ;

[0169] In one embodiment, the AAV serotype may be or may have a sequence as described in U.S. Publication No. US20160369298, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, a site-specific mutant capsid protein of AAV2 (SEQ ID NO: 97 of US20160369298; SEQ ID NO: 2086 herein) or a variant thereof, wherein the specific site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or a fragment thereof.

[0170] 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: 2087 herein), SPSGASN (SEQ ID NO: 2 of US20160369298; SEQ ID NO: 2088 herein), SHSGASN (SEQ ID NO: 3 of US20160369298; SEQ ID NO: 2089 herein), SRSGASN (SEQ ID NO: 4 of US20160369298; SEQ ID NO: 2090 herein), SKSGASN (SEQ ID NO: 5 of US20160369298; SEQ ID NO: 2091 herein), SNSGASN (SEQ ID NO: 6 of US20160369298; SEQ ID NO: 2092 herein), NO: 2092), SGSGASN (SEQ ID NO: 7 of US20160369298; SEQ ID NO: 2093 herein), SASGASN (SEQ ID NO: 8, 175 and 221 of US20160369298; SEQ ID NO: 2094 herein), SESGTSN (SEQ ID NO: 9 of US20160369298; SEQ ID NO: 2095 herein), STTGGSN (SEQ ID NO: 10 of US20160369298; SEQ ID NO: 2096 herein), SSAGSTN (SEQ ID NO: 11 of US20160369298; SEQ ID NO: 2097 herein), NNDSQA (SEQ ID NO: 12 of US20160369298; SEQ ID NO: 13 herein), NO: 2098), NNRNQA (SEQ ID NO: 13 of US20160369298; SEQ ID NO: 2099 herein), NNNKQA (SEQ ID NO: 14 of US20160369298; SEQ ID NO: 2100 herein), NAKRQA (SEQ ID NO: 15 of US20160369298; SEQ ID NO: 2101 herein), NDEHQA (SEQ ID NO: 16 of US20160369298; SEQ ID NO: 2102 herein), NTSQKA (SEQ ID NO: 17 of US20160369298);: 2103 herein), YYLSRTNTPSGTDTQSRLVFSQAGA (SEQ ID NO: 18 of US20160369298; SEQ ID NO: 2104 herein), YYLSRTNTDSGTETQSGLDFSQAGA (SEQ ID NO: 19 of US20160369298; SEQ ID NO: 2105 herein), YYLSRTNTESGTPTQSALEFSQAGA (SEQ ID NO: 20 of US20160369298; SEQ ID NO: 2106 herein), YYLSRTNTHSGTHTQSPLHFSQAGA (SEQ ID NO: 21 of US20160369298; SEQ ID NO: 2107 herein), YYLSRTNTSSGTITISHLIFSQAGA (SEQ ID NO: 22 of US20160369298; SEQ ID NO: 2208 herein), NO: 2108), YYLSRTNTRSGIMTKSSLMFSQAGA (SEQ ID NO: 23 of US20160369298; SEQ ID NO: 2109 herein), YYLSRTNTKSGRKTLSNLSFSQAGA (SEQ ID NO: 24 of US20160369298; SEQ ID NO: 2110 herein), YYLSRTNDGSGPVTPSKLRFSQRGA (SEQ ID NO: 25 of US20160369298; SEQ ID NO: 2111 herein), YYLSRTNAASGHATHSDLKFSQPGA (SEQ ID NO: 26 of US20160369298; SEQ ID NO: 2112 herein), YYLSRTNGQAGSLTMSELGFSQVGA (SEQ ID NO: 27 of US20160369298; SEQ ID NO: 271 herein), NO: 2113), YYLSRTNSTGGNQTTSQLLFSQLSA (SEQ ID NO: 28 of US20160369298; SEQ ID NO: 2114 herein), YFLSRTNNNTGLNTNSTLNFSQGRA (SEQ ID NO: 29 of US20160369298; SEQ ID NO: 2115 herein), SKTGADNNNSEYSWTG (SEQ ID NO: 30 of US20160369298; SEQ ID NO: 2116 herein), SKTDADNNNSEYSWTG (SEQ ID NO: 31 of US20160369298;: SEQ ID NO: 2117 herein), SKTEADNNNSEYSWTG (SEQ ID NO: 32 of US20160369298; SEQ ID NO: 2118 herein), SKTPADNNNSEYSWTG (SEQ ID NO: 33 of US20160369298; SEQ ID NO: 2119 herein), SKTHADNNNSEYSWTG (SEQ ID NO: 34 of US20160369298; SEQ ID NO: 2120 herein), SKTQADNNNSEYSWTG (SEQ ID NO: 35 of US20160369298; SEQ ID NO: 2121 herein), SKTIADNNNSEYSWTG (SEQ ID NO: 36 of US20160369298; SEQ ID NO: 2122 herein), SKTMADNNNSEYSWTG (SEQ ID NO: 37 of US20160369298; SEQ ID NO: 2123 herein), SKTHADNNNSEYSWTG (SEQ ID NO: 38 of US20160369298; SEQ ID NO: 2124 herein), SKTQADNNNSEYSWTG (SEQ ID NO: 39 of US20160369298; SEQ ID NO: 2125 herein), NO:37 of US20160369298; SEQ ID NO:2123 herein), SKTRADNNNSEYSWTG (SEQ ID NO:38 of US20160369298; SEQ ID NO:2124 herein), SKTNADNNNSEYSWTG (SEQ ID NO:39 of US20160369298; SEQ ID NO:2125 herein), SKTVGRNNNSEYSWTG (SEQ ID NO:40 of US20160369298; SEQ ID NO:2126 herein), SKTADRNNNSEYSWTG (SEQ ID NO:41 of US20160369298; SEQ ID NO:2127 herein), SKKLSQNNNSKYSWQG (SEQ ID NO:42 of US20160369298; SEQ ID NO:2128 herein), SKPTTGNNNSDYSWPG (SEQ ID NO:43 of US20160369298; SEQ ID NO:2129 herein), SKKLSQNNNSKYSWQG (SEQ ID NO:44 of US20160369298; SEQ ID NO:2130 herein), SKKLSQNNNSKYSWQG (SEQ ID NO:45 of US20160369298; SEQ ID NO:2131 herein), SKKLSQNNNSKYSWQG (SEQ ID NO:46 of US20160369298; SEQ ID NO:2132 herein), SKKTSGNNNSDYSWPG (SEQ ID NO:47 of US20160369298; SEQ ID NO:2133 herein), SKTGNNNSDYSWPG (SEQ ID NO:48 of US20160369298; NO:43; SEQ ID NO:2129 herein), STQKNENNNSNYSWPG (SEQ ID NO:44 of US20160369298; SEQ ID NO:2130 herein), HKDDEGKF (SEQ ID NO:45 of US20160369298; SEQ ID NO:2131 herein), HKDDNRKF (SEQ ID NO:46 of US20160369298; SEQ ID NO:2132 herein), HKDDTNKF (SEQ ID NO:47 of US20160369298;: SEQ ID NO: 2133 herein), HEDSDKNF (SEQ ID NO: 48 of US20160369298; SEQ ID NO: 2134 herein), HRDGADSF (SEQ ID NO: 49 of US20160369298; SEQ ID NO: 2135 herein), HGDNKSRF (SEQ ID NO: 50 of US20160369298; SEQ ID NO: 2136 herein), KQGSEKTNVDFEEV (SEQ ID NO: 51 of US20160369298; SEQ ID NO: 2137 herein), KQGSEKTNVDSEEV (SEQ ID NO: 52 of US20160369298; SEQ ID NO: 2138 herein), KQGSEKTNVDVEEV (SEQ ID NO: 53 of US20160369298; SEQ ID NO: 2139 herein), NO: 2139), KQGSDKTNVDDAGV (SEQ ID NO: 54 of US20160369298; SEQ ID NO: 2140 herein), KQGSSKTNVDPREV (SEQ ID NO: 55 of US20160369298; SEQ ID NO: 2141 herein), KQGSRKTNVDHKQV (SEQ ID NO: 56 of US20160369298; SEQ ID NO: 2142 herein), KQGSKGGNVDTNRV (SEQ ID NO: 57 of US20160369298; SEQ ID NO: 2143 herein), KQGSGEANVDNGDV (SEQ ID NO: 58 of US20160369298; SEQ ID NO: 2144 herein), KQDAAADNIDYDHV (SEQ ID NO: 59 of US20160369298; SEQ ID NO: 2150 herein), KQGSRKTNVDHKQV (SEQ ID NO: 51 of US20160369298; SEQ ID NO: 2151 herein), KQGSKGGNVDTNRV (SEQ ID NO: 52 of US20160369298; SEQ ID NO: 2152 herein), KQG NO:59; SEQ ID NO:2145 herein), KQSGTRSNAAASSV (SEQ ID NO:60 of US20160369298; SEQ ID NO:2146 herein), KENTNTNDTELTNV (SEQ ID NO:61 of US20160369298; SEQ ID NO:2147 herein), QRGNNVAATADVNT (SEQ ID NO:62 of US20160369298; SEQ ID NO:2148 herein), QRGNNEAATADVNT (SEQ ID NO:63 of US20160369298;: SEQ ID NO: 2149 herein), QRGNNPAATADVNT (SEQ ID NO: 64 of US20160369298; SEQ ID NO: 2150 herein), QRGNNHAATADVNT (SEQ ID NO: 65 of US20160369298; SEQ ID NO: 2151 herein), QEENNIAATPGVNT (SEQ ID NO: 66 of US20160369298; SEQ ID NO: 2152 herein), QPPNNMAATHEVNT (SEQ ID NO: 67 of US20160369298; SEQ ID NO: 2153 herein), QHHNNSAATTIVNT (SEQ ID NO: 68 of US20160369298; SEQ ID NO: 2154 herein), QTTNNRAAFNMVET (SEQ ID NO: 69 of US20160369298; SEQ ID NO: 2160 herein), NO: 2155), QKKNNNAASKKVAT (SEQ ID NO: 70 of US20160369298; SEQ ID NO: 2156 herein), QGGNNKAADDAVKT (SEQ ID NO: 71 of US20160369298; SEQ ID NO: 2157 herein), QAAKGGAADDAVKT (SEQ ID NO: 72 of US20160369298; SEQ ID NO: 2158 herein), QDDRAAAANESVDT (SEQ ID NO: 73 of US20160369298; SEQ ID NO: 2159 herein), QQQHDDAAYQRVHT (SEQ ID NO: 74 of US20160369298; SEQ ID NO: 2160 herein), QSSSSLAAVSTVQT (SEQ ID NO: 75 of US20160369298; SEQ ID NO: 2161 herein), NO: 2161), QNNQTTAAIRNVTT (SEQ ID NO: 76 of US20160369298; SEQ ID NO: 2162 herein), NYNKKSDNVDFT (SEQ ID NO: 77 of US20160369298; SEQ ID NO: 2163 herein), NYNKKSENVDFT (SEQ ID NO: 78 of US20160369298; SEQ ID NO: 2164 herein), NYNKKSLNVDFT (SEQ ID NO: 79 of US20160369298;: SEQ ID NO: 2165 herein), NYNKKSPNVDFT (SEQ ID NO: 80 of US20160369298; SEQ ID NO: 2166 herein), NYSKKSHCVDFT (SEQ ID NO: 81 of US20160369298; SEQ ID NO: 2167 herein), NYRKTIYVDFT (SEQ ID NO: 82 of US20160369298; SEQ ID NO: 2168 herein), NYKEKKDVHFT (SEQ ID NO: 83 of US20160369298; SEQ ID NO: 2169 herein), NYGHRAIVQFT (SEQ ID NO: 84 of US20160369298; SEQ ID NO: 2170 herein), NYANHQFVVCT (SEQ ID NO: 85 of US20160369298; SEQ ID NO: 2171 herein), NO: 2171), NYDDDPTGVLLT (SEQ ID NO: 86 of US20160369298; SEQ ID NO: 2172 herein), NYDDPTGVLLT (SEQ ID NO: 87 of US20160369298; SEQ ID NO: 2173 herein), NFEQQNSVEWT (SEQ ID NO: 88 of US20160369298; SEQ ID NO: 2174 herein), SQSGASN (SEQ ID NO: 89 and SEQ ID NO: 241 of US20160369298; SEQ ID NO: 