Adeno-associated virus variants

Abstract

The present disclosure relates to AAV variants, such as AAV2 variants. The AAV variants according to some embodiments disclosed herein include a VP1 variant having the amino acid sequence of any one selected from SEQ ID NOs: 02-31.

Description

Adeno-associated virus variant

[0001] The present disclosure relates to variants of adeno-associated virus and their use. Some embodiments of the present disclosure provide an AAV2 variant comprising a capsid variant.

[0002]

[0003] Adeno-associated virus (AAV) is a small, single-stranded DNA virus belonging to the family Parvoviridae. AAV is classified as a non-enveloped virus. AAV contains a capsid containing three viral proteins (e.g., VP1, VP2, and VP3) and single-stranded DNA, with the single-stranded DNA packaged within the capsid. Due to the broad tissue tropism and low pathogenicity of wild-type AAV, recombinant AAV vectors (rAAV vectors) have established themselves as the primary means for therapeutic gene delivery. To date, the FDA has approved numerous AAV therapies. Among these approved AAV therapies, Luxturna TM It is a therapeutic agent for treating RPE65-related retinal dystrophy using AAV serotype 2 (AAV2). Despite this success, the development of new AAV variants with high delivery rates to retinal cells remains important to expand clinical applications, and there is high demand in the market. Literature related to AAV variants and their development includes international patent application number PCT / US2020 / 059294.

[0004]

[0005] The development of novel AAV variants is important, and market demand is also high. The technical problem of the present disclosure is to provide novel AAV variants. Furthermore, the technical problem of the present disclosure is to provide AAV variants having excellent delivery efficacy to target sites.

[0006]

[0007] The present disclosure provides a novel AAV variant. An AAV variant according to some embodiments of the present disclosure comprises an AAV capsid variant comprising VP1 (or a VP1 variant) having an amino acid sequence of any one of SEQ ID NOs 02 to 31. The AAV variant of the present disclosure may have excellent delivery efficacy to a target site.

[0008]

[0009] The AAV variant of the present disclosure may have excellent delivery efficacy to a target site. For example, the AAV variant of the present disclosure may have excellent delivery efficacy to retinal tissue or RPE tissue.

[0010]

[0011] Figure 01 shows the results of the correlation analysis of three biological replicates of the AAV library.

[0012] Figure 02 shows the results of a diversity performance analysis between a random library and a machine learning-based library.

[0013] Figure 03 shows the analysis results of the RPE delivery function of wild-type AAV2 and 28 superior AAV variants selected through mouse subretinal injection experiments.

[0014] Figure 04 shows the analysis results of the RPE delivery function of wild-type AAV2 and five superior AAV variants selected through mouse intravitreal injection experiments.

[0015]

[0016] Some embodiments of the present disclosure provide an AAV variant.

[0017] In some embodiments, the AAV variant may include the following:

[0018] AAV capsid variant, wherein the AAV capsid variant comprises a VP1 variant having the amino acid sequences of SEQ ID NOs 02 to 31.

[0019] In some embodiments, the AAV capsid variant may be any one selected from the following:

[0020] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 02, a VP2 variant having the amino acid sequence of SEQ ID NO. 33, and a VP3 variant having the amino acid sequence of SEQ ID NO. 64;

[0021] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 03, a VP2 variant having the amino acid sequence of SEQ ID NO. 34, and a VP3 variant having the amino acid sequence of SEQ ID NO. 65;

[0022] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 04, a VP2 variant having the amino acid sequence of SEQ ID NO. 35, and a VP3 variant having the amino acid sequence of SEQ ID NO. 66;

[0023] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 05, a VP2 variant having the amino acid sequence of SEQ ID NO. 36, and a VP3 variant having the amino acid sequence of SEQ ID NO. 67;

[0024] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 06, a VP2 variant having the amino acid sequence of SEQ ID NO. 37, and a VP3 variant having the amino acid sequence of SEQ ID NO. 68;

[0025] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 07, a VP2 variant having the amino acid sequence of SEQ ID NO. 38, and a VP3 variant having the amino acid sequence of SEQ ID NO. 69;

[0026] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 08, a VP2 variant having the amino acid sequence of SEQ ID NO. 39, and a VP3 variant having the amino acid sequence of SEQ ID NO. 70;

[0027] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 09, a VP2 variant having the amino acid sequence of SEQ ID NO. 40, and a VP3 variant having the amino acid sequence of SEQ ID NO. 71;

[0028] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 10, a VP2 variant having the amino acid sequence of SEQ ID NO. 41, and a VP3 variant having the amino acid sequence of SEQ ID NO. 72;

[0029] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 11, a VP2 variant having the amino acid sequence of SEQ ID NO. 42, and a VP3 variant having the amino acid sequence of SEQ ID NO. 73;

[0030] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 12, a VP2 variant having the amino acid sequence of SEQ ID NO. 43, and a VP3 variant having the amino acid sequence of SEQ ID NO. 74;

[0031] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 13, a VP2 variant having the amino acid sequence of SEQ ID NO. 44, and a VP3 variant having the amino acid sequence of SEQ ID NO. 75;

[0032] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 14, a VP2 variant having the amino acid sequence of SEQ ID NO. 45, and a VP3 variant having the amino acid sequence of SEQ ID NO. 76;

[0033] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 15, a VP2 variant having the amino acid sequence of SEQ ID NO. 46, and a VP3 variant having the amino acid sequence of SEQ ID NO. 77;

[0034] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 16, a VP2 variant having the amino acid sequence of SEQ ID NO. 47, and a VP3 variant having the amino acid sequence of SEQ ID NO. 78;

[0035] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 17, a VP2 variant having the amino acid sequence of SEQ ID NO. 48, and a VP3 variant having the amino acid sequence of SEQ ID NO. 79;

[0036] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 18, a VP2 variant having the amino acid sequence of SEQ ID NO. 49, and a VP3 variant having the amino acid sequence of SEQ ID NO. 80;

[0037] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 19, a VP2 variant having the amino acid sequence of SEQ ID NO. 50, and a VP3 variant having the amino acid sequence of SEQ ID NO. 81;

[0038] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 20, a VP2 variant having the amino acid sequence of SEQ ID NO. 51, and a VP3 variant having the amino acid sequence of SEQ ID NO. 82;

[0039] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 21, a VP2 variant having the amino acid sequence of SEQ ID NO. 52, and a VP3 variant having the amino acid sequence of SEQ ID NO. 83;

[0040] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 22, a VP2 variant having the amino acid sequence of SEQ ID NO. 53, and a VP3 variant having the amino acid sequence of SEQ ID NO. 84;

[0041] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 23, a VP2 variant having the amino acid sequence of SEQ ID NO. 54, and a VP3 variant having the amino acid sequence of SEQ ID NO. 85;

[0042] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 24, a VP2 variant having the amino acid sequence of SEQ ID NO. 55, and a VP3 variant having the amino acid sequence of SEQ ID NO. 86;

[0043] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 25, a VP2 variant having the amino acid sequence of SEQ ID NO. 56, and a VP3 variant having the amino acid sequence of SEQ ID NO. 87;

[0044] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 26, a VP2 variant having the amino acid sequence of SEQ ID NO. 57, and a VP3 variant having the amino acid sequence of SEQ ID NO. 88;

[0045] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 27, a VP2 variant having the amino acid sequence of SEQ ID NO. 58, and a VP3 variant having the amino acid sequence of SEQ ID NO. 89;

[0046] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 28, a VP2 variant having the amino acid sequence of SEQ ID NO. 59, and a VP3 variant having the amino acid sequence of SEQ ID NO. 90;

[0047] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 29, a VP2 variant having the amino acid sequence of SEQ ID NO. 60, and a VP3 variant having the amino acid sequence of SEQ ID NO. 91;

[0048] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 30, a VP2 variant having the amino acid sequence of SEQ ID NO. 61, and a VP3 variant having the amino acid sequence of SEQ ID NO. 92; and

[0049] An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 31, a VP2 variant having the amino acid sequence of SEQ ID NO. 62, and a VP3 variant having the amino acid sequence of SEQ ID NO. 93.

[0050] In some embodiments, the AAV variant further comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid encoding a product of interest, and wherein the nucleic acid molecule may be packaged in the AAV capsid variant.

[0051] In some embodiments, the nucleic acid molecule further comprises a promoter, wherein the promoter may be operably linked to the nucleic acid encoding the product of interest. In some embodiments, the nucleic acid molecule further comprises two ITRs, wherein the promoter and the nucleic acid encoding the product of interest may be located between the two ITRs.

[0052] In some embodiments, the nucleic acid molecule may be single-stranded DNA.

[0053]

[0054] The AAV variant of the present disclosure may have enhanced RPE tissue or RPE cell delivery capabilities.

[0055] In some embodiments, the AAV capsid variant of the AAV variant may include a VP1 variant having an amino acid sequence selected from SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31, wherein the AAV variant may have enhanced RPE tissue or RPE cell delivery ability when administered to a subject via intravitreal injection.

[0056] In some embodiments, the AAV capsid variant of the AAV variant may include a VP1 variant having an amino acid sequence selected from SEQ ID NOs 02 to 29, and the AAV variant may have enhanced RPE tissue or RPE cell delivery ability when administered to a subject via subretinal injection.

[0057]

[0058] In some embodiments, an AAV variant comprising the following is provided:

[0059] AAV capsid variant,

[0060] At this time, the above AAV capsid variant comprises a VP1 variant having an amino acid sequence selected from SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31, and

[0061] At this time, the above AAV variant is an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject via intravitreal injection.

[0062] In some embodiments, an AAV variant comprising the following is provided:

[0063] AAV capsid variant,

[0064] At this time, the above AAV capsid variant comprises a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29, and

[0065] At this time, the above AAV variant is an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject via subretinal injection.

[0066]

[0067] Hereinafter, the content of the invention disclosed in this disclosure will be explained in more detail through embodiments and examples. The invention disclosed by this disclosure may be implemented in various ways and is not limited to the specific embodiments described herein.

[0068] A person skilled in the art to which the invention disclosed herein pertains would be able to conceive of various modifications and other embodiments of the content of the invention disclosed herein. Accordingly, the content of the invention disclosed herein is not limited to the specific embodiments or examples described herein, and modifications and other embodiments thereof should be understood to be included within the invention disclosed herein.

[0069]

[0070] Definition of Terms

[0071] Unless otherwise stated, all technical and scientific terms used in this disclosure have the meanings generally understood by those skilled in the art related to this disclosure. All publications, patents, and other references mentioned in this disclosure are incorporated by reference in their entirety.

[0072] The term “about” as used in the present disclosure means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0073] As used in this disclosure, the term "treatment" means alleviating, reducing, and / or inhibiting the progression of a disease, illness, and / or symptom, etc., and encompasses all such meanings. For example, treatment may mean a partial or complete recovery of a disease, illness, and / or symptomatic state to a normal state. For example, said treatment may mean a change in state in which symptoms are improved or beneficially altered by the compositions of this disclosure, or any act that causes symptoms to be improved or altered. In some cases, treatment may be interpreted to include a preventive mode.

[0074] As used in this disclosure, the terms "protein," "peptide," and "polypeptide" are used interchangeably and encompass naturally occurring proteins, etc. and unnaturally occurring proteins, etc. Unnaturally occurring proteins, etc. may be, for example, recombinant proteins, etc. or synthetic proteins, etc. For example, a protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. For example, a protein, peptide, or polypeptide may be naturally occurring. For example, a protein, peptide, or polypeptide may be recombinant or synthetic, or a combination of recombinant or synthetic and naturally occurring.

[0075] As used in this disclosure, the term “nucleic acid” encompasses naturally occurring nucleic acids and unnaturally occurring nucleic acids. Unnaturally occurring nucleic acids may be, for example, recombinant nucleic acids or synthetic nucleic acids. For example, nucleic acids may be naturally occurring, recombinant or synthetic, or any combination thereof. For example, nucleic acids may be naturally occurring. For example, nucleic acids may be recombinant or synthetic, or a combination of recombinant or synthetic and naturally occurring. In this disclosure, the term nucleic acid may be used to refer to the molecule itself or a part of a molecule. For example, nucleic acid may refer to a DNA molecule or a part or region thereof. For example, nucleic acid may refer to an RNA molecule or a part or region thereof. For example, nucleic acid may refer to a DNA-RNA hybrid molecule or a part or region thereof. Nucleic acid may be DNA, RNA, or a DNA-RNA hybrid, but is not limited thereto.

[0076] Unless otherwise described, nucleic acids or nucleic acid sequences disclosed in this disclosure (e.g., DNA sequences, RNA sequences, DNA / RNA hybrid sequences) should be understood as being described in the 5' to 3' direction.

[0077] Unless otherwise stated, amino acid sequences described in this disclosure are written in the direction from the N-terminal to the C-terminal using either a single-letter or three-letter amino acid notation. For example, when denoted as RNVP, it signifies a peptide in which arginine, asparagine, valine, and proline are linked in sequence from the N-terminal to the C-terminal. As another example, when denoted as Thr-Leu-Lys, it signifies a peptide in which threonine, leucine, and lysine are linked in sequence from the N-terminal to the C-terminal. For amino acids that cannot be represented by the single-letter notation, other letters are used for notation and further supplementary explanations are provided.

[0078] In this disclosure, the term "expression" as used in relation to nucleic acids, genes, products, or proteins is used to encompass the process of producing products, such as RNA and / or proteins, using information encoded in nucleic acids or genes.

[0079]

[0080] Known structure of AAV and wild-type AAV2 capsid

[0081] This section provides a description of AAV and AAV2 based on facts known in the art regarding AAV and AAV2 to aid the understanding of those skilled in the art, and the content of this section does not limit the disclosure, including aspects, embodiments, and examples of the present disclosure.