2175 herein), NNGSQA (SEQ ID NO: 90 of US20160369298; SEQ ID NO: 2176 herein), YYLSRTNTPSGTTTWSRLQFSQAGA (SEQ ID NO: 91 of US20160369298; SEQ ID NO: 92 herein), NO: 2177), SKTSADNNNSEYSWTG (SEQ ID NO: 92 of US20160369298; SEQ ID NO: 2178 herein), HKDDEEKF (SEQ ID NO: 93, 209, 214, 219, 224, 234, 239 and 244 of US20160369298; SEQ ID NO: 2179 herein), KQGSEKTNVDIEEV (SEQ ID NO: 94 of US20160369298; SEQ ID NO: 2180 herein), QRGNNQAATADVNT (SEQ ID NO: 95 of US20160369298;SEQ ID NO: 2181 of this article), NYNKKSVNVDFT (SEQ ID NO: 96 of US20160369298; SEQ ID NO: 2182 of this article), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSEYSWTGATKYH (SEQ ID NO: 106 of US20160369298; SEQ ID NO of this article) NO:2183), SASGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO:107 of US20160369298; SEQ ID NO:2184 of this article), SQSGASNYNTPSGTTTQSRLQFSTDGENNNSDFSWTGATKYH (SEQ ID NO:108 of US20160369298; SEQ ID of this article) NO:2185), SASGASNYNTPSGTTTQSRLQFSTSADNNNSEFSWPGATTYH (SEQ ID NO:109 of US20160369298; SEQ ID NO:2186 of this article), SQSGASNFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID of US20160369298 NO: 110; SEQ ID NO: 2187 of this article), SASGASNYNTPSGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 111 of US20160369298; SEQ ID NO: 2188 of this article), SQSGASNYNTPSGTTTQSRLQFSTSADNNNSDFSWTGATKYH (SEQ ID of US20160369298 NO:112; SEQ ID of this article NO: 2189), SGAGASFNSEGGSLTQSSLGFSTDGENNNSDFSWTGATKYH (SEQ ID NO: 113 of US20160369298; SEQ ID NO: 2190 herein), SGAGASN (SEQ ID NO: 176 of US20160369298; SEQ ID NO: 2191 herein), NSEGGSLTQSSLGFS (SEQ ID NOs: 177, 185, 193 and 202 of US20160369298; SEQ ID NO: 2192 herein), TDGENNNSDFS (SEQ ID NO: 178 of US20160369298;: 2193 herein), SEFSWPGATT (SEQ ID NO: 179 of US20160369298; SEQ ID NO: 2194 herein), TSADNNNSDFSWT (SEQ ID NO: 180 of US20160369298; SEQ ID NO: 2195 herein), SQSGASNY (SEQ ID NOs: 181, 187 and 198 of US20160369298; SEQ ID NO: 2196 herein), NTPSGTTTQSRLQFS (SEQ ID NOs: 182, 188, 191 and 199 of US20160369298; SEQ ID NO: 2197 herein), TSADNNNSEYSWTGATKYH (SEQ ID NO: 183 of US20160369298; SEQ ID NO: 2198 herein), NO: 2198), SASGASNF (SEQ ID NO: 184 of US20160369298; SEQ ID NO: 2199 herein), TDGENNNSDFSWTGATKYH (SEQ ID NOs: 186, 189, 194, 197 and 203 of US20160369298; SEQ ID NO: 2200 herein), SASGASNY (SEQ ID NOs: 190 and 195 of US20160369298; SEQ ID NO: 2201 herein), TSADNNNSEFSWPGATTYH (SEQ ID NO: 192 of US20160369298; SEQ ID NO: 2202 herein), NTPSGSLTQSSLGFS (SEQ ID NO: 196 of US20160369298; SEQ ID NO: 2207 herein), NO:2203), TSADNNNSDFSWTGATKYH (SEQ ID NO:200 of US20160369298; SEQ ID NO:2204 of this article), SGAGASNF (SEQ ID NO:201 of US20160369298; SEQ ID of this article) NO: 2205), CTCCAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACACAA (SEQ ID NO: 204 of US20160369298; SEQ ID NO: 2206 of this article), CTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAA (SEQ ID NO: 205 of US20160369298;: SEQ ID NO: 2207 herein), SAAGASN (SEQ ID NO: 206 of US20160369298; SEQ ID NO: 2208 herein), YFLSRTNTESGSTTQSTLRFSQAG (SEQ ID NO: 207 of US20160369298; SEQ ID NO: 2209 herein), SKTSADNNNSDFS (SEQ ID NOs: 208, 228 and 253 of US20160369298; SEQ ID NO: 2210 herein), KQGSEKTDVDIDKV (SEQ ID NO: 210 of US20160369298; SEQ ID NO: 2211 herein), STAGASN (SEQ ID NO: 211 of US20160369298; SEQ ID NO: 212 herein), NO: 2212), YFLSRTNTTSGIETQSTLRFSQAG (SEQ ID NO: 212 and SEQ ID NO: 247 of US20160369298; SEQ ID NO: 2213 herein), SKTDGENNNSDFS (SEQ ID NO: 213 and SEQ ID NO: 248 of US20160369298; SEQ ID NO: 2214 herein), KQGAAADDVEIDGV (SEQ ID NO: 215 and SEQ ID NO: 250 of US20160369298; SEQ ID NO: 2215 herein), SEAGASN (SEQ ID NO: 216 of US20160369298; SEQ ID NO: 2216 herein), YYLSRTNTPSGTTTQSRLQFSQAG (SEQ ID NO: 217, 232 and 242 of US20160369298; SEQ ID NO: 2214 herein), NO: 2217), SKTSADNNNSEYS (SEQ ID NOs: 218, 233, 238 and 243 of US20160369298; SEQ ID NO: 2218 herein), KQGSEKTNVDIEKV (SEQ ID NOs: 220, 225 and 245 of US20160369298; SEQ ID NO: 2219 herein), YFLSRTNDASGSDTKSTLLFSQAG (SEQ ID NO: 222 of US20160369298; SEQ ID NO: 2220 herein), STTPSENNNSEYS (SEQ ID NO: 223 of US20160369298;: SEQ ID NO: 2221 herein), SAAGATN (SEQ ID NO: 226 and SEQ ID NO: 251 of US20160369298; SEQ ID NO: 2222 herein), YFLSRTNGEAGSATLSELRFSQAG (SEQ ID NO: 227 of US20160369298; SEQ ID NO: 2223 herein), HGDDADRF (SEQ ID NO: 229 and SEQ ID NO: 254 of US20160369298; SEQ ID NO: 2224 herein), KQGAEKSDVEVDRV (SEQ ID NO: 230 and SEQ ID NO: 255 of US20160369298; SEQ ID NO: 2225 herein), KQDSGGDNIDIDQV (SEQ ID NO: 235 of US20160369298; SEQ ID NO: 236 herein). NO: 2226), SDAGASN (SEQ ID NO: 236 of US20160369298; SEQ ID NO: 2227 herein), YFLSRTNTEGGHDTQSTLRFSQAG (SEQ ID NO: 237 of US20160369298; SEQ ID NO: 2228 herein), KEDGGGSDVAIDEV (SEQ ID NO: 240 of US20160369298; SEQ ID NO: 2229 herein), SNAGASN (SEQ ID NO: 246 of US20160369298; SEQ ID NO: 2230 herein), and YFLSRTNGEAGSATLSELRFSQPG (SEQ ID NO: 252 of US20160369298; SEQ ID NO: 2231 herein). Nucleotide sequences that can encode amino acid mutation sites include the following, AGCVVMDCAGGARSCASCAAC (SEQ ID NO: 97 of US20160369298; SEQ ID NO: 2232 herein), AACRACRRSMRSMAGGCA (SEQ ID NO: 98 of US20160369298; SEQ ID NO: 2233 herein), CACRRGGACRRCRMSRRSARSTTT (SEQ ID NO: 99 of US20160369298;: SEQ ID NO: 2234 herein), TATTTCTTGAGCAGAACAAACRVCVVSRSCGGAMNCVHSACGMHSTCAVVSCTTVDS TTTTCTCAGSBCRGSGCG (SEQ ID NO: 100 of US20160369298; SEQ ID NO: 2235 herein), TCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCGTGGMMAGGA (SEQ ID NO: 101 of US20160369298; SEQ ID NO: 2236 herein), AAGSAARRCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTC (SEQ ID NO: 102 of US20160369298; SEQ ID NO: 2237 herein), CAGVVSVVSMRSRVCVNSGCAGCTDHCVVSRNSGTCVMSACA (SEQ ID NO: 103 of US20160369298; SEQ ID NO: 2238 herein), NO: 2238), AACTWCRVSVASMVSVHSDDTGTGSWSTKSACT (SEQ ID NO: 104 of US20160369298; SEQ ID NO: 2239 herein), TTGTTGAACATCACCACGTGACGCACGTTC (SEQ ID NO: 256 of US20160369298; SEQ ID NO: 2240 herein), TCCCCGTGGTTCTACTACATAATGTGGCCG (SEQ ID NO: 257 of US20160369298; SEQ ID NO: 2241 herein), TTCCACACTCCGTTTTGGATAATGTTGAAC (SEQ ID NO: 258 of US20160369298; SEQ ID NO: 2242 herein), AGGGACATCCCCAGCTCCATGCTGTGGTCG (SEQ ID NO: 259 of US20160369298; SEQ ID NO: 2243 herein). NO: 2243), AGGGACAACCCCTCCGACTCGCCCTAATCC (SEQ ID NO: 260 of US20160369298; SEQ ID NO: 2244 herein), TCCTAGTAGAAGACACCCTCTCACTGCCCG (SEQ ID NO: 261 of US20160369298;SEQ ID NO: 2245 of the present text), AGTACCATGTACACCCACTCTCCCAGTGCC (SEQ ID NO: 262 of US20160369298; SEQ ID NO: 2246 of the present text), ATATGGACGTTCATGCTGATCACCATACCG (SEQ ID NO: 263 of US20160369298; SEQ ID NO: 2247 of the present text), AGCAGGAGCTCCTTGGCCTCAGCGTGCGAG (SEQ ID NO: 264 of US20160369298; SEQ ID NO: 2248 of the present text), ACAAGCAGCTTCACTATGACAACCACTGAC (SEQ ID NO: 265 of US20160369298; SEQ ID NO: 2249 of the present text), CAGCCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGAGAGTCTCAAMAMMAVNSRVCSRSAACAACAACAGTRASTTCTCCTGGMMAGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCCGGACCAGCTATGGCAAGCCACRRGGACRRCRMSRRSARSTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGSAARRCRSCRVSRVARVCRATRYCGMSNHCRVMVRSGTCATGATTACAGACGAAGAGGAGATCTGGAC (SEQ ID NO: 266 of US20160369298; SEQ ID NO: 2250 of the present text), TGGGACAATGGCGGTCGTCTCTCAGAGTTKTKKT (SEQ ID NO: 267 of US20160369298; SEQ ID NO: 2251 of the present text), AGAGGACCKKTCCTCGATGGTTCATGGTGGAGTTA (SEQ ID NO: 268 of US20160369298; SEQ ID NO: 2252 of the present text), CCACTTAGGGCCTGGTCGATACCGTTCGGTG (SEQ ID NO: 269 of US20160369298; SEQ ID NO: 2253 of the present text) and TCTCGCCCCAAGAGTAGAAACCCTTCSTTYYG (SEQ ID NO: 270 of US20160369298; SEQ ID NO: 2254 of the present text).;