[0082] Adeno-associated virus (AAV) is known as a small virus belonging to the family Parvoviridae. Wild-type AAV contains approximately 4.7 to 4.8 kilobases of single-stranded DNA, which is packaged within the capsid. The AAV capsid is icosahedral in shape and contains a total of 60 units of VP1 capsid protein (also referred to as VP1 or VP1 subunit), VP2 capsid protein (also referred to as VP2 or VP2 subunit), and VP3 capsid protein (also referred to as VP3 or VP3 subunit). In other words, the AAV capsid is known to contain a total of 60 molecules of VPs. For example, the AAV capsid is known to be composed of 60 molecules of VPs. AAVs are currently widely used in the field of gene therapy due to their advantages over other viral vectors, such as low toxicity and the inclusion of over 150 accessible naturally occurring genotypes and serotypes. Since these serotypes differ in tropism, they can target most tissue and cell types for gene delivery. Recombinant AAVs (rAAVs) packaging a gene of interest (GOI) have been successfully studied in clinical trials for the treatment of various genetic diseases. Notably, Luxturna, Zolgensma, and Glybera have been approved by the FDA and / or EMA. In the application of these AAVs, the GOI intended to treat genetic diseases replaces the natural AAV genome for delivery to tissues and / or cells. Furthermore, rAAVs are one of the widely used research tools for transgene expression in tissue cultures and preclinical animal models. (Worner, T.P., Bennett, A., Habka, S., Snijder, J., Friese, O., Powers, T., ... & Heck, A.J. (2021).[Adeno-associated virus capsid assembly is divergent and stochastic. Nature communications, 12(1), 1642.] See, the full contents of which are incorporated herein by reference).

[0083] As previously mentioned, the capsid of AAV is known to contain a total of 60 molecules of VPs, including VP1, VP2, and VP3. In natural AAV, VP1, VP2, and VP3 are encoded by the cap-open reading frame (ORF). VPs are generated through alternative splicing of the mRNA and the use of alternate translation start codons. The sequence of VP3 is shared among all VPs, and this shared sequence is referred to as the VP3 common region. VP2 is known to be about 50aa to 70aa longer than VP3, and the region on the N-terminal side of this VP2 is referred to as the VP1 / VP2 common region. VP1 is known to be approximately 130 to 140 aa longer than VP2, and this region is referred to as the VP1 unique region (VP1u). Furthermore, the ratio of VP1, VP2, and VP3 among VPs in natural AAVs is known to be approximately 1:1:10. (See reference [Worner, TP, Bennett, A., Habka, S., Snijder, J., Friese, O., Powers, T., ... & Heck, AJ (2021). Adeno-associated virus capsid assembly is divergent and stochastic. Nature communications, 12(1), 1642.]).

[0084] Meanwhile, there are 11 well-known serotypes of AAV (AAV1 to AAV11). Among these, AAV2 (AAV serotype 2) has historically been the most well-characterized and is generally known to have adequately safe and efficient packaging and delivery capabilities.

[0085] Wild-type AAV2 and the capsid proteins of wild-type AAV2 capsid are well known in the art. Wild-type AAV2 VP1 (wild-type VP1) has the amino acid sequence of SEQ ID NO. 01. Wild-type AAV2 VP2 (wild-type VP2) has the amino acid sequence of SEQ ID NO. 32. Wild-type AAV2 VP3 (wild-type VP3) has the amino acid sequence of SEQ ID NO. 63.

[0086] In wild-type AAV2, the VP3 common region (533aa) is located at positions 203 through 735 in the amino acid sequence of wild-type VP1. That is, the amino acid sequence from amino acid residue 203 to amino acid residue 735 in the amino acid sequence of wild-type VP1 (i.e., the amino acid sequence at positions 203-735) corresponds to the VP3 common region.

[0087] In the amino acid sequence of wild-type VP2, the VP3 common region is located at positions 66 through 598. That is, the amino acid sequence located between amino acid residue 66 and amino acid residue 598 in the amino acid sequence of wild-type VP2 (i.e., the amino acid sequence at positions 66-598) corresponds to the VP3 common region.

[0088] For example, a wild-type AAV2 capsid contains VP1 of SEQ ID NO. 01. For example, a wild-type AAV2 capsid contains VP1 of SEQ ID NO. 01, VP2 of SEQ ID NO. 32, and VP3 of SEQ ID NO. 63.

[0089]

[0090] AAV (Adeno-associated virus) variant

[0091] Overview of AAV Variants

[0092] Some embodiments of the present disclosure provide an AAV variant.

[0093] The AAV variants of the present disclosure may be referred to as recombinant AAV variants, AAV variant vectors, or recombinant AAV variant vectors, and are not otherwise limited. For example, the AAV variants of the present disclosure may be referred to as engineered AAV or engineered AAV variants.

[0094] In some embodiments, the AAV variant of the present disclosure comprises an AAV capsid variant. In some embodiments, the AAV capsid variant may comprise a VP1 variant (e.g., also referred to as a VP1 capsid protein variant). In some embodiments, the AAV capsid variant may be referred to as a capsid, an AAV capsid, a capsid variant, an AAV2 capsid variant, etc. The names used to refer to the AAV capsid variant are not limited thereto and may be referred to by terms commonly used in the art.

[0095] In some embodiments, the AAV variant may further include a nucleic acid molecule. For example, the AAV variant may further include said nucleic acid molecule by packaging said nucleic acid molecule. In this case, said nucleic acid molecule may be packaged within the AAV capsid. For example, the nucleic acid molecule may be located within the AAV capsid, where the AAV capsid surrounds said nucleic acid molecule. In some embodiments, the nucleic acid molecule may be a recombinant nucleic acid molecule. In some embodiments, the nucleic acid molecule or the recombinant nucleic acid molecule may include the gene of interest.

[0096] In some embodiments, the AAV variant may be referred to as an AAV2 variant, an AAV2 variant vector, a recombinant AAV2 variant, or a recombinant AAV2 variant vector, but is not otherwise limited.

[0097]

[0098] AAV capsid variant

[0099] Some embodiments of the present disclosure provide an AAV capsid variant.

[0100] In some embodiments, as described above, the AAV variant of the present disclosure may include an AAV capsid variant (e.g., an AAV2 capsid variant).

[0101] In some embodiments, the AAV capsid variant may include a VP1 variant. In some embodiments, the AAV capsid variant may include a VP2 variant. In some embodiments, the AAV capsid variant may include a VP3 variant. In some embodiments, the AAV capsid variant may further include a VP2 variant in addition to the VP1 variant. In some embodiments, the AAV capsid variant may further include a VP3 variant in addition to the VP1 variant. In some embodiments, the AAV capsid variant may include a VP1 variant, a VP2 variant, and a VP3 variant.

[0102] In some embodiments, the AAV capsid variant comprises a total of 60 VPs, wherein the total 60 VPs may include VP1 variants, VP2 variants, and VP3 variants. In some embodiments, the ratio of VP1 variants, VP2 variants, and VP3 variants in the AAV capsid variant may be about 1:1:10, but is not limited thereto.

[0103]

[0104] VP1 variant

[0105] Some embodiments of the present disclosure provide VP1 variants. The VP1 variants of the present disclosure may be referred to as VP1 capsid protein variants or AAV (or AAV2) VP1 capsid protein variants, but are not limited thereto.

[0106] In some embodiments, the VP1 variant may comprise any one amino acid sequence selected from SEQ ID NOs 02 to 31. In some embodiments, the VP1 variant may be represented by any one amino acid sequence selected from SEQ ID NOs 02 to 31. Accordingly, in some embodiments, the AAV capsid variant may comprise any one amino acid sequence selected from SEQ ID NOs 02 to 31.

[0107] In some embodiments, the VP1 variant may have the amino acid sequence of any one of the following selected sequence numbers:

[0108] Sequence No. 02; Sequence No. 03; Sequence No. 04; Sequence No. 05; Sequence No. 06; Sequence No. 07; Sequence No. 08; Sequence No. 09; Sequence No. 10; Sequence No. 11; Sequence No. 12; Sequence No. 13; Sequence No. 14; Sequence No. 15; Sequence No. 16; Sequence No. 17; Sequence No. 18; Sequence No. 19; Sequence No. 20; Sequence No. 21; Sequence No. 22; Sequence No. 23; Sequence No. 24; Sequence No. 25; Sequence No. 26; Sequence No. 27; Sequence No. 28; Sequence No. 29; Sequence No. 30; and Sequence No. 31.

[0109]

[0110] VP2 variant

[0111] Some embodiments of the present disclosure provide VP2 variants. The VP2 variants of the present disclosure may be referred to as VP2 capsid protein variants or AAV (or AAV2) VP2 capsid protein variants, but are not limited thereto.

[0112] In some embodiments, the VP2 variant may comprise any one amino acid sequence selected from SEQ ID NOs 33 to 62. In some embodiments, the VP2 variant may be represented by any one amino acid sequence selected from SEQ ID NOs 33 to 62. Accordingly, in some embodiments, the AAV capsid variant may comprise any one amino acid sequence selected from SEQ ID NOs 33 to 62.

[0113] In some embodiments, the VP2 variant may have the amino acid sequence of any one of the following selected sequence numbers:

[0114] Sequence No. 33; Sequence No. 34; Sequence No. 35; Sequence No. 36; Sequence No. 37; Sequence No. 38; Sequence No. 39; Sequence No. 40; Sequence No. 41; Sequence No. 42; Sequence No. 43; Sequence No. 44; Sequence No. 45; Sequence No. 46; Sequence No. 47; Sequence No. 48; Sequence No. 49; Sequence No. 50; Sequence No. 51; Sequence No. 52; Sequence No. 53; Sequence No. 54; Sequence No. 55; Sequence No. 56; Sequence No. 57; Sequence No. 58; Sequence No. 59; Sequence No. 60; Sequence No. 61; and Sequence No. 62.

[0115]

[0116] VP3 variant

[0117] Some embodiments of the present disclosure provide a VP3 variant (e.g., a variant of the VP3 capsid protein). The VP3 variant of the present disclosure may be referred to as a VP3 capsid protein variant or an AAV (or AAV2) VP3 capsid protein variant, but is not limited thereto.

[0118] In some embodiments, the VP3 variant may comprise any one amino acid sequence selected from SEQ ID NOs 64 to 93. In some embodiments, the VP3 variant may be represented by any one amino acid sequence selected from SEQ ID NOs 64 to 92. Accordingly, in some embodiments, the AAV capsid variant may comprise any one amino acid sequence selected from SEQ ID NOs 64 to 93.

[0119] In some embodiments, the VP3 variant may have the amino acid sequence of any one of the following selected sequence numbers:

[0120] Sequence No. 64; Sequence No. 65; Sequence No. 66; Sequence No. 67; Sequence No. 68; Sequence No. 69; Sequence No. 70; Sequence No. 71; Sequence No. 72; Sequence No. 73; Sequence No. 74; Sequence No. 75; Sequence No. 76; Sequence No. 77; Sequence No. 78; Sequence No. 79; Sequence No. 80; Sequence No. 81; Sequence No. 82; Sequence No. 83; Sequence No. 84; Sequence No. 85; Sequence No. 86; Sequence No. 87; Sequence No. 88; Sequence No. 89; Sequence No. 90; Sequence No. 91; Sequence No. 92; and Sequence No. 93.

[0121]

[0122] Characteristics of VP1, VP2, and VP3 variants

[0123] The VP1 variant of the present disclosure may be a variant of wild-type AAV2 VP1 (wild-type VP1). Accordingly, the VP1 variant of the present disclosure may be expressed as being derived from the VP1 of wild-type AAV2. In some embodiments, the VP1 variant of the present disclosure may be obtained by introducing one or more mutations (e.g., amino acid substitution mutations) into the amino acid sequence at positions 561–588 (the amino acid sequence of 28aa) of the VP1 of wild-type AAV2 (wild-type VP1). The amino acid sequence at positions 561–588 of wild-type VP1 is located within the VP3 common region of wild-type AAV2.

[0124] Wild-type VP1 is represented by SEQ ID NO. 01, and the amino acid sequence at position 561-588 of wild-type VP1 is represented by SEQ ID NO. 94. The VP1 variant of the present disclosure may include any one amino acid sequence selected from SEQ ID NOs 95 to 124, which is different from the amino acid sequence at position 561-588 of wild-type VP1 (SEQ ID NO. 01) (SEQ ID NO. 94). In this case, any one amino acid sequence selected from SEQ ID NOs 95 to 124 may be located at position 561-588 in the VP1 variant.

[0125] In some embodiments, a VP1 variant may be one in which the amino acid sequence at positions 561-588 of wild-type VP1 (SEQ NO. 94) is manipulated or altered to any one of SEQ NOs 95 to 124. Such a VP1 variant may be obtained by substituting or altering the amino acid sequence at positions 561-588 of wild-type VP1 (SEQ NO. 01) to any one of SEQ NOs 95 to 124, or by substituting or altering one or more amino acid residues included in the amino acid sequence at positions 561-588 to other amino acid residues, and is not otherwise limited. Any one of SEQ NOs 95 to 124 may be referred to as a mutant sequence or a manipulated sequence. In some embodiments, the VP1 variant comprises any one of the engineered sequences selected from SEQ ID NOs 95 to 124, wherein the mutant sequence may be located at positions 561-588 relative to the amino acid sequence of wild-type VP1 (SEQ ID NO 01).

[0126]

[0127] The VP2 variant of the present disclosure may be a variant of wild-type AAV VP2 (wild-type VP2). Accordingly, the VP2 variant of the present disclosure may be expressed as being derived from wild-type AAV2 VP2. In some embodiments, the VP2 variant of the present disclosure may be obtained by introducing one or more mutations (e.g., amino acid substitution mutations) into the amino acid sequence at positions 424–451 of wild-type AAV2 VP2.

[0128] Wild-type VP2 is represented by SEQ ID NO. 32, and the amino acid sequence at position 424-451 of wild-type VP2 is represented by SEQ ID NO. 94. The VP2 variant of the present disclosure may include any one amino acid sequence selected from SEQ ID NOs 95 to 124, which is different from the amino acid sequence at position 424-451 of wild-type VP2 (SEQ ID NO. 32) (SEQ ID NO. 94). In this case, any one amino acid sequence selected from SEQ ID NOs 95 to 124 may be located at position 424-451 of the VP2 variant.

[0129] In some embodiments, the VP2 variant may be a wild-type VP2 variant in which the amino acid sequence at positions 424-451 (SEQN 94) is manipulated or altered to any one of SEQN 95 to 124. Such a VP2 variant may be obtained by substituting or altering the amino acid sequence at positions 424-451 of the wild-type VP2 to any one of SEQN 95 to 124, or by substituting or altering one or more amino acid residues included in the amino acid sequence at positions 424-451 to other amino acid residues, and is not otherwise limited. Any one of SEQN 95 to 124 may be referred to as a mutant sequence or a manipulated sequence. In some embodiments, the VP2 variant comprises any one of SEQN 95 to 124 of the manipulated sequence, wherein the manipulated sequence may be located at positions 424-451 relative to the amino acid sequence of the wild-type VP2.

[0130]

[0131] The VP3 variant of the present disclosure may be a variant of wild-type AAV2 VP3 (wild-type VP3). Accordingly, the VP3 variant of the present disclosure may be expressed as being derived from wild-type AAV2 VP3. In some embodiments, the VP3 variant of the present disclosure may be obtained by introducing one or more mutations (e.g., amino acid substitution mutations) into the amino acid sequence at positions 359–386 of wild-type AAV2 VP3.

[0132] Wild-type VP3 is represented by SEQ ID NO. 63, and the amino acid sequence at positions 359-386 of wild-type VP3 is represented by SEQ ID NO. 94. The VP3 variant of the present disclosure may include any one amino acid sequence selected from SEQ ID NOs 95 to 124, which is different from the amino acid sequence at positions 359-386 of wild-type VP3 (SEQ ID NO. 63) (SEQ ID NO. 94). In this case, any one amino acid sequence selected from SEQ ID NOs 95 to 124 may be located at positions 359-386 in the VP3 variant.

[0133] In some embodiments, the VP3 variant may be a wild-type VP3 variant in which the amino acid sequence at positions 359-386 (SEQN 94) is manipulated or altered to any one of SEQN 95 to 124. Such a VP3 variant may be obtained by substituting or altering the amino acid sequence at positions 359-386 of the wild-type VP3 to any one of SEQN 95 to 124, or by substituting or altering one or more amino acid residues included in the amino acid sequence at positions 359-386 to other amino acid residues, and is not otherwise limited. Any one of SEQN 95 to 124 may be referred to as a mutant sequence or a manipulated sequence. In some embodiments, the VP3 variant comprises any one of SEQN 95 to 124 of the manipulated sequence, wherein the manipulated sequence may be located at positions 359-386 relative to the amino acid sequence of the wild-type VP3.

[0134]

[0135] Examples of AAV capsid variants

[0136] In some embodiments, the AAV capsid variant comprises a VP1 variant, wherein the VP1 variant may have any one amino acid sequence selected from SEQ ID NOs 02 to 31.

[0137] In some embodiments, the AAV capsid variant may be any one selected from AAV capsid variants 1 to 30 disclosed in this disclosure. In some embodiments, AAV capsid variant 1 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 02. In some embodiments, AAV capsid variant 2 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 03. In some embodiments, AAV capsid variant 3 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 04. In some embodiments, AAV capsid variant 4 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 05. In some embodiments, AAV capsid variant 5 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 06. In some embodiments, AAV capsid variant 6 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 07. In some embodiments, AAV capsid variant 7 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 08. In some embodiments, AAV capsid variant 8 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 09. In some embodiments, AAV capsid variant 9 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 10. In some embodiments, AAV capsid variant 10 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 11. In some embodiments, AAV capsid variant 11 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 12. In some embodiments, AAV capsid variant 12 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 13. In some embodiments, AAV capsid variant 13 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 14.In some embodiments, AAV capsid variant 14 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 15. In some embodiments, AAV capsid variant 15 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 16. In some embodiments, AAV capsid variant 16 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 17. In some embodiments, AAV capsid variant 17 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 18. In some embodiments, AAV capsid variant 18 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 19. In some embodiments, AAV capsid variant 19 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 20. In some embodiments, AAV capsid variant 20 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 21. In some embodiments, AAV capsid variant 21 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 22. In some embodiments, AAV capsid variant 22 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 23. In some embodiments, AAV capsid variant 23 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 24. In some embodiments, AAV capsid variant 24 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 25. In some embodiments, AAV capsid variant 25 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 26. In some embodiments, AAV capsid variant 26 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 27. In some embodiments, AAV capsid variant 27 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 28.In some embodiments, AAV capsid variant 28 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 29. In some embodiments, AAV capsid variant 29 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 30. In some embodiments, AAV capsid variant 30 may include a VP1 variant having the amino acid sequence of SEQ ID NO. 31.

[0138] In some embodiments, a capsid variant (e.g., AAV capsid variant 1) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 02 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 33 and a VP3 variant having the amino acid sequence of SEQ ID NO. 64.

[0139] In some embodiments, a capsid variant (e.g., AAV capsid variant 2) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 03 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 34 and a VP3 variant having the amino acid sequence of SEQ ID NO. 65.

[0140] In some embodiments, a capsid variant (e.g., AAV capsid variant 3) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 04 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 35 and a VP3 variant having the amino acid sequence of SEQ ID NO. 66.

[0141] In some embodiments, a capsid variant (e.g., AAV capsid variant 4) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 05 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 36 and a VP3 variant having the amino acid sequence of SEQ ID NO. 67.

[0142] In some embodiments, a capsid variant (e.g., AAV capsid variant 5) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 06 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 37 and a VP3 variant having the amino acid sequence of SEQ ID NO. 68.

[0143] In some embodiments, a capsid variant (e.g., AAV capsid variant 6) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 07 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 38 and a VP3 variant having the amino acid sequence of SEQ ID NO. 69.

[0144] In some embodiments, a capsid variant (e.g., AAV capsid variant 7) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 08 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 39 and a VP3 variant having the amino acid sequence of SEQ ID NO. 70.

[0145] In some embodiments, a capsid variant (e.g., AAV capsid variant 8) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 09 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 40 and a VP3 variant having the amino acid sequence of SEQ ID NO. 71.

[0146] In some embodiments, a capsid variant (e.g., AAV capsid variant 9) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 10 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 41 and a VP3 variant having the amino acid sequence of SEQ ID NO. 72.

[0147] In some embodiments, a capsid variant (e.g., AAV capsid variant 10) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 11 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 42 and a VP3 variant having the amino acid sequence of SEQ ID NO. 73.

[0148] In some embodiments, a capsid variant (e.g., AAV capsid variant 11) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 12 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 43 and a VP3 variant having the amino acid sequence of SEQ ID NO. 74.

[0149] In some embodiments, a capsid variant (e.g., AAV capsid variant 12) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 13 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 44 and a VP3 variant having the amino acid sequence of SEQ ID NO. 75.

[0150] In some embodiments, a capsid variant (e.g., AAV capsid variant 13) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 14 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 45 and a VP3 variant having the amino acid sequence of SEQ ID NO. 76.

[0151] In some embodiments, a capsid variant (e.g., AAV capsid variant 14) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 15 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 46 and a VP3 variant having the amino acid sequence of SEQ ID NO. 77.

[0152] In some embodiments, a capsid variant (e.g., AAV capsid variant 15) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 16 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 47 and a VP3 variant having the amino acid sequence of SEQ ID NO. 78.

[0153] In some embodiments, a capsid variant (e.g., AAV capsid variant 16) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 17 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 48 and a VP3 variant having the amino acid sequence of SEQ ID NO. 79.

[0154] In some embodiments, a capsid variant (e.g., AAV capsid variant 17) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 18 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 49 and a VP3 variant having the amino acid sequence of SEQ ID NO. 80.

[0155] In some embodiments, a capsid variant (e.g., AAV capsid variant 18) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 19 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 50 and a VP3 variant having the amino acid sequence of SEQ ID NO. 81.

[0156] In some embodiments, a capsid variant (e.g., AAV capsid variant 19) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 20 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 51 and a VP3 variant having the amino acid sequence of SEQ ID NO. 82.

[0157] In some embodiments, a capsid variant (e.g., AAV capsid variant 20) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 21 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 52 and a VP3 variant having the amino acid sequence of SEQ ID NO. 83.

[0158] In some embodiments, a capsid variant (e.g., AAV capsid variant 21) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 22 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 53 and a VP3 variant having the amino acid sequence of SEQ ID NO. 84.

[0159] In some embodiments, a capsid variant (e.g., AAV capsid variant 22) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 23 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 54 and a VP3 variant having the amino acid sequence of SEQ ID NO. 85.

[0160] In some embodiments, a capsid variant (e.g., AAV capsid variant 23) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 24 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 55 and a VP3 variant having the amino acid sequence of SEQ ID NO. 86.

[0161] In some embodiments, a capsid variant (e.g., AAV capsid variant 24) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 25 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 56 and a VP3 variant having the amino acid sequence of SEQ ID NO. 87.

[0162] In some embodiments, a capsid variant (e.g., AAV capsid variant 25) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 26 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 57 and a VP3 variant having the amino acid sequence of SEQ ID NO. 88.

[0163] In some embodiments, a capsid variant (e.g., AAV capsid variant 26) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 27 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 58 and a VP3 variant having the amino acid sequence of SEQ ID NO. 89.

[0164] In some embodiments, a capsid variant (e.g., AAV capsid variant 27) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 28 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 59 and a VP3 variant having the amino acid sequence of SEQ ID NO. 90.

[0165] In some embodiments, a capsid variant (e.g., AAV capsid variant 28) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 29 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 60 and a VP3 variant having the amino acid sequence of SEQ ID NO. 91.

[0166] In some embodiments, a capsid variant (e.g., AAV capsid variant 29) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 30 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 61 and a VP3 variant having the amino acid sequence of SEQ ID NO. 92.

[0167] In some embodiments, a capsid variant (e.g., AAV capsid variant 30) comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 31 may further comprise either or both of a VP2 variant having the amino acid sequence of SEQ ID NO. 62 and a VP3 variant having the amino acid sequence of SEQ ID NO. 93.

[0168] In some embodiments, the AAV capsid variant may be any one selected from the following:

[0169] An AAV capsid variant (e.g., AAV capsid variant 1) comprising VP1 having the amino acid sequence of SEQ ID NO. 02 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 33 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 64 (e.g., VP3 variant);

[0170] An AAV capsid variant (e.g., AAV capsid variant 2) comprising VP1 having the amino acid sequence of SEQ ID NO. 03 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 34 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 65 (e.g., VP3 variant);

[0171] An AAV capsid variant (e.g., AAV capsid variant 3) comprising VP1 having the amino acid sequence of SEQ ID NO. 04 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 35 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 66 (e.g., VP3 variant);

[0172] An AAV capsid variant (e.g., AAV capsid variant 4) comprising VP1 having the amino acid sequence of SEQ ID NO. 05 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 36 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 67 (e.g., VP3 variant);

[0173] An AAV capsid variant (e.g., AAV capsid variant 5) comprising VP1 having the amino acid sequence of SEQ ID NO. 06 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 37 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 68 (e.g., VP3 variant);

[0174] An AAV capsid variant (e.g., AAV capsid variant 6) comprising VP1 having the amino acid sequence of SEQ ID NO. 07 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 38 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 69 (e.g., VP3 variant);

[0175] An AAV capsid variant (e.g., AAV capsid variant 7) comprising VP1 having the amino acid sequence of SEQ ID NO. 08 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 39 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 70 (e.g., VP3 variant);

[0176] An AAV capsid variant (e.g., AAV capsid variant 8) comprising VP1 having the amino acid sequence of SEQ ID NO. 09 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 40 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 71 (e.g., VP3 variant);

[0177] An AAV capsid variant (e.g., AAV capsid variant 9) comprising VP1 having the amino acid sequence of SEQ ID NO. 10 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 41 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 72 (e.g., VP3 variant);

[0178] An AAV capsid variant (e.g., AAV capsid variant 10) comprising VP1 having the amino acid sequence of SEQ ID NO. 11 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 42 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 73 (e.g., VP3 variant);

[0179] An AAV capsid variant (e.g., AAV capsid variant 11) comprising VP1 having the amino acid sequence of SEQ ID NO. 12 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 43 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 74 (e.g., VP3 variant);

[0180] An AAV capsid variant (e.g., AAV capsid variant 12) comprising VP1 having the amino acid sequence of SEQ ID NO. 13 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 44 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 75 (e.g., VP3 variant);

[0181] An AAV capsid variant (e.g., AAV capsid variant 13) comprising VP1 having the amino acid sequence of SEQ ID NO. 14 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 45 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 76 (e.g., VP3 variant);

[0182] An AAV capsid variant (e.g., AAV capsid variant 14) comprising VP1 having the amino acid sequence of SEQ ID NO. 15 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 46 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 77 (e.g., VP3 variant);

[0183] An AAV capsid variant (e.g., AAV capsid variant 15) comprising VP1 having the amino acid sequence of SEQ ID NO. 16 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 47 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 78 (e.g., VP3 variant);

[0184] An AAV capsid variant (e.g., AAV capsid variant 16) comprising VP1 having the amino acid sequence of SEQ ID NO. 17 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 48 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 79 (e.g., VP3 variant);

[0185] An AAV capsid variant (e.g., AAV capsid variant 17) comprising VP1 having the amino acid sequence of SEQ ID NO. 18 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 49 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 80 (e.g., VP3 variant);

[0186] An AAV capsid variant (e.g., AAV capsid variant 18) comprising VP1 having the amino acid sequence of SEQ ID NO. 19 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 50 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 81 (e.g., VP3 variant);

[0187] An AAV capsid variant (e.g., AAV capsid variant 19) comprising VP1 having the amino acid sequence of SEQ ID NO. 20 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 51 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 82 (e.g., VP3 variant);

[0188] An AAV capsid variant (e.g., AAV capsid variant 20) comprising VP1 having the amino acid sequence of SEQ ID NO. 21 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 52 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 83 (e.g., VP3 variant);

[0189] An AAV capsid variant (e.g., AAV capsid variant 21) comprising VP1 having the amino acid sequence of SEQ ID NO. 22 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 53 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 84 (e.g., VP3 variant);

[0190] An AAV capsid variant (e.g., AAV capsid variant 22) comprising VP1 having the amino acid sequence of SEQ ID NO. 23 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 54 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 85 (e.g., VP3 variant);

[0191] An AAV capsid variant (e.g., AAV capsid variant 23) comprising VP1 having the amino acid sequence of SEQ ID NO. 24 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 55 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 86 (e.g., VP3 variant);

[0192] An AAV capsid variant (e.g., AAV capsid variant 24) comprising VP1 having the amino acid sequence of SEQ ID NO. 25 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 56 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 87 (e.g., VP3 variant);

[0193] An AAV capsid variant (e.g., AAV capsid variant 25) comprising VP1 having the amino acid sequence of SEQ ID NO. 26 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 57 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 88 (e.g., VP3 variant);

[0194] An AAV capsid variant (e.g., AAV capsid variant 26) comprising VP1 having the amino acid sequence of SEQ ID NO. 27 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 58 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 89 (e.g., VP3 variant);

[0195] An AAV capsid variant (e.g., AAV capsid variant 27) comprising VP1 having the amino acid sequence of SEQ ID NO. 28 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 59 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 90 (e.g., VP3 variant);

[0196] An AAV capsid variant (e.g., AAV capsid variant 28) comprising VP1 having the amino acid sequence of SEQ ID NO. 29 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 60 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 91 (e.g., VP3 variant);

[0197] An AAV capsid variant (e.g., AAV capsid variant 29) comprising VP1 having the amino acid sequence of SEQ ID NO. 30 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 61 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 92 (e.g., VP3 variant); and

[0198] An AAV capsid variant (e.g., AAV capsid variant 30) comprising VP1 having the amino acid sequence of SEQ ID NO. 31 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 62 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 93 (e.g., VP3 variant).