[0171] In some embodiments, the AAV serotype may include an ocular cell targeting peptide described in International Patent Publication 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. In addition, 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: 2255 herein) or AAV9 (SEQ ID NO: 11 of WO2016134375; SEQ ID NO: 2256 herein). In some embodiments, modifications, such as insertions, are made at AAV2 protein P34-A35, T138-A139, A139-P140, G453-T454, N587-R588, and / or R588-Q589. In certain embodiments, the insertion is made at D384, G385, 1560, 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: 2257 herein) or GETRAPL (SEQ ID NO: 4 of WO2016134375; SEQ ID NO: 2258 herein).

[0172] In some embodiments, the AAV serotype can be modified as described in U.S. Publication No. US20170145405, the contents of which are incorporated herein by reference in their entirety. The AAV serotype can include a modified AAV2 (e.g., a modification at Y444F, Y500F, Y730F, and / or S662V), a modified AAV3 (e.g., a modification at Y705F, Y731F, and / or T492V), and a modified AAV6 (e.g., a modification at S663 V and / or T492V).

[0173] In some embodiments, the AAV serotype can be modified as described in International Publication 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).

[0174] In some embodiments, the AAV serotype may comprise an engineered epitope as described in International Patent Publication No. WO2017015102, the contents of which are incorporated herein by reference in their entirety, comprising amino acids SPAKFA (SEQ ID NO: 24 of WO2017015102; SEQ ID NO: 2259 herein) or NKDKLN (SEQ ID NO: 2 of WO2017015102; SEQ ID NO: 2260 herein). The epitope may be inserted in the region of amino acids 665 to 670 based on the numbering of the VP1 capsid of AAV8 (SEQ ID NO: 3 of WO2017015102) and / or residues 664 to 668 of AAV3B (SEQ ID NO: 3).

[0175] In one embodiment, 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 that may comprise a substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500, 528-534, 547-552, 588-597, 709-710, 716-722 of AAV1 (in any combination) or at the equivalent amino acid residues of AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. The amino acid substitution may be, but is not limited to, any amino acid sequence described in WO2017058892.In one embodiment, the AAV may comprise amino acid substitutions at the following 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); NO:5) 244N, 246Q, 248R, 249E, 250I, 251K, 252S, 253G, 254S, 255V, 256D, 263Y, 377E, 378N, 453L, 456R, 532Q, 533P, 535N, 536P, 537G, 538T, 539T, 540A, 541T, 542Y, 543L, 546N, 653V, 654P, 656S, 697Q, 698F, 704D, 705S, 706T, 707G, 708E, 709Y and / or 710R (in any combination); AAV5 (SEQ ID NO: WO2017058892 NO:5) 248R, 316V, 317Q, 318D, 319S, 443N, 530N, 531S, 532Q 533P, 534A, 535N, 540A, 541T, 542Y, 543L, 545G, 546N, 697Q, 704D, 706T, 708E, 709Y and / or 710R (in any combination); 264S, 266G, 269N, 272H, 457Q, 588S and / or 589I (in any combination) of AAV6 (SEQ ID NO:6WO2017058892); AAV8 (SEQ ID NO:8WO2017058892) of 457T, 459N, 496G, 499N, 500N, 589Q, 590N and / or 592A (in any combination); 451I, 452N, 453G, 454S, 455G, 456Q, 457N and / or 458Q (in any combination) of AAV9 (SEQ ID NO:9WO2017058892).

[0176] In some embodiments, the AAV may comprise the amino acid sequence of 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 independently, but are not limited to, 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 and AAV12. In some embodiments, the AAV may comprise a deletion of at least one amino acid at position 156, 157, or 158 of VP1 or position 19, 20, or 21 of VP2, wherein the AAV may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0177] In one embodiment, 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. In some embodiments, the AAV serotype is PHP.B or AAV9. In some embodiments, the AAV serotype is paired with the synapsin promoter to enhance neuronal transduction compared to the use of more common promoters (i.e., CBA or CMV).

[0178] In one embodiment, peptides contained in an AAV serotype can be identified by isolating human splenocytes, restimulating the splenocytes in vitro with individual peptides spanning the amino acid sequence of the AAV capsid protein, IFN-γ ELISpot using the individual peptides for in vitro restimulation, bioinformatics analysis to determine the restriction of a given allele of the 15-mer recognized by the IFN-γ ELISpot, identification of candidate reactive 9-mer epitopes for a given allele, synthesis of candidate 9-mers, performing a second IFN-γ ELISpot screening of splenocytes from subjects carrying the specific allele to which the expected recognized AAV epitope binds, determining AAV capsid reactive CD8+ T cell epitopes, and determining the frequency with which subjects respond to a given AAV epitope.

[0179] AAV particles containing regulatory polynucleotides encoding siRNA molecules can be prepared or derived from a variety of AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ8 and AAV-DJ. In some cases, AAVs of different serotypes can be mixed together or mixed with other types of viruses to produce chimeric AAV particles. As a non-limiting example, AAV particles are derived from AAV9 serotypes.

[0180] Viral genome

[0181] In one embodiment, as shown, the AAV particle comprises a viral genome having a payload region.

[0182] In one embodiment, the viral genome may comprise Figure 1 The payload region 110 is located within the viral genome 100. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. Between the 5' ITR 120 and the payload region 110, a promoter region 130 may be present. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0183] In one embodiment, the viral genome 100 may include Figure 2 The components shown. The payload region 110 is located within the viral genome 100. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. Between the 5' ITR 120 and the payload region 110, there may be a promoter region 130. Between the promoter region 130 and the payload region 110, there may be an intron region 140. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0184] In one embodiment, the viral genome 100 may include Figure 3 The components shown. At least one inverted terminal repeat (ITR) 120 may be present at the 5' and / or 3' end of the viral genome 100. In the viral genome 100, there may be an enhancer region 150, a promoter region 130, an intron region 140 and a payload region 110. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0185] In one embodiment, the viral genome 100 may include Figure 4The components shown. There may be at least one inverted terminal repeat (ITR) 120 at the 5' and / or 3' end of the viral genome 100. In the viral genome 100, there may be an enhancer region 150, a promoter region 130, an intron region 140, a payload region 110 and a polyadenylation signal sequence region 160. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0186] In one embodiment, the viral genome 100 may include Figure 5 Components shown. At the 5' and / or 3' end of the viral genome 100, there may be at least one inverted terminal repeat (ITR) 120. In the viral genome 100, there may be at least one MCS region 170, one enhancer region 150, one promoter region 130, one intron region 140, one payload region 110 and one polyadenylation signal sequence region 160. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0187] In one embodiment, the viral genome 100 may include Figure 6 At the 5' and / or 3' end of the viral genome 100, there may be at least one inverted terminal repeat (ITR) 120. Within the viral genome 100, there may be at least one MCS region 170, one enhancer region 150, one promoter region 130, at least one exon region 180, at least one intron region 140, one payload region 110, and one polyadenylation signal sequence region 160. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0188] In one embodiment, the viral genome 100 may include Figure 7 and 8 In the viral genome 100, there may be at least one promoter region 130 and one payload region 110. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0189] In one embodiment, the viral genome 100 may include Fig. 9 In the viral genome 100, there may be at least one promoter region 130, one payload region 110 and one polyadenylation signal sequence region 160. In one embodiment, the payload region may include at least one regulatory polynucleotide.

[0190] Viral genome size

[0191] In one embodiment, the viral genome comprising the payload described herein can be a single-stranded or double-stranded viral genome. The size of the viral genome can be small, medium, large or maximum size. In addition, the viral genome can include a promoter and a polyA tail.

[0192] In one embodiment, the viral genome comprising the payload described herein can be a small single-stranded viral genome. The size of the small single-stranded viral genome can be 2.7 to 3.5 kb, such as about 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 and 3.5 kb. As a non-limiting example, the size of the small single-stranded viral genome can be 3.2 kb. In addition, the viral genome can include a promoter and a polyA tail.

[0193] In one embodiment, the viral genome comprising the payload described herein can be a small double-stranded viral genome. The size of the small double-stranded viral genome can be 1.3 to 1.7 kb, such as about 1.3, 1.4, 1.5, 1.6 and 1.7 kb. As a non-limiting example, the size of the small double-stranded viral genome can be 1.6 kb. In addition, the viral genome can include a promoter and a polyA tail.

[0194] In one embodiment, the viral genome comprising the payload described herein can be a medium single-stranded viral genome. The size of the medium single-stranded viral genome can be 3.6 to 4.3 kb, such as about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb. As a non-limiting example, the size of the medium single-stranded viral genome can be 4.0 kb. In addition, the viral genome can include a promoter and a poly A tail.

[0195] In one embodiment, the viral genome comprising the payload described herein can be a medium double-stranded viral genome. The size of the medium double-stranded viral genome can be 1.8 to 2.1 kb, such as about 1.8, 1.9, 2.0 and 2.1 kb. As a non-limiting example, the size of the medium double-stranded viral genome can be 2.0 kb. In addition, the viral genome can include a promoter and a polyA tail.

[0196] In one embodiment, the viral genome comprising the payload described herein can be a large single-stranded viral genome. The size of the large single-stranded viral genome may 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 size of the large single-stranded viral genome can be 4.7 kb. As another non-limiting example, the size of the large single-stranded viral genome can be 4.8 kb. As another non-limiting example, the size of the large single-stranded viral genome can be 6.0 kb. In addition, the viral genome can include a promoter and a poly A tail.

[0197] In one embodiment, the viral genome comprising the payload described herein can be a large double-stranded viral genome. The size of the large double-stranded viral genome can be 2.2 to 3.0 kb, such as 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 viral genome can be 2.4 kb. In addition, the viral genome can include a promoter and a polyA tail.

[0198] Viral genome components: Inverted terminal repeats (ITRs)

[0199] The AAV particles of the present invention comprise a viral genome having at least one ITR region and a payload region. In one embodiment, the viral genome has two ITRs. The two ITRs are located on the flanks of the payload region, at the 5' and 3' ends. The ITRs serve as the origin of replication comprising a replication recognition site. The ITRs comprise sequence regions that can be complementary and symmetrically arranged. The ITRs incorporated into the viral genome of the present invention can consist of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0200] The ITRs may be derived from the same serotype as the capsid, selected from any serotype listed in Table 1 or a derivative thereof. The ITRs may have a different serotype than the capsid. In one embodiment, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In one embodiment, the ITRs have the same serotype as each other. In another embodiment, the ITRs have different serotypes. Non-limiting examples include zero, one, or two ITRs having the same serotype as the capsid. In one embodiment, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0201] In some embodiments, the ITR has a length of about 100-150 nucleotides. The ITR has a length of about 100-105 nucleotides, a length of about 106-110 nucleotides, a length of about 111-115 nucleotides, a length of about 116-120 nucleotides, a length of about 121-125 nucleotides, a length of about 126-130 nucleotides, a length of about 131-135 nucleotides, a length of about 136-140 nucleotides, a length of about 141-145 nucleotides, or a length of about 146-150 nucleotides. In one embodiment, the ITR has a length of about 140-142 nucleotides. The non-limiting examples of ITR length are 102, 140, 141, 142, 145 nucleotides, and nucleotides with at least 95% identity thereto.