[0199]

[0200] Capsid surface modification

[0201] In some embodiments, capsid surface modifications may be added to AAV capsid variants. For example, AAV capsid variants may be modified to include probes such as chemical functional groups, peptides, and aptamers on their surface, but are not limited thereto. Such capsid surface modifications may be performed to further enhance the delivery activity of the AAV variant to a target and / or to increase the stability of the AAV variant, but are not otherwise limited.

[0202]

[0203] Factors that AAV variants may additionally include

[0204] Overview of additional factors AAV variants may include

[0205] In some embodiments, the AAV variant of the present disclosure (e.g., AAV2 variant) may further include one or more additional elements in addition to the aforementioned AAV capsid variant.

[0206] In some embodiments, the AAV variant may further comprise a nucleic acid molecule. The nucleic acid molecule may be referred to as a recombinant nucleic acid molecule, a nucleic acid construct, a recombinant nucleic acid construct, an expression cassette, a gene expression cassette, a gene expression cassette of interest, an AAV genome, a recombinant AAV genome, or an engineered AAV genome, but is not limited thereto, and may be referred to by any term used in the art. In this case, the nucleic acid molecule may be located inside the AAV capsid variant, surrounded by the AAV capsid variant.

[0207] In some embodiments, the nucleic acid molecule of such AAV variant may contain a gene or nucleic acid (e.g., a gene of interest) encoding a product of interest (e.g., a protein of interest). Depending on the design of the AAV variant and the nucleic acid molecule, the product of interest may be generated or expressed at a target site (e.g., a target cell) within the target to which the AAV variant is delivered.

[0208] In some embodiments, the AAV variant may further comprise one or more selected from a nucleic acid encoding the product of interest, a promoter, and an ITR. For example, the nucleic acid encoding the product of interest, the promoter, and the ITR may be located within a packaged nucleic acid molecule of the AAV variant.

[0209]

[0210] Nucleic acid encoding product of interest

[0211] In some embodiments, the AAV variant may comprise a nucleic acid encoding one or more products of interest. The nucleic acid encoding the products of interest may comprise, for example, a gene of interest. The nucleic acid encoding the products of interest may comprise, for example, a nucleic acid encoding a protein of interest.

[0212] In some embodiments, the nucleic acid encoding the product of interest may comprise an Open Reading Frame (ORF) sequence of the product of interest (e.g., protein of interest) or the gene encoding it, or a sequence complementary thereto. In some embodiments, the nucleic acid encoding the product of interest may comprise a coding sequence (CDS) of the product of interest or the gene encoding the product of interest, or a sequence complementary thereto. In some embodiments, the nucleic acid encoding the product of interest may comprise a cDNA sequence of the product of interest or the gene encoding the product of interest, or a sequence complementary thereto.

[0213] In some embodiments, the nucleic acid encoding the product of interest may be a therapeutic gene.

[0214] In some embodiments, the product of interest may be a substance for treating a disease or condition of interest.

[0215] In some embodiments, the product of interest may be a protein of interest. For example, the protein of interest may be a protein to be expressed in a subject for therapeutic purposes. In some embodiments, the protein of interest may be a specific protein (e.g., a therapeutic protein) to treat a disease or condition of interest.

[0216] In some embodiments, the disease or condition of interest is, for example, achromatopsia, Usher syndrome, diabetic retinopathy (proliferative diabetic retinopathy), diabetic macular edema, geographic atrophy, retinopathy of premature, Leber's hereditary optic neuropathy, central retinal vein occlusion, proliferative vitreoretinopathy, retinal cone dystrophy, Behcet's disease, Bietti's crystallin dystrophy, Best vitelliform macular dystrophy, Best disease, choroideremia, It may be retinal detachment, uveitis, glaucoma, retinitis pigmentosa (RP), macular degeneration [e.g., age-related macular degeneration (AMD)], RPE65-associated retinal dystrophy, Stargardt disease (SD), retinoschisis, or Leber congenital amaurosis (LCA).

[0217] The disease or condition of interest may be, for example, an eye disease or condition, a retinal disease or condition, or an RPE-related disease or condition.

[0218] In some embodiments, the protein of interest may be a protein for treating an eye disease or condition. In some embodiments, the eye disease or condition may be a disease or condition caused by a problem in the eye tissue. For example, the eye disease or condition may be caused by a functional abnormality of the eye tissue, a structural abnormality of the eye tissue, or an abnormality of at least some of the cells constituting the eye tissue.

[0219] In some embodiments, the protein of interest may be a protein for treating a retinal disease or disease. In some embodiments, the retinal disease or disease may be a disease or disease caused by a problem in retinal tissue or retinal cells. For example, the retinal disease or disease may be caused by a dysfunction of retinal tissue, a structural abnormality of retinal tissue, or an abnormality of at least some of the cells constituting the retinal tissue. In some embodiments, the protein of interest may be a protein for treating a disease or disease related to retinal cells [e.g., neurons of the retina, photoreceptors, bipolar cells, horizontal cells, amacrine, ganglion cells, etc.] or a disease or disease related to retinal pigment epithelium (RPE) cells.

[0220] In some embodiments, the protein of interest may be a protein for treating a retinal pigment epithelial-related disease or condition. In some embodiments, the retinal pigment epithelial-related disease or condition may be a disease or condition caused by a problem in the retinal pigment epithelial tissue or retinal pigment epithelial cells. For example, the retinal pigment epithelial-related disease or condition may be caused by dysfunction of the retinal pigment epithelial tissue, structural abnormalities of the retinal pigment epithelial tissue, or abnormalities of at least some of the cells constituting the retinal pigment epithelial tissue. In some embodiments, the retinal pigment epithelial-related disease may be a disease caused by RPE dysfunction. In some embodiments, the retinal pigment epithelium-associated disease may be retinitis pigmentosa (RP), macular degeneration [e.g., age-related macular degeneration (AMD)], RPE65-associated retinal dystrophy, Stargardt disease (SD), retinoschisis, or Leber congenital amaurosis type 2 (LCA2).

[0221] In some embodiments, the disease or condition may be Leber congenital amaurosis (LCA). To date, 18 genes associated with LCA have been identified, and mutations in these genes are generally known to cause LCA. In some embodiments, LCA may be LCA1, LCA2, LCA3, LCA4, LCA5, LCA6, LCA7, LCA8, LCA9, LCA10, LCA11, LCA12, LCA13, LCA14, LCA15, LCA16, LCA17, or LCA18. Among these, LCA2 is known to be caused by a functional defect in RPE 65.

[0222] In some embodiments, retinal pigment epithelial-related disease may be an hereditary retinal disease (IRD).

[0223] In some embodiments, the disease or illness may be a disease or illness of a mammal. In some embodiments, the disease or illness may be a disease or illness of a human. In some embodiments, the disease or illness may be a disease or illness of a mouse, dog, horse, cat, rat, pig, rabbit, sheep, monkey, chimpanzee, or cattle.

[0224] In some embodiments, the proteins of interest are ABCA4 (e.g., the protein encoded in the ABCA4 gene), PDE6B (e.g., the protein encoded in the PDE6B gene), RPGR (e.g., the protein encoded in the RPGR gene), ACHM3A (e.g., the protein encoded in the ACHM3A gene), ACHM3B (e.g., the protein encoded in the ACHM3B gene), RLBP1 (e.g., the protein encoded in the RLBP1 gene), CEP290 (e.g., the protein encoded in the CEP290 gene), CLN3 (e.g., the protein encoded in the CLN3 gene), CRX (e.g., the protein encoded in the CRX gene), GUCA1A (e.g., the protein encoded in the GUCA1A gene), GUCY2D (e.g., the protein encoded in the GUCY2D gene), NMNAT1 (e.g., the protein encoded in the NMNAT1 gene), TULP1 (e.g., the protein encoded in the TULP1 gene), LRAT (e.g., Protein encoded in the LRAT gene), MERTK (e.g., protein encoded in the MERTK gene), RDH12 (e.g., protein encoded in the RDH12 gene), AIPL1 (e.g., protein encoded in the AIPL1 gene), IMPDH1 (e.g., protein encoded in the IMPDH1 gene), RPGRIP1 (e.g., protein encoded in the RPGRIP1 gene), CRB1 (e.g., protein encoded in the CRB1 gene), NR2E3 (e.g., protein encoded in the NR2E3 gene), RPGR (e.g., protein encoded in the RPGR gene), EYS (e.g., protein encoded in the EYS gene), ADIPOR1 (e.g., protein encoded in the ADIPOR1 gene), ARL3 (e.g., protein encoded in the ARL3 gene), CA4 (e.g., protein encoded in the CA4 gene), HK1 (e.g., protein encoded in the HK1 gene), KLHL7 (e.g., protein encoded in the KLHL7 gene),NRL (e.g., protein encoded in the NRL gene), PRPF3 (e.g., protein encoded in the PRPF3 gene), PRPF4 (e.g., protein encoded in the PRPF4 gene), PRPF6 (e.g., protein encoded in the PRPF6 gene), PRPF8 (e.g., protein encoded in the PRPF8 gene), PRPF31 (e.g., protein encoded in the PRPF31 gene), ROM1 (e.g., protein encoded in the ROM1 gene), RP1 (e.g., protein encoded in the RP1 gene), RP9 (e.g., protein encoded in the RP9 gene), SAG (e.g., protein encoded in the SAG gene), SNRNP200 (e.g., protein encoded in the SNRNP200 gene), SPP2 (e.g., protein encoded in the SPP2 gene), TOPORS (e.g., protein encoded in the TOPORS gene), PRPH2 (e.g., protein encoded in the PRPH2 gene), SEMA4A (e.g., protein encoded in the SEMA4A gene Protein), RIMS1 (e.g., protein encoded in the RIMS1 gene), PIPNM3 (e.g., protein encoded in the PIPNM3 gene), UNC119 (e.g., protein encoded in the UNC119 gene), PROM1 (e.g., protein encoded in the PROM1 gene), FSCN2 (e.g., protein encoded in the FSCN2 gene), GUCA1B (e.g., protein encoded in the GUCA1B gene), C1QTNF5 (e.g., protein encoded in the C1QTNF5 gene), CTNNA (e.g., protein encoded in the CTNNA gene), EFEMP1 (e.g., protein encoded in the EFEMP1 gene), ELOVL4 (e.g., protein encoded in the ELOVL4 gene), HMCN1 (e.g., protein encoded in the HMCN1 gene), IMPG1 (e.g., protein encoded in the IMPG1 gene), OTX2 (e.g., protein encoded in the OTX2 gene), PRDM13 (e.g. Listen,It may be the protein encoded in the PRDM13 gene), RP1L1 (e.g., the protein encoded in the RP1L1 gene), TIMP3 (e.g., the protein encoded in the TIMP3 gene), RPE 65 (e.g., the protein encoded in the RPE 65 gene), Bestrophin, Rhodopsin, Lebercilin, Usherin, retinoschisin, Norrin, complement factor H, complement factor I, complement factor P, CD59, anti-VEGF antibodies (e.g., sevacizumab, ranibizumab, bebacizumab, brolucizumab), anti-C5 antibodies (e.g., tesidolumab, ravulizumab, eculizumab), anti-C3 antibodies, anti-TNF antibodies (e.g., adalimumab, infliximab, golimumab), or anti-ANGPTL3 antibodies (e.g., evinacumab).

[0225] For example, the protein of interest may be a protein for genetic manipulation of a genetic engineering system. The protein for genetic manipulation may include a Cas protein of a CRISPR / Cas system. The Cas protein may be, for example, Cas9 or a variant thereof such as spCas9 or cjCas9, Cas12 or a variant thereof such as Cpf1 (Cas12a), C2c1 (Cas12b), or C2c3 (Cas12c), Cas13 or a variant thereof such as C2c2 (Cas13a), C2c4 (Cas13b), C2c7 (Cas13c), or Cas13d, but is not limited thereto. In some embodiments, the protein for genetic manipulation may include a base editor of a base editing system or a prime editor of a prime editing system or a transcription factor regulator, but is not limited thereto. Genetic engineering systems and genetically engineered proteins are described in detail in the literature [Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Computational and structural biotechnology journal, 18, 2401-2415.], the entire contents of which are incorporated herein by reference.

[0226] For example, the protein of interest may be a fluorescent protein such as GFP (green fluorescent protein) or luciferase. These fluorescent proteins may be used for screening, but are not limited thereto.

[0227] In some embodiments, one or more nuclear localization sequences (NLS) may be added to the product of interest or the protein of interest. For example, the genetically engineered protein may be a genetically engineered protein with one or more nuclear localization sequences added, but is not limited thereto.

[0228] In some embodiments, the product of interest may be an RNA of interest. For example, the product of interest may be a guide RNA or pegRNA of a gene engineering system (see reference [Xu, Y., & Li, Z. (2020). CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy. Computational and structural biotechnology journal, 18, 2401-2415.]). In some embodiments, the product of interest may be a siRNA or a miRNA.