[0202] In one embodiment, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which can be located near the 5' end of the flip ITR in the expression vector. In another embodiment, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which can be located near the 3' end of the flip ITR in the expression vector. In yet another embodiment, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which can be located near the 5' end of the flop ITR in the expression vector. In yet another embodiment, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which can be located near the 3' end of the flop ITR in the expression vector. In one embodiment, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which can be located between the 5' end of the flip ITR and the 3' end of the flop ITR in the expression vector. In one embodiment, the AAV particle comprises a nucleic acid sequence encoding a siRNA molecule, which can be located between the 3' end of the flip ITR and the 5' end of the flop ITR in the expression vector (e.g., between the 5' end of the flip ITR and the 3' end of the flop ITR or between the 3' end of the flop ITR and the 5' end of the flip ITR). As a non-limiting example, the AAV particle comprises a nucleic acid sequence encoding a siRNA molecule, which 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 the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector. As a non-limiting example, the AAV particle comprises a nucleic acid sequence encoding a siRNA molecule, which 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 upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector. As another non-limiting example, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule which can 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 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector.As another non-limiting example, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule which can 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 upstream of the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in an expression vector. As a non-limiting example, the AAV particle comprises a nucleic acid sequence encoding a siRNA molecule, which can 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 upstream of the 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector. As another non-limiting example, the AAV particle comprises a nucleic acid sequence encoding a siRNA molecule, which can 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 5' or 3' end of the ITR (e.g., Flip or Flop ITR) in the expression vector.

[0203] Viral genome components: promoters

[0204] In one embodiment, the payload region of the viral genome comprises at least one element that enhances transgene target specificity and expression (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 transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences and upstream enhancers (USEs), CMV enhancers, and introns.

[0205] Those skilled in the art will recognize that expression of the polypeptides of the present invention in 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).

[0206] In one embodiment, a promoter is considered effective when it drives expression of a polypeptide encoded in the payload region of the viral genome of an AAV particle.

[0207] In one embodiment, the promoter is one that is believed to be effective in driving expression of a regulatory polynucleotide.

[0208] In one embodiment, the promoter is one that is considered efficient when it drives expression in the cell to be targeted.

[0209] In one embodiment, the promoter drives the expression of the payload in the target tissue for a period of time. The expression driven by the promoter can last 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 ... days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more. Expression can last 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.

[0210] In one embodiment, the promoter drives expression of the payload for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 or more years.

[0211] The promoter may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters, and mammalian promoters. In some embodiments, the promoter may be a human promoter. In some embodiments, the promoter may be truncated.

[0212] Promoters that drive or initiate expression in most tissues include, but are not limited to, human elongation factor 1 alpha subunit (EF1α), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (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, muscle-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters, which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0213] Non-limiting examples of muscle-specific promoters include the mammalian muscle creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the mammalian troponin I (TNNI2) promoter, and the mammalian skeletal α-actin (ASKA) promoter (see, e.g., U.S. Patent Publication No. US20110212529, the contents of which are herein incorporated by reference in their entirety).

[0214] 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), Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (neurofilament light, NFL) or neurofilament heavy (neurofilament heavy, NFH), β-globin minigene nβ2, proenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoter. 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.

[0215] In one embodiment, the promoter may be less than 1 kb. The length of the promoter may be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 50 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more 800 nucleotides. The promoter may be 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 in length.

[0216] In one embodiment, the promoter can be a combination of two or more components of the same or different starting or parent promoters, such as, but not limited to, CMV and CBA. The length of each component can be 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 581, 582, 583, 584, 585, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 701 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more. The length of each component can be 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. In one embodiment, the promoter is a combination of a 382 nucleotide CMV-enhancer sequence and a 260 nucleotide CBA-promoter sequence.

[0217] In one embodiment, the viral genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0218] Yu et al. (Molecular Pain 2011, 7:63; the contents of which are incorporated herein by reference in their entirety) evaluated the expression of eGFP in rat DRG cells and primary DRG cells under the CAG, EFIα, PGK and UBC promoters using lentiviral vectors and found that UBC showed weaker expression than the other three promoters, and only 10-12% glial expression was observed for all promoters. Soderblom et al. (E. Neuro 2015; the contents of which are incorporated herein by reference in their entirety) evaluated the expression of eGFP in AAV8 with CMV and UBC promoters and in AAV2 with CMV promoter after motor cortex injection. Intranasal administration of plasmids containing UBC or EFIα promoters showed stronger sustained airway expression than expression with CMV promoter (see, e.g., Gill 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 hereby incorporated by reference in their entirety) evaluated HβH constructs with hGUSB promoter, HSV-1LAT promoter, and NSE promoter, and found that HβH constructs showed weaker expression than NSE in mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, the contents of which are hereby incorporated by reference in their entirety) evaluated the long-term effects of HβH vectors after intracerebroventricular injection in newborn mice and found that they continued to be expressed for at least one year. Xu et al. (Gene Therapy 2001, 8, 1323-1332; the contents of which are incorporated herein by reference in their entirety) found that when NFL and NFH promoters were used, expression was low in all brain regions 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 promoter activity was NSE (1.8 kb), EF, NSE (0.3 kb), GFAP, CMV, hENK, PPE, NFL, and NFH in descending order. NFL is a 650 nucleotide promoter, while NFH is a 920 nucleotide promoter, and neither of them is present in the liver, but NFH is abundant in sensory proprioceptive neurons, the brain, and the spinal cord, and NFH is present in the heart.Scn8a is a 470-nucleotide promoter that is expressed throughout the DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus, and hypothalamus (see, e.g., Drews et al., Identification of evolutionary conserved, functional noncoding elements in the promoter region of the sodium channel gene SCN8A, Mamm Genome (2007) 18:723-731; and Raymond et al., Expression of Alternatively Spliced ​​Sodium Channel α-subunit genes, Journal of Biological Chemistry (2004) 279(44)46234-46241; the entire contents of which are incorporated herein by reference in their entirety).

[0219] Any of the promoters taught by Yu, Soderblom, Gill, Husain, Passini, Xu, Drews, or Raymond, supra, may be used in the present invention.

[0220] In one embodiment, the promoter is not cell-specific.

[0221] In one embodiment, the promoter is the ubiquitin c (UBC) promoter. The UBC promoter may have a size of 300-350 nucleotides. As a non-limiting example, the UBC promoter is 332 nucleotides.

[0222] In one embodiment, the promoter is a β-glucuronidase (GUSB) promoter. The size of the GUSB promoter can be 350-400 nucleotides. As a non-limiting example, the GUSB promoter is 378 nucleotides.

[0223] In one embodiment, the promoter is a neurofilament light peptide (NFL) promoter. The NFL promoter can have a size of 600-700 nucleotides. As a non-limiting example, the NFL promoter is 650 nucleotides. As a non-limiting example, the construct can be an AAV promoter-CMV / globin intron regulatory polynucleotide-RBG, wherein the AAV can be self-complementary and the AAV can be a DJ serotype.

[0224] In one embodiment, the promoter is a neurofilament heavy peptide (FH) promoter. The NFH promoter may have a size of 900-950 nucleotides. As a non-limiting example, the NFH promoter is 920 nucleotides. As a non-limiting example, the construct may be AAV-promoter-CMV / globin intron-regulatory polynucleotide-RBG, wherein the AAV may be self-complementary and the AAV may be a DJ serotype.

[0225] In one embodiment, the promoter is a scn8a promoter. The size of the scn8a promoter can be 450-500 nucleotides. As a non-limiting example, the scn8a promoter is 470 nucleotides. As a non-limiting example, the construct can be an AAV promoter-CMV / globin intron regulatory polynucleotide-RBG, wherein the AAV can be self-complementary and the AAV can be a DJ serotype.

[0226] In one embodiment, the viral genome comprises a Pol III promoter.

[0227] In one embodiment, the viral genome comprises a P1 promoter.

[0228] In one embodiment, the viral genome comprises a FXN promoter.

[0229] In one embodiment, the promoter is the phosphoglycerate kinase 1 (PGK) promoter.

[0230] In one embodiment, the promoter is the chicken beta-actin (CBA) promoter.

[0231] In one embodiment, the promoter is the CAG promoter, which is a construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-actin (CBA) promoter.

[0232] In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.

[0233] In one embodiment, the viral genome comprises an H1 promoter.

[0234] In one embodiment, the viral genome comprises a U6 promoter.

[0235] In one embodiment, the promoter is a liver or skeletal muscle promoter. Non-limiting examples of liver promoters include human alpha-1-antitrypsin (hAAT) and thyroxine binding globulin (TBG). Non-limiting examples of skeletal muscle promoters include desmin, MCK, or synthetic C5-12.

[0236] In one embodiment, the promoter is an RNApol III promoter. As a non-limiting example, the RNApol III promoter is U6. As a non-limiting example, the RNApol III promoter is H1.

[0237] In one embodiment, the viral genome comprises two promoters. As non-limiting examples, the promoters are the EF1α promoter and the CMV promoter.

[0238] In one embodiment, the viral genome comprises an enhancer element, a promoter and / or a 5'UTR intron. The enhancer element, also referred to herein as an "enhancer", may be, but is not limited to, a CMV enhancer, the promoter may be, but is not limited to, a CMV, CBA, UBC, GUSB, NSE, synapsin, MeCP2, and GFAP promoter, and the 5'UTR / intron may be, but is not limited to, SV40 and CBA-MVM. As non-limiting examples, 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 (11) U6 promoter.

[0239] In one embodiment, the viral genome comprises an engineered promoter.

[0240] In another embodiment, the viral genome comprises a promoter from a naturally expressed protein.

[0241] Viral genome components: untranslated regions (UTRs)

[0242] 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, and the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal.

[0243] Features commonly found in abundantly expressed genes in a particular target organ can be engineered into UTRs to enhance stability and protein production. As a non-limiting example, a 5'UTR from an mRNA 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 invention to enhance expression in hepatocyte cell lines or the liver.

[0244] Without wishing to be bound by theory, the wild-type 5' untranslated region (UTR) includes features that play a role in translation initiation. It is generally known that the Kozak sequence involved in the process of ribosome initiation of translation of many genes is often contained in the 5'UTR. The Kozak sequence has a consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) 3 bases upstream of the start codon (ATG), followed by another 'G'.

[0245] In one embodiment, the 5'UTR in the viral genome includes a Kozak sequence.

[0246] In one embodiment, the 5'UTR in the viral genome does not include a Kozak sequence.

[0247] Without wishing to be bound by theory, it is known that the wild-type 3'UTR has a stretch of adenosine and uridine embedded therein. These AU-rich features are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995, the contents of which are incorporated herein by reference in their entirety): Class I AREs, such as but not limited to c-Myc and MyoD, contain several dispersed copies of the AUUUA motif in the U-rich region. Class II AREs, such as but not limited to GM-CSF and TNF-a, have two or more overlapping UUAUUUA (U / A) (U / A) 9-mers. Class III AREs are less clearly defined, such as but not limited to c-Jun and myogenin. These U-rich regions do not contain the AUUUA motif. It is known that most proteins that bind to AREs destabilize messengers, and members of the ELAV family (most notably HuR) have been shown to increase the stability of mRNA. HuR binds to all three types of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding and thereby stabilization of the message in vivo.

[0248] The introduction, removal or modification of 3'UTR AU-rich elements (AREs) can be used to regulate 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 production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability and thereby increase translation and production of the resulting protein.

[0249] In one embodiment, the 3'UTR of the viral genome may include an oligo(dT) sequence for templated addition of a poly-A tail.

[0250] In one embodiment, the viral genome may include at least one miRNA seed, binding site or complete sequence. MicroRNA (or miRNA or miR) is a non-coding RNA of 19-25 nucleotides that binds to a nucleic acid target site and downregulates gene expression by reducing the stability of the nucleic acid molecule or inhibiting translation. The microRNA sequence contains a "seed" region, i.e., a sequence in the 2-8 region of a mature microRNA that has perfect Watson-Crick complementarity with the miRNA target sequence of the nucleic acid.

[0251] In one embodiment, the virus can be genetically engineered to contain, alter or remove at least one miRNA binding site, sequence or seed region.

[0252] Any UTR from any gene known in the art can be introduced into the viral genome of the AAV particle. These UTRs or parts thereof can be placed in the same direction as in the gene in which they are selected, or the direction or position can be changed. In one embodiment, the UTR used in the viral genome of the AAV particle can be reversed, shortened, extended, made with one or more other 5'UTRs or 3'UTRs known in the art. As used herein, the term "change" when referring to UTR means that the UTR has been changed in some way relative to a reference sequence. For example, 3' or 5'UTR can be changed relative to a wild-type or native UTR by a change in direction or position as described above, or 3' or 5'UTR can be changed by including additional nucleotides, deletions of nucleotides, exchanges of nucleotides or transposition.

[0253] In one embodiment, the viral genome of the AAV particle comprises at least one artificial UTR that is not a variant of a wild-type UTR.

[0254] In one embodiment, the viral genome of the AAV particle comprises a UTR selected from a family of transcripts whose proteins have a common function, structure, feature, or property.

[0255] Viral genome components: polyadenylation sequence

[0256] In one embodiment, the viral genome of the AAV particle of the invention comprises at least one polyadenylation sequence.The viral genome of the AAV particle may comprise a polyadenylation sequence between the 3' end of the payload encoding sequence and the 5' end of the 3' ITR.

[0257] In one embodiment, the polyadenylation sequence or "polyA 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 02, 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 and 500 nucleotides.

[0258] In one embodiment, the polyadenylation sequence is 50-100 nucleotides in length.

[0259] In one embodiment, the polyadenylation sequence is 50-150 nucleotides in length.

[0260] In one embodiment, the polyadenylation sequence is 50-160 nucleotides in length.

[0261] In one embodiment, the polyadenylation sequence is 50-200 nucleotides in length.

[0262] In one embodiment, the polyadenylation sequence is 60-100 nucleotides in length.

[0263] In one embodiment, the polyadenylation sequence is 60-150 nucleotides in length.

[0264] In one embodiment, the polyadenylation sequence is 60-160 nucleotides in length.

[0265] In one embodiment, the polyadenylation sequence is 60-200 nucleotides in length.

[0266] In one embodiment, the polyadenylation sequence is 70-100 nucleotides in length.

[0267] In one embodiment, the polyadenylation sequence is 70-150 nucleotides in length.

[0268] In one embodiment, the polyadenylation sequence is 70-160 nucleotides in length.

[0269] In one embodiment, the polyadenylation sequence is 70-200 nucleotides in length.

[0270] In one embodiment, the polyadenylation sequence is 80-100 nucleotides in length.

[0271] In one embodiment, the polyadenylation sequence is 80-150 nucleotides in length.

[0272] In one embodiment, the polyadenylation sequence is 80-160 nucleotides in length.

[0273] In one embodiment, the polyadenylation sequence is 80-200 nucleotides in length.

[0274] In one embodiment, the polyadenylation sequence is 90-100 nucleotides in length.

[0275] In one embodiment, the polyadenylation sequence is 90-150 nucleotides in length.

[0276] In one embodiment, the polyadenylation sequence is 90-160 nucleotides in length.

[0277] In one embodiment, the polyadenylation sequence is 90-200 nucleotides in length.

[0278] In one embodiment, the AAV particles include a nucleic acid sequence encoding an siRNA molecule, which may be located upstream of a polyadenylation sequence in an expression vector. In addition, the AAV particles include a nucleic acid sequence encoding an siRNA molecule, which may be located downstream of a promoter (e.g., but not limited to, CMV, U6, CAG, CBA, or a CBA promoter with an SV40 intron or a human beta globin intron) in an expression vector. As a non-limiting example, the AAV particles include a nucleic acid sequence encoding an siRNA molecule, which may 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. As another non-limiting example, the AAV particles comprise a nucleic acid sequence encoding a siRNA molecule, which in the expression vector 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 a promoter and / or upstream of a polyadenylation sequence. As a non-limiting example, the AAV particles comprise a nucleic acid sequence encoding an siRNA molecule, which may be located within the first 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more than 25% of nucleotides downstream of a promoter and / or upstream of a polyadenylation sequence in an expression vector. As another non-limiting example, the AAV particles comprise a nucleic acid sequence encoding an siRNA molecule, which 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 nucleotides downstream of a promoter and / or upstream of a polyadenylation sequence in an expression vector.

[0279] In one embodiment, the AAV particle comprises a rabbit globin polyadenylation (poly A) signal sequence.

[0280] In one embodiment, the AAV particle comprises a human growth hormone polyadenylation (poly A) signal sequence.

[0281] Viral genome components: introns

[0282] In one embodiment, the payload region includes at least one element that enhances expression, such as one or more introns or portions thereof. 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).

[0283] In one embodiment, the length of an intron or an intron portion can be 100-500 nucleotides. The length of an intron can be 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. The intron may be 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 in length.

[0284] In one embodiment, the AAV viral genome may comprise a promoter, such as, but not limited to, CMV or U6. As a non-limiting example, the promoter of the AAV comprising the nucleic acid sequence of the siRNA molecule of the present invention is the CMV promoter. As another non-limiting example, the promoter of the AAV comprising the nucleic acid sequence of the siRNA molecule of the present invention is the U6 promoter.