[0229] In some embodiments, the product of interest may be the rep protein of AAV. In some embodiments, the gene of interest may comprise a nucleic acid encoding the rep protein (replication protein) of AAV. Rep proteins are associated with the replication, packaging, and viral assembly of the AAV genome and are well known in the art (see [Li, C., & Samulski, RJ (2020). Engineering adeno-associated virus vectors for gene therapy. Nature Reviews Genetics, 21(4), 255-272.]; and [Matsuzaka, Y., & Yashiro, R. (2024). Therapeutic Application and Structural Features of Adeno-Associated Virus Vector. Current Issues in Molecular Biology, 46(8), 8464-8498.]). The nucleic acid encoding the rep protein may encode one or more of the rep78 protein, rep68 protein, rep52 protein, and rep40, but is not limited thereto. For example, nucleic acids encoding rep proteins may encode rep78 proteins, rep68 proteins, rep52 proteins, and rep40. Rep proteins may include, but are not limited to, one or more of rep78 proteins, rep68 proteins, rep52 proteins, and rep40 proteins.

[0230] In some embodiments, the product of interest may be the cap of the AAV (e.g., the cap of an AAV variant of the present disclosure). In some embodiments, the gene of interest may comprise a nucleic acid encoding the cap. In some embodiments, the nucleic acid encoding the cap may comprise, but is not limited to, a nucleic acid encoding one or more of the VP1 variant, VP2 variant, and VP3 variant of the AAV variant of the present disclosure. In some embodiments, the nucleic acid encoding the cap may comprise a nucleic acid encoding the VP1 variant of the present disclosure.

[0231] In some embodiments, the product of interest, the protein of interest, or the gene of interest may be of human origin, of non-human mammalian origin such as mouse, dog, horse, cat, rat, pig, rabbit, sheep, monkey, chimpanzee, or cow, of archaea, bacteria, or viruses, but is not limited thereto.

[0232]

[0233] Additional elements for regulating the expression of products of interest, etc.

[0234] In some embodiments, the AAV variant may include, in addition to the nucleic acid encoding the product of interest (e.g., the gene of interest), one or more additional elements for regulating the expression of the product of interest. In some embodiments, one or more additional elements may each be independently selected from a promoter, an enhancer, a polyadenylation signal, a Kozak consensus sequence, an inverted terminal repeat (ITR), a long terminal repeat (LTR), a terminator, an origin of replication, a multicloning site (MCS), an internal ribosome entry site (IRES), poly A, and 2A self-cleaving peptides. In some embodiments, the AAV variant may further comprise, in addition to the nucleic acid encoding the product of interest, one or more selected from a promoter, an enhancer, a polyadenylation signal, a Kozak common sequence, an ITR, an LTR, a terminator, a replication origin, a multiple cloning site, an internal ribosome inflow site, poly A, and a 2A self-cleaving peptide.

[0235] In some embodiments, the additional element may be a control element (or regulatory element). In this disclosure, a control element may be used as a term referring to an element, region, or DNA sequence that is operably connected to a nucleic acid element (e.g., a nucleic acid encoding a product of interest) to enable or assist in the transcription and / or expression of the nucleic acid element at a specific location (e.g., within a cell). Examples of control elements in prokaryotic cells may include a promoter, an operator sequence, and a ribosome binding site, etc. Examples of control elements in eukaryotic cells may include a promoter, a polyadenylation sequence or signal [e.g., a bovine growth hormone polyadenylation signal (bGh poly A signal)], and an enhancer, etc. The control elements of this disclosure may include, but are not limited to, a promoter, an enhancer, an intron splicing signal, a polyadenylation sequence, and an ITR, etc. A promoter is a DNA sequence located adjacent to a nucleic acid (e.g., a gene of interest) encoding a product of interest, and is known to initiate or promote the transcription and / or expression of the nucleic acid encoding the product of interest. A promoter is generally operably linked to the nucleic acid encoding the product of interest. For example, a promoter may be linked upstream or to the 5' side of the nucleic acid encoding the product of interest. An enhancer is known as a component designed to enhance the activity of a promoter or increase the potential for transcription or expression of a specific gene, and is known to be located independently of the promoter's position.

[0236] In some embodiments, the AAV variant may further include a promoter capable of initiating the expression of a nucleic acid encoding the product of interest. The promoter may be present within the aforementioned nucleic acid molecule.

[0237] In some embodiments, the promoter is an SV40 early promoter, an LTR (mouse mammary tumor virus long terminal repeat) promoter, an Ad MLP (adenovirus major late) promoter, an HSV (herpes simplex virus) promoter, a CMV (cytomegalovirus) promoter, an RSV (rous sarcoma virus) promoter, a Ubc promoter, an EF1a promoter, an MNDU3 promoter, a U6 promoter, an H1 promoter, a 7SK promoter, a CBA promoter, a PGK promoter, an NES promoter, a GFAP promoter, a CaMKII promoter, an NSE promoter, a SYN1 promoter, or a CAG promoter, an RPE 65 gene promoter, a human retinal binding protein;It may be a CRALBP gene promoter, a murine 11-cis-retinol dehydrogenase (RDH) gene promoter, a rhodopsin promoter, a rhodopsin kinase promoter, a tissue inhibitor of metalloproteinase 3 (Timp3) promoter, a photoreceptor retinol binding protein promoter and vitelliform macular dystrophy 2 promoter, an interphotoreceptor retinoid-binding protein (IRBP) promoter, an opsin promoter, a retinitis pigmentosa gene promoter, a human cone arrestin promoter, a cellular retinaldehyde-binding protein promoter, a vitelliform macular dystrophy promoter, a neural retina-specific leucine zipper protein promoter, a glial fibrillary acidic protein promoter, a retinoschisin promoter, or a homeodomain protein promoter.;

[0238] In some embodiments, the promoter may be a target site, tissue, or cell-specific promoter of the subject. For example, the promoter may be an ocular tissue-specific promoter. In some embodiments, ocular tissue-specific promoters include the RPE 65 gene promoter, the human retinal binding protein (CRALBP) gene promoter, the murine 11-cis-retinol dehydrogenase (RDH) gene promoter, the rhodopsin promoter, the rhodopsin kinase promoter, the tissue inhibitor of metalloproteinase 3 (Timp3) promoter, the photoreceptor retinol binding protein promoter and vitelliform macular dystrophy 2 promoter, the interphotoreceptor retinoid-binding protein (IRBP) promoter, the opsin promoter, the retinitis pigmentosa gene promoter, the human cone arrestin promoter, the cellular retinaldehyde-binding protein promoter, the vitelliform macular dystrophy promoter, the neural retina-specific leucine zipper protein promoter, the glial fibrillary acidic protein promoter, and the retinoschisin The promoter may be a homeodomain protein promoter, but is not limited thereto. In some embodiments, the promoter may be an RPE-specific promoter.

[0239] In some embodiments, the promoter may be of human, mouse, dog, horse, cat, rat, pig, rabbit, sheep, monkey, chimpanzee, cow, horse, bacterial, or viral origin, but is not limited thereto. The promoter may be used by artificially synthesizing or manipulating it.

[0240] In some embodiments, one or more additional elements may be operablely linked to the coding region. For example, a promoter and / or enhancer may be operablely linked to a nucleic acid encoding the product of interest (e.g., a gene of interest). The operablely linked element or nucleic acid may be linked to the nucleic acid encoding the product of interest either sequentially or through a linker (e.g., an oligonucleotide linker, etc.). The promoter and / or enhancer may be operablely linked to the nucleic acid encoding the product of interest and may influence the transcription of the nucleic acid encoding the product of interest. In some embodiments, the promoter may be linked to the 5' end or the 3' end of the nucleic acid encoding the product of interest. Preferably, the promoter may be linked toward the 5' end of the nucleic acid encoding the product of interest, whereby the promoter may be operablely linked to the nucleic acid encoding the product of interest. The term "operably linked" means that the described components are in a relationship in which they can function in an intended manner. For example, if a promoter facilitates the transcription of a coding sequence, then this promoter is operably linked to the coding sequence.

[0241] In some embodiments, the AAV variant may further include one or more inverted terminal repeat (ITR) sequences. One or more ITRs may be located within the aforementioned nucleic acid molecule. In some embodiments, the AAV variant may include one or two ITRs. For example, the nucleic acid molecule may include two ITRs. In the nucleic acid molecule of the recombinant AAV, the two ITRs are each located at opposite ends of the nucleic acid molecule, and between the two ITRs, a region containing a promoter and a nucleic acid encoding the product of interest may be located. One or more ITRs may each be selected independently. For example, if two ITRs are present, the two ITRs may be identical or different from each other. In some embodiments, if two ITRs are present, the two ITRs may be symmetrical to each other. In some embodiments, the length of each ITR may independently be 50 nt to 300 nt, but is not limited thereto. For example, the length of the ITR may be 145 nt. In some embodiments, the ITR may be of wild-type AAV or a variant thereof. In some embodiments, the ITR may be an ITR derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, or a known AAV, or a variant thereof, but is not limited thereto.

[0242]

[0243] Forms of nucleic acid molecules

[0244] As described above, the AAV variant may further include a nucleic acid molecule (e.g., a recombinant nucleic acid molecule). The nucleic acid molecule may be packaged within the AAV capsid variant. For example, the nucleic acid molecule may be located inside the AAV capsid variant. In some embodiments, the nucleic acid molecule may be single-stranded DNA, single-stranded RNA, or a single-stranded DNA-RNA hybrid. Preferably, the nucleic acid molecule may be single-stranded DNA.

[0245] In some embodiments, the nucleic acid molecule may comprise a nucleic acid encoding a product of interest. In some embodiments, the nucleic acid molecule may comprise a nucleic acid encoding a product of interest and a promoter operably linked to the nucleic acid encoding a product of interest. In some embodiments, the nucleic acid molecule may comprise a nucleic acid encoding a product of interest, a promoter operably linked to the nucleic acid encoding a product of interest, and one or more ITRs (e.g., one or two ITRs). For example, the nucleic acid molecule may comprise two ITRs, and the nucleic acid encoding a product of interest and / or the promoter may be located between the ITRs within the two nucleic acid molecules. For example, within the nucleic acid molecule, the ITR (e.g., 5' ITR), the promoter, the nucleic acid encoding a product of interest, and the ITR (e.g., 3' ITR) may be located in the 5' to 3' direction in the order described. In some embodiments, the nucleic acid molecule may further comprise, but is not limited to, a poly-A tail, etc. For example, within a nucleic acid molecule, an ITR (e.g., 5' ITR), a promoter, a nucleic acid encoding the product of interest, poly A, and an ITR (e.g., 3' ITR) may be located in the described order. For example, a nucleic acid molecule may be represented by any one of the following structures:

[0246] 5' end-[ITR]-[promoter]-[nucleic acid encoding the product of interest]-[ITR]-3' end; and

[0247] 5' end-[ITR]-[promoter]-[nucleic acid encoding product of interest]-[poly A]-[ITR]-3' end.

[0248] In some embodiments, the nucleic acid molecule may include one or more nucleic acids encoding a product of interest. For example, the nucleic acid molecule may include a nucleic acid encoding a single product of interest. For example, the nucleic acid molecule may include nucleic acids encoding two or three products of interest. For example, the nucleic acid molecule may include a nucleic acid encoding a first product of interest and a nucleic acid encoding a second product of interest. In this case, the expression of the nucleic acid encoding the first product of interest and the nucleic acid encoding the second product of interest may be regulated by one promoter or by two promoters. For example, the nucleic acid encoding the first product of interest may be linked to the first promoter, and the nucleic acid encoding the second product of interest may be linked to the second promoter. For example, the nucleic acid molecule may further include two ITRs, and the nucleic acid encoding the first product of interest, the first promoter, the nucleic acid encoding the second product of interest, and the second promoter may be located between the two ITRs within the nucleic acid molecule.

[0249] In some embodiments, the length of the nucleic acid molecule may be 3.0 kb (kilobase) to 6.0 kb. For example, the length of the nucleic acid molecule may be 4.0 kb to 5.3 kb. For example, the length of the nucleic acid molecule may be 4.5 kb to 5.2 kb. In certain embodiments, the length of the nucleic acid molecule may be about 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5.0 kb, 5.1 kb, or 5.2 kb, but is not limited thereto.

[0250] In some embodiments, nucleic acid molecules may be codon-optimized. In some embodiments, one or more of the nucleic acid elements included in an AAV variant may be codon-optimized. For example, a nucleic acid encoding a product of interest may be codon-optimized. Codon optimization refers to modifying the codons that make up a nucleic acid sequence so that the codons are best suited for expression in a specific system (e.g., a specific species or group of species). For example, codon optimization for a nucleic acid sequence does not change the amino acid sequence of the encoded protein. For example, a nucleic acid sequence may be optimized for more efficient expression in mammalian cells. Various codon optimization methods are known in the art. For example, methods disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148 may be referenced for codon optimization, but are not limited thereto.

[0251]

[0252] Benefits of AAV Variants

[0253] In some embodiments, the AAV variants of the present disclosure may have the ability to deliver to a desired target site. For example, the AAV variants of the present disclosure may have an enhanced or superior delivery ability to a desired target site. Such enhanced or superior delivery ability may be caused by AAV capsid variants, or capsid protein variants such as VP1 variants, VP2 variants, or VP3 variants. Accordingly, it will be understood by those skilled in the art that the contents described in the advantages section of the AAV variants of the present disclosure may apply not only to the AAV variants of the present disclosure but also to AAV capsid variants, or capsid protein variants such as VP1 variants, VP2 variants, or VP3 variants.

[0254] In some embodiments, the AAV variant of the present disclosure may have a target site delivery capability. In some embodiments, the AAV variant of the present disclosure may accumulate at the target site. In some embodiments, the AAV variant of the present disclosure may have a target site tropism. In some embodiments, the AAV variant of the present disclosure may have a target site-specific delivery capability.

[0255] In some embodiments, the AAV variant of the present disclosure may have an enhanced (or superior) target site delivery ability compared to wild-type AAV (e.g., wild-type AAV2). In some embodiments, the AAV variant of the present disclosure may have an enhanced (or superior) target site transport ability. In some embodiments, the AAV variant of the present disclosure may have an enhanced (or superior) target site tropism. In some embodiments, the target site may be ocular tissue (or ocular cells), retinal tissue (or retinal cells), or RPE tissue (or RPE cells).

[0256] In some embodiments, the AAV variant of the present disclosure may have enhanced ocular tissue delivery capabilities. In some embodiments, the AAV variant of the present disclosure may have enhanced retinal tissue (or retinal cell) delivery capabilities. In some embodiments, the AAV variant of the present disclosure may have enhanced RPE tissue (or RPE cell) delivery capabilities. For example, the AAV variant may have enhanced or superior ocular tissue, retinal tissue, or RPE tissue delivery capabilities compared to wild-type AAV2.

[0257] In some embodiments, the AAV variants of the present disclosure may have enhanced delivery capabilities to ocular tissue, retinal tissue, or RPE tissue when administered to subjects (e.g., humans, mice, or rats). For example, the AAV variants may accumulate in ocular tissue, retinal tissue, or RPE tissue when administered to subjects. For example, the degree to which the AAV variants accumulate in ocular tissue, retinal tissue, or RPE tissue may be superior to that of wild-type AAV2.

[0258] In some embodiments, the AAV variants of the present disclosure may have enhanced delivery capabilities to retinal tissue or retinal cells when administered to a subject by intravitreal injection. For example, when the AAV variant is administered to a subject via intravitreal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better to retinal tissue or retinal cells. For example, when the AAV variant is administered to a subject via intravitreal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better or more to retinal tissue or retinal cells than when administered via wild-type AAV2.

[0259] In some embodiments, the AAV variant of the present disclosure may have enhanced delivery ability to RPE tissue or RPE cells when administered to a subject by intravitreal injection. For example, when the AAV variant of the present disclosure is administered to a subject by intravitreal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better to RPE tissue or RPE cells. For example, when the AAV variant is administered to a subject by intravitreal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better or more to RPE tissue or RPE cells than when administered via wild-type AAV2. In some embodiments, the AAV variant having enhanced delivery ability to RPE tissue or RPE cells when administered to a subject by intravitreal injection may include an AAV capsid variant comprising a VP1 variant having any one of the amino acid sequences of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31. For example, an AAV capsid variant of an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject by intravitreal injection may be any one of AAV capsid variant 9, AAV capsid variant 10, AAV capsid variant 14, AAV capsid variant 29, and AAV capsid variant 30.

[0260] In some embodiments, the AAV variant of the present disclosure may have enhanced delivery ability to retinal tissue or retinal cells when administered to a subject by subretinal injection. For example, when the AAV variant of the present disclosure is administered to a subject via subretinal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better to retinal tissue or retinal cells. For example, when the AAV variant is administered to a subject via subretinal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better or more to retinal tissue or retinal cells than when administered via wild-type AAV2.

[0261] In some embodiments, the AAV variant of the present disclosure may have enhanced delivery ability to RPE tissue or RPE cells when administered to a subject by subretinal injection. For example, when the AAV variant of the present disclosure is administered to a subject by subretinal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better to RPE tissue or RPE cells. For example, when the AAV variant is administered to a subject by subretinal injection, the nucleic acid encoding the product of interest within the AAV variant may be delivered better or more to RPE tissue or RPE cells than when administered via wild-type AAV2. In some embodiments, the AAV variant having enhanced delivery ability to RPE tissue or RPE cells when administered to a subject by subretinal injection may include an AAV capsid variant comprising a VP1 variant having an amino acid sequence of any one of SEQ ID NOs 02 to 29. For example, an AAV capsid variant of an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject by subretinal injection may be any one of AAV capsid variant 01 to AAV capsid variant 28.

[0262] In some embodiments, the AAV variant may have enhanced ocular tissue tropism. In some embodiments, the AAV variant may have enhanced retinal tissue tropism. In some embodiments, the AAV variant may have enhanced retinal tissue tropism upon subretinal or intravitreal administration. In some embodiments, the AAV variant may have enhanced RPE tissue tropism. In some embodiments, the AAV variant may have enhanced RPE tissue tropism upon subretinal or intravitreal administration.

[0263] AAV variants with such excellent delivery capabilities can be used for the delivery of therapeutic genes for diseases or diseases associated with target sites (e.g., RPE), but are not limited thereto.

[0264]

[0265] Production of AAV variants

[0266] The method or protocol for producing the AAV variant or recombinant AAV variant of the present disclosure may refer to AAV production methods widely known in the art.

[0267] For example, a popular method for producing recombinant AAV involves the triple transformation of HeLa (HeK293) cells. These cells possess continuously expressed adenovirus (adv) E1a and E1b genes. Hek293 cells are triple-transformed into a trans-plasmid expressing the rep and cap genes, a cis-plasmid encoding the gene to be packaged into the AAV capsid, and a helper plasmid containing other helper-functioning adv genes, such as the E2A, E4, and VA rNa genes, which are essential for replication, mRNA processing, and translation, respectively. Furthermore, HeK293 cells can be tuned to grow in suspension to expand culture size and increase production yield. (See reference [Wang, D., Tai, PW, & Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews Drug discovery, 18(5), 358-378.])

[0268] Methods or protocols for the preparation of recombinant AAV may be referenced in the literature [Wang, D., Tai, PW, & Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nature reviews Drug discovery, 18(5), 358-378.]; [Green, MR, & Sambrook, J. (2012). Molecular cloning. A Laboratory Manual 4th, 448.] and [Clement, N., & Grieger, JC (2016). Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Molecular therapy Methods & clinical development, 3.], the full contents of which are incorporated by reference into the present disclosure.

[0269] Below, an example of a method for manufacturing an AAV variant is described.

[0270] For example, a method for manufacturing an AAV vector is,

[0271] A first plasmid comprising a nucleic acid encoding a product of interest (e.g., a sequence intended for delivery or expression) (e.g., this may be referred to as a transfer plasmid or a trans-plasmid), wherein the transfer plasmid may further comprise an ITR and a promoter in addition to the nucleic acid portion encoding the product of interest;

[0272] A second plasmid comprising a rep gene (e.g., a nucleic acid encoding a rep protein) and / or a cap gene (e.g., a nucleic acid encoding a cap protein) (e.g., this may be referred to as a Rep-Cap plasmid or a cis-plasmid); and

[0273] Optionally, a third plasmid containing a helper gene (e.g., this may be referred to as a helper plasmid)

[0274] Introducing into a host cell; and

[0275] It may include obtaining (or extracting) an AAV vector (e.g., an AAV variant vector) from the above host cell.

[0276] Here, the cap gene may comprise a nucleic acid encoding an AAV capsid protein variant according to some embodiments of the present disclosure. For example, the cap gene may comprise a nucleic acid encoding a VP1 variant. For example, the cap gene may comprise a nucleic acid encoding any one amino acid sequence selected from SEQ ID NOs 02 to 31. For example, the cap gene may comprise a nucleic acid encoding a VP1 variant, a VP2 variant, and a VP3 variant.

[0277] Here, the host cell into which the three types of plasmids or two types of plasmids (e.g., the first plasmid and the second plasmid) are introduced may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell. In some embodiments, the host cell may be a mammalian-derived cell. In some embodiments, the host cell may be, for example, HEK293, HEK293T, Huh-7, HeLa, HepG2, Hep1A, SV40, CHO, COS, MeWo, NIH3T3, A549, PERC6, HT1180, 293 AAV cell, monocyte, or dendritic cell.

[0278] After introducing the aforementioned three or two types of plasmids into a host cell, the researcher may culture the host cell in an appropriate manner (e.g., according to a protocol provided by the host cell manufacturer or a known protocol) to obtain an AAV vector from the host cell. After culture, an AAV vector containing the desired sequence may be obtained from the host cell.

[0279] The introduction of a plasmid into a host cell may be performed by appropriately selecting methods known in the art. For example, electroporation, gene gun, sonication, magnetofection, microinjection, transient cell compression or squeezing, cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection, calcium phosphate precipitation, lipofection, PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection, etc., may be used, but are not limited thereto.

[0280] At this time, the order in which the plasmid is introduced into the host cell is not particularly restricted.

[0281] The above AAV vector may be prepared in the form of exosomes or microparticles, but is not limited thereto. The method of extracting the AAV vector may be performed by a method known in the art.

[0282] For example, methods for extracting AAV vectors may include centrifugation, precipitation, immunoprecipitation, affinity chromatography, filtration, using magnetic beads coated with specific antibodies or actamers, freezing / thawing, sonication, commercial kits, etc.

[0283] The AAV vector may optionally be concentrated by methods known in the art. For example, the concentration may be performed using immunomagnetic capture, organic flocculation, PEG (polyethylene glycol) precipitation, etc., but is not limited thereto.

[0284] Additionally, further quality tests may be optionally performed. For example, the quality tests may include, but are not limited to, titer measurement, sterility tests for bacteria and fungi, and mycoplasma detection tests.

[0285] Furthermore, mammalian cell-free platforms for the production of recombinant AAV or AAV vectors can also be applied for the production of recombinant AAV. The most commonly used mammalian cell-free platforms are based on recombinant baculoviruses, known as baculovirus expression vectors (BEVs). These BEVs infect Spodoptera frugiperda (sf9) insect cells for vector production. (See reference [Kondratov, O., Marsic, D., Crosson, SM, Mendez-Gomez, HR, Moskalenko, O., Mietzsch, M., ... & Zolotukhin, S. (2017). Direct head-to-head evaluation of recombinant adeno-associated viral vectors manufactured in human versus insect cells. Molecular Therapy, 25(12), 2661-2675.])

[0286] Methods for manufacturing AAV variants are not limited to those disclosed in this disclosure, and AAV variants may be manufactured through methods known in the art or newly developed methods / platforms.

[0287]

[0288] Production of AAV variants - Recombinant nucleic acid molecules containing nucleic acids encoding capsid protein variants

[0289] Some embodiments of the present disclosure provide a nucleic acid encoding a capsid protein variant and a nucleic acid molecule containing the same (e.g., a recombinant nucleic acid molecule). The nucleic acid encoding the capsid protein variant and the recombinant nucleic acid molecule containing the same may be used for the production of the aforementioned AAV variant. An example of a recombinant nucleic acid molecule containing the nucleic acid encoding the capsid protein variant is the aforementioned second plasmid used in a method for manufacturing an AAV vector. The nucleic acid encoding the capsid protein variant may be referred to as the cap gene.

[0290] In some embodiments, the capsid protein variant may be a VP1 variant according to some embodiments of the present disclosure. In some embodiments, the cap gene may comprise a nucleic acid sequence encoding any one amino acid sequence selected from SEQ ID NOs 02 to 31.

[0291] Some embodiments of the present disclosure provide a recombinant nucleic acid molecule comprising a cap gene comprising a nucleic acid sequence encoding any one amino acid sequence selected from SEQ ID NOs 02 to 31.

[0292] In some embodiments, the recombinant nucleic acid molecule may further include a nucleic acid encoding a rep protein (i.e., a rep gene) in addition to the cap gene. The rep protein may be, for example, one or more of rep78, rep68, rep52, and rep40. In some embodiments, four types of rep proteins may be produced from a single rep gene. The four rep proteins may be rep78, rep68, rep52, and rep40.

[0293] In some embodiments, the recombinant nucleic acid molecule may further comprise one or more promoters. For example, the recombinant nucleic acid molecule may further comprise a promoter operably linked to the cap gene and / or a promoter operably linked to the rep gene. In some embodiments, the promoters may be selected independently, and specific examples of promoters are described in detail in the preceding paragraphs, including the section "Adeno-associated virus (AAV) variants" and the subsection "Additional elements for controlling the expression of products of interest, etc." of this disclosure.

[0294] In some embodiments, the recombinant nucleic acid molecule may be a DNA molecule. For example, the recombinant nucleic acid molecule may be a plasmid, but is not limited thereto. The recombinant nucleic acid molecule may be referred to as a cap plasmid or a Rep-Cap plasmid.

[0295]

[0296] Production of AAV variants - Engineered host cells for AAV variant production

[0297] Some embodiments of the present disclosure provide engineered host cells for the production of AAV variants. The engineered host cells of the present disclosure are host cells transfected with a recombinant nucleic acid molecule (e.g., Rep-Cap plasmid) comprising a nucleic acid encoding at least a capsid protein variant, and can be used for the production of AAV variants.

[0298] In some embodiments, the engineered host cell may comprise a recombinant nucleic acid molecule comprising a nucleic acid encoding a capsid protein variant according to some embodiments of the present disclosure. For example, the engineered host cell may comprise a recombinant nucleic acid molecule comprising a cap gene comprising a nucleic acid sequence encoding any one of the amino acid sequences selected from SEQ ID NOs 02 to 31. The engineered host cell may be produced by a method comprising introducing a recombinant nucleic acid molecule into the host cell, and is not otherwise limited. The method of introducing the nucleic acid molecule is described in detail in the preceding paragraph.

[0299] In some embodiments, the engineered host cell may further comprise a first plasmid containing a nucleic acid encoding a product of interest and / or a third plasmid containing a helper gene, in addition to a recombinant nucleic acid molecule (e.g., a second plasmid) containing a nucleic acid encoding a capsid protein variant.

[0300]

[0301] Composition or kit containing an AAV variant

[0302] Some embodiments of the present disclosure provide a composition comprising the aforementioned AAV variant. Some embodiments of the present disclosure provide a kit comprising an AAV variant. In some embodiments, the composition or kit comprising an AAV variant may comprise an AAV variant according to some embodiments of the present disclosure. The composition or kit comprising an AAV variant may be used to deliver a nucleic acid encoding an AAV variant or a product of interest to a target site, and / or to treat a disease of interest of the target. The use of the AAV variant or an AAV variant vector is described in detail below.

[0303]

[0304] Use of AAV variant vectors

[0305] Overview of the Use of AAV Variant Vectors

[0306] In some embodiments, the AAV variant or AAV variant vector of the present disclosure may be used to deliver a nucleic acid encoding a product of interest to a target site. In some embodiments, the AAV variant vector of the present disclosure may be used to express a nucleic acid encoding a product of interest at a target site. The target site may be referred to, for example, as a target site. The uses of recombinant AAV or AAV vector are well known in the art and are not limited to those described in this section.