[0285] In one embodiment, the AAV viral genome may comprise a CMV promoter.

[0286] In one embodiment, the AAV viral genome may comprise a U6 promoter.

[0287] In one embodiment, the AAV viral genome may comprise CMV and U6 promoters.

[0288] In one embodiment, the AAV viral genome may comprise an H1 promoter.

[0289] In one embodiment, the AAV viral genome may comprise a CBA promoter.

[0290] In one embodiment, the encoded siRNA molecule can be located downstream of a promoter in an expression vector, such as, but not limited to, CMV, U6, H1, CBA, CAG, or a CBA promoter with introns (e.g., SV40 or other introns known in the art). In addition, the encoded siRNA molecule can also be located upstream of a polyadenylation sequence in an expression vector. As a non-limiting example, the encoded siRNA molecule 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 amino acids downstream of a promoter and / or upstream of a polyadenylation sequence in an expression vector. As a non-limiting example, the encoded siRNA molecule can 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 promoter and / or upstream of the polyadenylation sequence in the expression vector. As a non-limiting example, the encoded siRNA molecule can 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. As a non-limiting example, the encoded siRNA molecule 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 nucleotides downstream of the promoter and / or upstream of the polyadenylation sequence in the expression vector.

[0291] Viral genome components: filler sequences

[0292] In one embodiment, the viral genome comprises one or more stuffer sequences.

[0293] In one embodiment, the viral genome comprises one or more stuffer sequences to make the length of the viral genome an optimal size for packaging. As a non-limiting example, the viral genome comprises at least one stuffer sequence to make the length of the viral genome about 2.3 kb. As a non-limiting example, the viral genome comprises at least one stuffer sequence to make the length of the viral genome about 4.6 kb.

[0294] In one embodiment, the viral genome comprises one or more stuffing sequences to reduce the possibility that the hairpin structure of the vector genome (e.g., a regulatory polynucleotide as described herein) can be read as an inverted terminal repeat (ITR) during expression / or packaging. As a non-limiting example, the viral genome comprises at least one stuffing sequence so that the length of the viral genome is about 2.3 kb. As a non-limiting example, the viral genome comprises at least one stuffing sequence so that the length of the viral genome is about 4.6 kb.

[0295] In one embodiment, the viral genome is a single-stranded (ss) viral genome and comprises one or more stuffer sequences having a length of about 0.1 kb-3.8 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 In some embodiments, the full-length filler sequence in the vector genome is 3.1kb. As a non-limiting example, the full-length filler sequence in the vector genome is 2.7kb. As a non-limiting example, the full-length filler sequence in the vector genome is 0.8kb. As a non-limiting example, the full-length filler sequence in the vector genome is 0.4kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.8kb. As a non-limiting example, each stuffer sequence in the vector genome is 0.4 kb in length.

[0296] In one embodiment, the viral genome is a self-complementary (sc) viral genome and comprises one or more filler sequences having a length of about 0.1 kb-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. As a non-limiting example, the full-length filler sequence in the vector genome is 0.8 kb. As a non-limiting example, the full-length filler sequence in the vector genome is 0.4 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.8 kb. As a non-limiting example, the length of each filler sequence in the vector genome is 0.4 kb.

[0297] In one embodiment, the viral genome comprises any portion of the stuffer 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 stuffer sequence.

[0298] In one embodiment, the viral genome is a single-stranded (ss) viral genome and comprises one or more stuffing sequences so that the length of the viral genome is about 4.6 kb. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 3' end of the 5'ITR sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 5' end of the promoter sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 5' end of the 3'ITR sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located between two intron sequences. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located within an intron sequence. As a non-limiting example, the viral genome comprises two stuffing sequences, and the first stuffing sequence is located at the 3' end of the 5'ITR sequence, and the second stuffing sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located at the 5' end of the promoter sequence, and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 5' end of the 5' ITR sequence.

[0299] In one embodiment, the viral genome is a self-complementary (sc) viral genome and comprises one or more stuffing sequences so that the length of the viral genome is about 2.3 kb. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 3' end of the 5'ITR sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 5' end of the promoter sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located at the 5' end of the 3'ITR sequence. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located between two intron sequences. As a non-limiting example, the viral genome comprises at least one stuffing sequence, and the stuffing sequence is located within an intron sequence. As a non-limiting example, the viral genome comprises two stuffing sequences, and the first stuffing sequence is located at the 3' end of the 5'ITR sequence, and the second stuffing sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located at the 5' end of the promoter sequence, and the second stuffer sequence is located at the 3' end of the polyadenylation signal sequence. As a non-limiting example, the viral genome comprises two stuffer sequences, and the first stuffer sequence is located at the 3' end of the 5' ITR sequence, and the second stuffer sequence is located at the 5' end of the 5' ITR sequence.

[0300] In one embodiment, the viral genome may include one or more stuffing sequences between one of the multiple regions of the viral genome. In one embodiment, the stuffing region may be located before a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region. In one embodiment, the stuffing region may be located after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region. In one embodiment, the stuffing region may be located before and after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, an enhancer region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and / or an exon region.

[0301] In one embodiment, the viral genome may include one or more stuffing sequences that fork at least one region of the viral genome. The forked region of the viral genome may include 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 5' end of the stuffing sequence region. As a non-limiting example, the stuffing sequence may fork at least one region so that 10% of the region is located at the 5' end of the stuffing sequence and 90% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 20% of the region is located at the 5' end of the stuffing sequence and 80% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 30% of the region is located at the 5' end of the stuffing sequence and 70% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 40% of the region is located at the 5' end of the stuffing sequence and 60% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 50% of the region is located at the 5' end of the stuffing sequence and 50% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 60% of the region is located at the 5' end of the stuffing sequence and 40% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 70% of the region is located at the 5' end of the stuffing sequence and 30% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may fork at least one region so that 80% of the region is located at the 5' end of the stuffing sequence and 20% of the region is located at the 3' end of the stuffing sequence. As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 90% of the region is located at the 5' end of the stuffing sequence and 10% of the region is located at the 3' end of the stuffing sequence.

[0302] In one embodiment, the viral genome comprises a stuffer sequence after the 5' ITR.

[0303] In one embodiment, the viral genome comprises a stuffing sequence after the promoter region. In one embodiment, the viral genome comprises a stuffing sequence after the payload region. In one embodiment, the viral genome comprises a stuffing sequence after the intron region. In one embodiment, the viral genome comprises a stuffing sequence after the enhancer region. In one embodiment, the viral genome comprises a stuffing sequence after the polyadenylation signal sequence region. In one embodiment, the viral genome comprises a stuffing sequence after the MCS region. In one embodiment, the viral genome comprises a stuffing sequence after the exon region.

[0304] In one embodiment, the viral genome comprises a stuffing sequence before the promoter region. In one embodiment, the viral genome comprises a stuffing sequence before the payload region. In one embodiment, the viral genome comprises a stuffing sequence before the intron region. In one embodiment, the viral genome comprises a stuffing sequence before the enhancer region. In one embodiment, the viral genome comprises a stuffing sequence before the polyadenylation signal sequence region. In one embodiment, the viral genome comprises a stuffing sequence before the MCS region. In one embodiment, the viral genome comprises a stuffing sequence before the exon region.

[0305] In one embodiment, the viral genome comprises a stuffer sequence before the 3' ITR.

[0306] In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the promoter region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the payload region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the intron region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the enhancer region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the polyadenylation signal sequence region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the 5'ITR and the MCS region.

[0307] In one embodiment, a stuffer sequence may be located between two regions, such as but not limited to a 5' ITR and an exonic region.

[0308] In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the payload region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the intron region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the enhancer region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the polyadenylation signal sequence region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the MCS region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the exon region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to the promoter region and the 3'ITR.

[0309] In one embodiment, the stuffing sequence may be located between two regions, such as, but not limited to, a payload region and an intron region. In one embodiment, the stuffing sequence may be located between two regions, such as, but not limited to, a payload region and an enhancer region. In one embodiment, the stuffing sequence may be located between two regions, such as, but not limited to, a payload region and a polyadenylation signal sequence region. In one embodiment, the stuffing sequence may be located between two regions, such as, but not limited to, a payload region and an MCS region. In one embodiment, the stuffing sequence may be located between two regions, such as, but not limited to, a payload region and an exon region.

[0310] In one embodiment, a stuffer sequence may be located between two regions, such as but not limited to the payload region and the 3' ITR.

[0311] In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an intron region and an enhancer region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an intron region and a polyadenylation signal sequence region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an intron region and a MCS region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an intron region and an exon region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an intron region and a 3'ITR. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an enhancer region and a polyadenylation signal sequence region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an enhancer region and a MCS region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an enhancer region and an exon region. In one embodiment, the stuffing sequence may be located between two regions, such as but not limited to an enhancer region and a 3'ITR.