[0307] In some embodiments, the AAV variant vector of the present disclosure has excellent ocular tissue delivery capability and can be used to deliver a nucleic acid encoding a product of interest to ocular tissue and to express it in the ocular tissue. Furthermore, the AAV variant vector of the present disclosure can deliver a nucleic acid encoding a product for treating an ocular disease (e.g., a nucleic acid encoding an ocular disease therapeutic protein) to ocular tissue and enable it to be expressed in the ocular tissue. The AAV variant vector of the present disclosure can be used to treat an ocular disease.

[0308] In some embodiments, the AAV variant vector of the present disclosure has excellent RPE tissue delivery capability and can be used to deliver nucleic acids encoding a product of interest to RPE tissue and to express it in RPE tissue. Furthermore, the AAV variant vector of the present disclosure can deliver nucleic acids encoding a product for treating RPE-related diseases (e.g., nucleic acids encoding a therapeutic protein for RPE-related diseases) to RPE tissue and enable it to be expressed in RPE tissue. The AAV variant vector of the present disclosure can be used to treat RPE-related diseases.

[0309] In the present disclosure, the term AAV variant vector is used to refer to an AAV variant that essentially comprises a nucleic acid (e.g., a gene of interest) encoding a product of interest. For example, an AAV variant vector comprises an AAV capsid variant and a nucleic acid molecule, wherein the nucleic acid molecule may comprise a nucleic acid encoding a product of interest. The AAV variant, the AAV capsid variant, the nucleic acid molecule, and the nucleic acid encoding a product of interest are described in detail in the present disclosure, including in the section “AAV variants” of the present disclosure.

[0310]

[0311] Delivery method of nucleic acids encoding a product of interest using an AAV variant vector

[0312] Some embodiments of the present disclosure provide a method for delivering nucleic acids encoding a product of interest using an AAV variant vector. Depending on the purpose of the method and the elements included in the AAV variant vector, the method for delivering nucleic acids encoding a product of interest of the present disclosure may be expressed as, but is not limited to, a method for delivering a gene of interest, a method for delivering a recombinant nucleic acid molecule, a method for delivering a heterologous nucleic acid, a method for delivering an AAV variant or an AAV variant vector, and a method for delivering an expression cassette.

[0313] Some embodiments of the present disclosure provide a method for delivering a nucleic acid encoding a product of interest. In some embodiments, the method for delivering a nucleic acid encoding a product of interest may include administering an AAV variant vector of the present disclosure to a subject. The AAV variant vector may be administered to the subject by being included in a composition (e.g., in the form of a composition) or by being included in a pharmaceutical composition (e.g., in the form of a pharmaceutical composition), but is not limited thereto. In some embodiments, the method for delivering a nucleic acid encoding a product of interest may be a method for delivering a nucleic acid encoding a product of interest to a target site of a subject.

[0314] In some embodiments, the subject may be a vertebrate, including mammals. In some embodiments, the subject may be a human. In some embodiments, the subject may be a non-human mammal. Non-human mammals may be, for example, a mouse, dog, horse, cat, rat, pig, rabbit, sheep, monkey, chimpanzee, or cow, but are not limited thereto. In some embodiments, the subject may be a non-human primate, but is not limited thereto.

[0315] In some embodiments, the target site may be ocular tissue. In some embodiments, the target site may be retinal tissue. In some embodiments, the target site may be RPE tissue. In some embodiments,

[0316] The target site may be specified as a target cell. In some embodiments, the target site may be a cell of ocular tissue. In some embodiments, the target site may be a retinal cell. In some embodiments, the target site may be an RPE cell.

[0317] The AAV variant vector of the present disclosure may be administered via various routes. In some embodiments, the AAV variant vector of the present disclosure may be administered to a subject subretinally, supracorbitally, or into the vitreous cavity. In some embodiments, the AAV variant vector of the present disclosure may be administered by subretinal injection, supracorbital injection, or intravitreal injection. In specific embodiments, the AAV variant vector of the present disclosure may be administered to a subject by subretinal injection. In specific embodiments, the AAV variant vector of the present disclosure may be administered to a subject by intravitreal injection.

[0318] In some embodiments, a method for delivering a nucleic acid encoding a product of interest to a target RPE tissue is provided, comprising: administering an AAV variant vector of the present disclosure to a target via subretinal injection or intravitreal injection.

[0319] In some embodiments, a method for delivering a nucleic acid encoding a product of interest to a target RPE tissue is provided, comprising: administering an AAV variant vector to a target via subretinal injection, wherein the AAV variant vector comprises an AAV capsid variant comprising a VP1 variant having any one amino acid sequence selected from SEQ ID NOs 02 to 29.

[0320] In some embodiments, a method for delivering a nucleic acid encoding a product of interest to a target RPE tissue is provided: administering an AAV variant vector to a target via intravitreal injection, wherein the AAV variant vector comprises an AAV capsid variant comprising a VP1 variant having an amino acid sequence selected from any one of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31.

[0321]

[0322] Treatment method for a disease of interest (or condition) using AAV variant vectors

[0323] Some embodiments of the present disclosure provide a method for treating a disease of interest (or disease) using an AAV variant vector. The product of interest of the nucleic acid encoding the product of interest of the AAV variant vector used in this section may preferably be one or more of a protein (e.g., a therapeutic protein) for treating the disease of interest (or disease), a genetically engineered protein, and a guide RNA, but is not limited thereto.

[0324] Some embodiments of the present disclosure provide a method for treating a disease of interest. In some embodiments, the method for treating the disease of interest may include administering an AAV variant vector to a subject. Herein, the AAV variant vector may be administered to a subject by being included in a composition (e.g., in the form of a composition) or by being included in a pharmaceutical composition (e.g., in the form of a pharmaceutical composition), but is not limited thereto. A suitable buffer, such as Phosphate-buffered saline (PBS), may be used to prepare a composition or pharmaceutical composition containing an AAV variant vector, but is not otherwise limited thereto. In some embodiments, a composition or pharmaceutical composition containing an AAV variant vector may further include, but is not limited to, stabilizers and / or excipients suitable for use with and for use with the AAV vector. In some embodiments, the excipients may be one or more selected from ionic salt excipients, sucrose, and surfactants. The ionic salt excipient may be, for example, one or more selected from potassium diphosphate, potassium phosphate, sodium chloride, anhydrous sodium diphosphate, sodium phosphate hexahydrate, sodium diphosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl), amino acid, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, and calcium citrate, but is not limited thereto. The surfactant may be one or more selected from, for example, poloxamer (e.g., poloxamer 188) and polysorbate (e.g., polysorbate 20, polysorbate 80), but is not limited thereto.

[0325] The disease or condition of interest may be, for example, an eye-related disease or condition, a retinal tissue-related disease or condition, or an RPE tissue-related disease or condition, but is not limited thereto. The disease or condition of interest may be, for example, a disease or condition related to retinal cells [e.g., neurons of the retina, photoreceptors, bipolar cells, horizontal cells, amacrine, ganglion cells, etc.] or a disease or condition related to retinal pigment epithelial cells. In some embodiments, a disease related to retinal pigment epithelium may be a disease caused by RPE dysfunction.In some embodiments, the disease or condition of interest is achromatopsia, Usher syndrome, diabetic retinopathy (proliferative diabetic retinopathy), diabetic macular edema, geographic atrophy, retinopathy of premature, Leber's hereditary optic neuropathy, central retinal vein occlusion, proliferative vitreoretinopathy, retinal cone dystrophy, Behcet's disease, Bietti's crystallin dystrophy, Best vitelliform macular dystrophy, Best disease, choroideremia, retinal detachment It may be detachment), uveitis, glaucoma, retinitis pigmentosa (RP), macular degeneration [e.g., age-related macular degeneration (AMD)], RPE65-associated retinal dystrophy, Stargardt disease (SD), retinoschisis, or Leber congenital amaurosis (LCA).

[0326] In some embodiments, an AAV variant vector or a composition containing it may be administered to a subject at an appropriate dose. For example, the dosage of the AAV variant vector or a composition containing it may be determined by considering, but is not limited to, one or more selected from the type of disease, site of administration, body weight, age, and stage of disease progression. Furthermore, the dosage may be appropriately determined within the scope of medical judgment in correspondence with a reasonable benefit / risk ratio and within a range free from other problems such as excessive toxicity, irritation, or allergic reactions. In some embodiments, the AAV variant vector or a composition containing it may be administered together with other elements or other substances, and is not otherwise limited. The substance administered together with the AAV variant vector or a composition containing it may be an element or substance that can help treat the disease of interest.

[0327]

[0328] Exemplary embodiment

[0329] In the following, exemplary embodiments of the invention provided according to some embodiments of the present disclosure are provided. The invention provided by the present disclosure is not limited to the examples below.

[0330]

[0331] Exemplary embodiment of an AAV variant

[0332] A01. AAV variants including the following:

[0333] AAV capsid variant,

[0334] At this time, the above AAV capsid variant includes a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 31.

[0335] A02. In A01, the AAV variant is an AAV variant selected from any one of the following:

[0336] An AAV capsid variant (e.g., AAV capsid variant 1) comprising VP1 having the amino acid sequence of SEQ ID NO. 02 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 33 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 64 (e.g., VP3 variant);

[0337] An AAV capsid variant (e.g., AAV capsid variant 2) comprising VP1 having the amino acid sequence of SEQ ID NO. 03 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 34 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 65 (e.g., VP3 variant);

[0338] An AAV capsid variant (e.g., AAV capsid variant 3) comprising VP1 having the amino acid sequence of SEQ ID NO. 04 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 35 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 66 (e.g., VP3 variant);

[0339] An AAV capsid variant (e.g., AAV capsid variant 4) comprising VP1 having the amino acid sequence of SEQ ID NO. 05 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 36 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 67 (e.g., VP3 variant);

[0340] An AAV capsid variant (e.g., AAV capsid variant 5) comprising VP1 having the amino acid sequence of SEQ ID NO. 06 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 37 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 68 (e.g., VP3 variant);

[0341] An AAV capsid variant (e.g., AAV capsid variant 6) comprising VP1 having the amino acid sequence of SEQ ID NO. 07 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 38 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 69 (e.g., VP3 variant);

[0342] An AAV capsid variant (e.g., AAV capsid variant 7) comprising VP1 having the amino acid sequence of SEQ ID NO. 08 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 39 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 70 (e.g., VP3 variant);

[0343] An AAV capsid variant (e.g., AAV capsid variant 8) comprising VP1 having the amino acid sequence of SEQ ID NO. 09 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 40 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 71 (e.g., VP3 variant);

[0344] An AAV capsid variant (e.g., AAV capsid variant 9) comprising VP1 having the amino acid sequence of SEQ ID NO. 10 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 41 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 72 (e.g., VP3 variant);

[0345] An AAV capsid variant (e.g., AAV capsid variant 10) comprising VP1 having the amino acid sequence of SEQ ID NO. 11 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 42 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 73 (e.g., VP3 variant);

[0346] An AAV capsid variant (e.g., AAV capsid variant 11) comprising VP1 having the amino acid sequence of SEQ ID NO. 12 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 43 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 74 (e.g., VP3 variant);

[0347] An AAV capsid variant (e.g., AAV capsid variant 12) comprising VP1 having the amino acid sequence of SEQ ID NO. 13 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 44 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 75 (e.g., VP3 variant);

[0348] An AAV capsid variant (e.g., AAV capsid variant 13) comprising VP1 having the amino acid sequence of SEQ ID NO. 14 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 45 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 76 (e.g., VP3 variant);

[0349] An AAV capsid variant (e.g., AAV capsid variant 14) comprising VP1 having the amino acid sequence of SEQ ID NO. 15 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 46 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 77 (e.g., VP3 variant);

[0350] An AAV capsid variant (e.g., AAV capsid variant 15) comprising VP1 having the amino acid sequence of SEQ ID NO. 16 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 47 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 78 (e.g., VP3 variant);

[0351] An AAV capsid variant (e.g., AAV capsid variant 16) comprising VP1 having the amino acid sequence of SEQ ID NO. 17 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 48 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 79 (e.g., VP3 variant);

[0352] An AAV capsid variant (e.g., AAV capsid variant 17) comprising VP1 having the amino acid sequence of SEQ ID NO. 18 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 49 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 80 (e.g., VP3 variant);

[0353] An AAV capsid variant (e.g., AAV capsid variant 18) comprising VP1 having the amino acid sequence of SEQ ID NO. 19 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 50 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 81 (e.g., VP3 variant);

[0354] An AAV capsid variant (e.g., AAV capsid variant 19) comprising VP1 having the amino acid sequence of SEQ ID NO. 20 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 51 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 82 (e.g., VP3 variant);

[0355] An AAV capsid variant (e.g., AAV capsid variant 20) comprising VP1 having the amino acid sequence of SEQ ID NO. 21 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 52 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 83 (e.g., VP3 variant);

[0356] An AAV capsid variant (e.g., AAV capsid variant 21) comprising VP1 having the amino acid sequence of SEQ ID NO. 22 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 53 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 84 (e.g., VP3 variant);

[0357] An AAV capsid variant (e.g., AAV capsid variant 22) comprising VP1 having the amino acid sequence of SEQ ID NO. 23 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 54 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 85 (e.g., VP3 variant);

[0358] An AAV capsid variant (e.g., AAV capsid variant 23) comprising VP1 having the amino acid sequence of SEQ ID NO. 24 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 55 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 86 (e.g., VP3 variant);

[0359] An AAV capsid variant (e.g., AAV capsid variant 24) comprising VP1 having the amino acid sequence of SEQ ID NO. 25 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 56 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 87 (e.g., VP3 variant);

[0360] An AAV capsid variant (e.g., AAV capsid variant 25) comprising VP1 having the amino acid sequence of SEQ ID NO. 26 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 57 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 88 (e.g., VP3 variant);

[0361] An AAV capsid variant (e.g., AAV capsid variant 26) comprising VP1 having the amino acid sequence of SEQ ID NO. 27 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 58 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 89 (e.g., VP3 variant);

[0362] An AAV capsid variant (e.g., AAV capsid variant 27) comprising VP1 having the amino acid sequence of SEQ ID NO. 28 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 59 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 90 (e.g., VP3 variant);

[0363] An AAV capsid variant (e.g., AAV capsid variant 28) comprising VP1 having the amino acid sequence of SEQ ID NO. 29 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 60 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 91 (e.g., VP3 variant);

[0364] An AAV capsid variant (e.g., AAV capsid variant 29) comprising VP1 having the amino acid sequence of SEQ ID NO. 30 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 61 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 92 (e.g., VP3 variant); and

[0365] An AAV capsid variant (e.g., AAV capsid variant 30) comprising VP1 having the amino acid sequence of SEQ ID NO. 31 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 62 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 93 (e.g., VP3 variant).