[0312] In one embodiment, the stuffer sequence may be located between two regions, such as but not limited to the polyadenylation signal sequence region and the MCS region. In one embodiment, the stuffer sequence may be located between two regions, such as but not limited to the polyadenylation signal sequence region and the exon region. In one embodiment, the stuffer sequence may be located between two regions, such as but not limited to the polyadenylation signal sequence region and the 3'ITR.

[0313] In one embodiment, the stuffer sequence may be located between two regions, such as but not limited to the MCS region and the exon region. In one embodiment, the stuffer sequence may be located between two regions, such as but not limited to the MCS region and the 3' ITR.

[0314] In one embodiment, a stuffer sequence may be located between two regions, such as but not limited to an exon region and a 3' ITR.

[0315] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the intron region and the enhancer region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the promoter region, and the second stuffer sequence may be located between the MCS region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5' ITR and the promoter region, and the second stuffer sequence may be located between the exon region and the 3' ITR.

[0316] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the payload region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the payload region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0317] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the intron region and the enhancer region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the intron region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the intron region, and the second stuffer sequence may be located between the MCS region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5' ITR and the intron region, and the second stuffer sequence may be located between the exon region and the 3' ITR.

[0318] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the intron region and the enhancer region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the enhancer region, and the second stuffer sequence may be located between the MCS region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5' ITR and the enhancer region, and the second stuffer sequence may be located between the exon region and the 3' ITR.

[0319] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0320] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the intron region and the enhancer region.In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the MCS region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the MCS region, and the second stuffer sequence may be located between the MCS region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5' ITR and the MCS region, and the second stuffer sequence may be located between the exon region and the 3' ITR.

[0321] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the payload region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the promoter region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the intron region and the enhancer region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the 5'ITR and the exon region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5'ITR and the exon region, and the second stuffer sequence may be located between the MCS region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the 5' ITR and the exon region, and the second stuffer sequence may be located between the exon region and the 3' ITR.

[0322] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the enhancer region and the MCS region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the payload region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0323] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the enhancer region and the exon region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the intron region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the intron region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the intron region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the intron region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the intron region, and the second stuffing sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the intron region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0324] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the enhancer region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the enhancer region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the enhancer region, and the second filler sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the enhancer region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the enhancer region, and the second filler sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the exon region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the enhancer region, and the second stuffing sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the enhancer region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0325] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the MCS region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0326] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the enhancer region and the exon region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the exon region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the exon region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the exon region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the exon region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the exon region, and the second stuffing sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the exon region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0327] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the MCS region, and the second filler sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the MCS region, and the second filler sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the MCS region, and the second filler sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the MCS region, and the second filler sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may include two filler sequences, the first filler sequence may be located between the promoter region and the MCS region, and the second filler sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the exon region.In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the MCS region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the MCS region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the MCS region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the MCS region, and the second stuffing sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffing sequences, the first stuffing sequence may be located between the promoter region and the MCS region, and the second stuffing sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the MCS region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0328] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the intron region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the payload region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the enhancer region and the MCS region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may contain two stuffer sequences, the first stuffer sequence may be located between the promoter region and the 3'ITR, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0329] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the intron region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0330] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the enhancer region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0331] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0332] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the MCS region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0333] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the exon region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0334] In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the intron region and the enhancer region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the intron region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the intron region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the intron region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the intron region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the enhancer region and the 3'ITR region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the exon region. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the polyadenylation signal sequence region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the MCS region and the exon region.In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the MCS region and the 3'ITR. In one embodiment, the viral genome may comprise two stuffer sequences, the first stuffer sequence may be located between the payload region and the 3'ITR region, and the second stuffer sequence may be located between the exon region and the 3'ITR.

[0335] In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the intron region and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the polyadenylation signal sequence region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the intron region and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the MCS region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the intron region and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the exon region. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the intron region and the enhancer region, and the second stuffing sequence may be located between the enhancer region and the 3'ITR. In one embodiment, the viral genome may include two stuffing sequences, the first stuffing sequence may be located between the intron region and the enhancer region, and the second stuffing sequence may be located between the polyadenylation signal se...

Claims

1. An AAV viral genome comprising a first inverted terminal repeat (ITR), optionally linked to a promoter comprising a nucleic acid encoding a regulatory polynucleotide encoding a sense strand sequence and an antisense strand sequence, and a second ITR; wherein the encoded antisense strand sequence comprises at least 19 consecutive nucleotides from a nucleotide sequence of any one of SEQ ID NOs: 918, 920, 914-917, 919, or 921-1013, and the encoded sense strand sequence comprises at least 19 consecutive nucleotides from a nucleotide sequence of any one of SEQ ID NOs: 1079, 1082, 1044, 1014-1043, 1045-1078, 1080, 1081, or 1083-1160, and wherein the AAV viral genome further comprises one or more of the following: i) a first ITR sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 1380 or 1381-1383, ii) a promoter comprising the nucleotide sequence of any one of SEQ ID NOs: 1410, 1411-1414, or iii) a second ITR sequence comprising the nucleotide sequence of any one of SEQ ID NO: 1382, 1380, 1381 or 1383.

2. The AAV viral genome according to claim 1, comprising in sequence: A: i) a 5'ITR sequence region, wherein the 5'ITR sequence region comprises the nucleotide sequence of SEQ ID NO: 1380 or 1381; ii) an enhancer sequence region, wherein the enhancer sequence region comprises the nucleotide sequence of SEQ ID NO: 1408 or 1409; iii) a promoter, wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 1410-1414; iv) a first intron sequence region, wherein the first intron sequence region comprises the nucleotide sequence of any one of SEQ ID NOs: 1417-1419; v) a regulatory polynucleotide sequence region, wherein the regulatory polynucleotide sequence region comprises the nucleotide sequence of any one of SEQ ID NOs: 1262, 1347, 1250, 1183-1249, 1251-1261, 1263-1279 and 1346; vi) a polyadenylation (polyA) signal sequence region, wherein the polyA signal sequence region comprises the nucleotide sequence of any one of SEQ ID NOs: 1420-1423; and vii) a 3'ITR sequence region, wherein the 3'ITR sequence region comprises the nucleotide sequence of SEQ ID NO: 1382 or 1383; or B: i) a 5'ITR sequence region comprising the nucleotide sequence of SEQ ID NO: 1380; ii) a first multiple cloning site (MCS) sequence region comprising a nucleotide sequence of 1384; iii) an enhancer sequence region comprising the nucleotide sequence of SEQ ID NO: 1408; iv) a promoter sequence region comprising the nucleotide sequence of SEQ ID NO: 1410; v) an intron sequence region comprising the nucleotide sequence of SEQ ID NO: 1417; vi) a regulatory polynucleotide sequence region, wherein the regulatory polynucleotide sequence region comprises the nucleotide sequence of any one of SEQ ID NOs: 1262, 1347 or 1250; vii) a second MCS sequence region comprising a nucleotide sequence of TCGAG; viii) a polyA signal sequence region comprising the nucleotide sequence of SEQ ID NO: 1420; and ix) a 3'ITR sequence region comprising the nucleotide sequence of SEQ ID NO:1382.

3. An AAV particle comprising the AAV viral genome and capsid of claim 1 or 2, wherein the capsid comprises an AAV1 capsid or a variant thereof, an AAV5 capsid or a variant thereof, or an AAV9 capsid or a variant thereof.

4. A vector comprising a nucleic acid containing the AAV viral genome of claim 1 or 2.

5. A cell comprising the AAV viral genome of claim 1 or 2 or the AAV particle of claim 3, optionally wherein the cell is a mammalian cell, a medium spiny neuron, a cortical neuron or an astrocyte.

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

7. Use of the AAV particle of claim 3 in the preparation of a medicament for inhibiting the expression of the HTT gene in a cell, optionally wherein the cell is: i) mammalian cells; ii) CNS cells; iii) neurons; iv) medium spiny neurons or cortical neurons; v) astrocytes; and / or vi) the cell is in a subject, optionally wherein the subject has Huntington's disease (HD) or has been diagnosed with Huntington's disease.

8. Use of the AAV particle of claim 3 in the preparation of a medicament for treating Huntington's disease (HD) in a subject.

9. The use according to claim 8, wherein the expression of HTT gene, mRNA and / or protein in CNS cells and / or CNS regions is inhibited, optionally wherein: i) CNS cells include neurons, medium spiny neurons, astrocytes or a combination thereof; ii) the CNS region is a forebrain region, a midbrain region, a putamen region, a striatum region, a cortical region, a motor cortical region, a somatosensory cortical region, a temporal cortical region, or a combination thereof; and / or iii) The CNS region is the putamen region.

10. The use according to claim 8 or 9, wherein the medicament is formulated for intravenous administration.

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