[0366] A03. AAV variants including the following:

[0367] AAV capsid variant,

[0368] At this time, the above AAV capsid variant includes a VP1 variant, and

[0369] At this time, the VP1 variant comprises any one amino acid sequence selected from SEQ ID NOs 95 to 124, and

[0370] At this time, any one amino acid sequence selected from SEQ ID NOs 95 to 124 is located at position 561-588 based on the VP1 amino acid sequence of wild-type AAV2.

[0371] A04. An AAV variant in any one of A01 to A03, wherein the AAV variant further comprises a nucleic acid molecule (e.g., a recombinant nucleic acid molecule), wherein the nucleic acid molecule comprises a nucleic acid encoding a product of interest.

[0372] A05. In A04, the nucleic acid molecule further comprises a promoter, wherein the promoter is operably linked to the nucleic acid encoding the product of interest, an AAV variant.

[0373] A06. In A05, the nucleic acid molecule further comprises two ITRs, wherein the nucleic acid encoding the promoter and the product of interest is an AAV variant located between the two ITRs.

[0374] A07. An AAV variant in any one of A04 to A06, wherein the nucleic acid molecule is single-stranded DNA.

[0375] A08. In any one of A04 to A07, the product of interest is an AAV variant that is a product for treating a disease of interest.

[0376] A09. In A08, the product of interest is an AAV variant, which is a protein for treating the disease of interest.

[0377] A10. An AAV variant characterized in that, in any one of A01 to A09, the AAV variant is delivered to retinal tissue or retinal cells.

[0378] A11. In any one of A01 to A10, the AAV variant is an AAV variant having excellent delivery ability (or targeting ability) to retinal tissue or retinal cells.

[0379] A12. In any one of A01 to A11, the AAV variant is an AAV variant having an enhanced ability to deliver to retinal tissue or retinal cells (or targeting ability) compared to wild-type AAV2.

[0380] A13. In any one of A01 to A12, the AAV variant is an AAV variant having retinal tissue or retinal cell trophism.

[0381] A14. An AAV variant characterized in that, in any one of A01 to A09, the AAV variant is delivered to an RPE tissue or an RPE cell.

[0382] A15. In any one of A01 to A09 and A14, the AAV variant is an AAV variant having excellent RPE tissue or RPE cell delivery ability (or targeting ability).

[0383] A16. In any one of A01 to A09 and A14 to A15, the AAV variant is an AAV variant having an enhanced ability to deliver to RPE tissues or RPE cells (or targeting ability) compared to wild-type AAV2.

[0384] A17. In any one of A01 to A09 and A14 to A16, the AAV variant is an AAV variant having an RPE tissue or RPE cell tropism.

[0385] A18. In any one of A01 to A09 and A14 to A17, the AAV variant has excellent RPE tissue or RPE cell delivery ability (or targeting ability) when administered to a subject via intravitreal injection.

[0386] A19. An AAV variant according to A18, wherein the AAV capsid variant of the AAV variant is an AAV capsid variant comprising a VP1 variant having an amino acid sequence selected from any one of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31.

[0387] A20. In any one of A01 to A09 and A14 to A17, the AAV variant has excellent RPE tissue or RPE cell delivery ability (or targeting ability) when administered to a subject via subretinal injection.

[0388] A21. An AAV variant according to A20, wherein the AAV capsid variant of the AAV variant is an AAV capsid variant comprising a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29.

[0389]

[0390] Exemplary embodiments of a method for delivering a nucleic acid encoding an AAV variant or a product of interest

[0391] B01. A method for delivering an AAV variant to a target site of a target comprising the following:

[0392] Administer any one of A01 to A09 AAV variants to the subject.

[0393] B02. A method according to B01, wherein the AAV variant is administered to a subject by subretinal injection, supracorbital injection, or intravitreal injection.

[0394] B03. A method in which, in any one of B01 to B02, the target site is retinal tissue or retinal cell.

[0395] B04. A method in which, in any one of B01 to B02, the target site is an RPE tissue or an RPE cell.

[0396] B05. A method in which, in any one of B01 to B04, the subject is a human or non-human mammal.

[0397] B06. A method in which, in any one of B01 to B05, the AAV variant is administered to a subject by subretinal injection, wherein the AAV capsid variant of the AAV variant comprises an AAV capsid variant having a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29.

[0398] B07. A method in which, in any one of B01 to B05, the AAV variant is administered to a subject by intravitreal injection, wherein the AAV capsid variant of the AAV variant comprises an AAV capsid variant having a VP1 variant having an amino acid sequence selected from SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31.

[0399] B08. A method for delivering a nucleic acid encoding a product of interest to a target site of a subject comprising the following: administering any one of A04 to A09 AAV variants to the subject.

[0400] B09. A method according to B08, wherein the AAV variant is administered to a subject by subretinal injection, supracorbital injection, or intravitreal injection.

[0401] B10. A method in which, in any one of B08 to B09, the target site is retinal tissue or retinal cell.

[0402] B11. A method in which, in any one of B08 to B09, the target site is an RPE tissue or an RPE cell.

[0403] B12. A method in which, in any one of B08 to B11, the subject is a human or non-human mammal.

[0404] B13. A method in which, in any one of B08 to B12, the AAV variant is administered to a subject by subretinal injection, wherein the AAV capsid variant of the AAV variant comprises an AAV capsid variant having a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29.

[0405] B14. A method in which, in any one of B08 to B12, the AAV variant is administered to a subject by intravitreal injection, wherein the AAV capsid variant of the AAV variant comprises an AAV capsid variant having a VP1 variant having an amino acid sequence selected from SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31.

[0406]

[0407] Method to treat diseases of interest using AAV variants

[0408] C01. A method for treating the disease or condition of interest of a subject including the following:

[0409] Administer any one of A08 to A09 AAV variants to the subject.

[0410] C02. In C01, the above-mentioned disease of interest is a retinal disease or a disease, method.

[0411] C03. A method in which, in any one of C01 to C02, the disease of interest is an RPE-related disease or illness.

[0412] C04. A method in any one of C01 to C03, wherein the subject is a human or non-human mammal.

[0413] C05. A method in which, in any one of C01 to C04, the AAV variant is administered to a subject via subretinal injection, supracorbital injection, or intravitreal injection.

[0414]

[0415] Exemplary embodiment of an AAV capsid variant

[0416] D01. An AAV capsid variant comprising VP1 (e.g., a VP1 variant) having an amino acid sequence selected from any one of SEQ ID NOs 02 to 31.

[0417] D02. In D01, the AAV capsid variant further comprises a VP2 (e.g., a VP2 variant) having an amino acid sequence selected from any one of SEQ ID NOs 33 to 62.

[0418] D03. An AAV capsid variant in any one of D01 to D02, wherein the AAV capsid variant further comprises a VP3 (e.g., a VP3 variant) having an amino acid sequence selected from any one of SEQ ID NOs 64 to 93.

[0419] D04. AAV capsid variant, wherein the AAV capsid variant is any one selected from the following:

[0420] An AAV capsid variant (e.g., AAV capsid variant 1) comprising VP1 having the amino acid sequence of SEQ ID NO. 02 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 33 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 64 (e.g., VP3 variant);

[0421] An AAV capsid variant (e.g., AAV capsid variant 2) comprising VP1 having the amino acid sequence of SEQ ID NO. 03 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 34 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 65 (e.g., VP3 variant);

[0422] An AAV capsid variant (e.g., AAV capsid variant 3) comprising VP1 having the amino acid sequence of SEQ ID NO. 04 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 35 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 66 (e.g., VP3 variant);

[0423] An AAV capsid variant (e.g., AAV capsid variant 4) comprising VP1 having the amino acid sequence of SEQ ID NO. 05 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 36 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 67 (e.g., VP3 variant);

[0424] An AAV capsid variant (e.g., AAV capsid variant 5) comprising VP1 having the amino acid sequence of SEQ ID NO. 06 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 37 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 68 (e.g., VP3 variant);

[0425] An AAV capsid variant (e.g., AAV capsid variant 6) comprising VP1 having the amino acid sequence of SEQ ID NO. 07 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 38 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 69 (e.g., VP3 variant);

[0426] An AAV capsid variant (e.g., AAV capsid variant 7) comprising VP1 having the amino acid sequence of SEQ ID NO. 08 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 39 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 70 (e.g., VP3 variant);

[0427] An AAV capsid variant (e.g., AAV capsid variant 8) comprising VP1 having the amino acid sequence of SEQ ID NO. 09 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 40 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 71 (e.g., VP3 variant);

[0428] An AAV capsid variant (e.g., AAV capsid variant 9) comprising VP1 having the amino acid sequence of SEQ ID NO. 10 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 41 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 72 (e.g., VP3 variant);

[0429] An AAV capsid variant (e.g., AAV capsid variant 10) comprising VP1 having the amino acid sequence of SEQ ID NO. 11 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 42 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 73 (e.g., VP3 variant);

[0430] An AAV capsid variant (e.g., AAV capsid variant 11) comprising VP1 having the amino acid sequence of SEQ ID NO. 12 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 43 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 74 (e.g., VP3 variant);

[0431] An AAV capsid variant (e.g., AAV capsid variant 12) comprising VP1 having the amino acid sequence of SEQ ID NO. 13 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 44 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 75 (e.g., VP3 variant);

[0432] An AAV capsid variant (e.g., AAV capsid variant 13) comprising VP1 having the amino acid sequence of SEQ ID NO. 14 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 45 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 76 (e.g., VP3 variant);

[0433] An AAV capsid variant (e.g., AAV capsid variant 14) comprising VP1 having the amino acid sequence of SEQ ID NO. 15 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 46 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 77 (e.g., VP3 variant);

[0434] An AAV capsid variant (e.g., AAV capsid variant 15) comprising VP1 having the amino acid sequence of SEQ ID NO. 16 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 47 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 78 (e.g., VP3 variant);

[0435] An AAV capsid variant (e.g., AAV capsid variant 16) comprising VP1 having the amino acid sequence of SEQ ID NO. 17 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 48 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 79 (e.g., VP3 variant);

[0436] An AAV capsid variant (e.g., AAV capsid variant 17) comprising VP1 having the amino acid sequence of SEQ ID NO. 18 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 49 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 80 (e.g., VP3 variant);

[0437] An AAV capsid variant (e.g., AAV capsid variant 18) comprising VP1 having the amino acid sequence of SEQ ID NO. 19 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 50 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 81 (e.g., VP3 variant);

[0438] An AAV capsid variant (e.g., AAV capsid variant 19) comprising VP1 having the amino acid sequence of SEQ ID NO. 20 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 51 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 82 (e.g., VP3 variant);

[0439] An AAV capsid variant (e.g., AAV capsid variant 20) comprising VP1 having the amino acid sequence of SEQ ID NO. 21 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 52 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 83 (e.g., VP3 variant);

[0440] An AAV capsid variant (e.g., AAV capsid variant 21) comprising VP1 having the amino acid sequence of SEQ ID NO. 22 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 53 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 84 (e.g., VP3 variant);

[0441] An AAV capsid variant (e.g., AAV capsid variant 22) comprising VP1 having the amino acid sequence of SEQ ID NO. 23 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 54 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 85 (e.g., VP3 variant);

[0442] An AAV capsid variant (e.g., AAV capsid variant 23) comprising VP1 having the amino acid sequence of SEQ ID NO. 24 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 55 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 86 (e.g., VP3 variant);

[0443] An AAV capsid variant (e.g., AAV capsid variant 24) comprising VP1 having the amino acid sequence of SEQ ID NO. 25 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 56 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 87 (e.g., VP3 variant);

[0444] An AAV capsid variant (e.g., AAV capsid variant 25) comprising VP1 having the amino acid sequence of SEQ ID NO. 26 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 57 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 88 (e.g., VP3 variant);

[0445] An AAV capsid variant (e.g., AAV capsid variant 26) comprising VP1 having the amino acid sequence of SEQ ID NO. 27 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 58 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 89 (e.g., VP3 variant);

[0446] An AAV capsid variant (e.g., AAV capsid variant 27) comprising VP1 having the amino acid sequence of SEQ ID NO. 28 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 59 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 90 (e.g., VP3 variant);

[0447] An AAV capsid variant (e.g., AAV capsid variant 28) comprising VP1 having the amino acid sequence of SEQ ID NO. 29 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 60 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 91 (e.g., VP3 variant);

[0448] An AAV capsid variant (e.g., AAV capsid variant 29) comprising VP1 having the amino acid sequence of SEQ ID NO. 30 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 61 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 92 (e.g., VP3 variant); and

[0449] An AAV capsid variant (e.g., AAV capsid variant 30) comprising VP1 having the amino acid sequence of SEQ ID NO. 31 (e.g., VP1 variant), VP2 having the amino acid sequence of SEQ ID NO. 62 (e.g., VP2 variant), and VP3 having the amino acid sequence of SEQ ID NO. 93 (e.g., VP3 variant).

[0450]

[0451] Exemplary embodiments of VP1, VP2, and VP3 capsid protein variants

[0452] E01. A capsid protein variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 31.

[0453] E02. In E01, the capsid protein variant having any one amino acid sequence selected from SEQ ID NOs 02 to 31 is an AAV2 VP1 capsid protein variant.

[0454] E03. A capsid protein variant having an amino acid sequence selected from any one of SEQ ID NOs 33 to 62.

[0455] E04. In E03, the capsid protein variant having any one amino acid sequence selected from SEQ ID NOs 33 to 62 is an AAV2 VP2 capsid protein variant.

[0456] E05. A capsid protein variant having an amino acid sequence selected from any one of SEQ ID NOs 64 to 93.

[0457] E06. In E05, the capsid protein variant having any one amino acid sequence selected from SEQ ID NOs 64 to 93 is an AAV2 VP3 capsid protein variant.

[0458]

[0459] Exemplary embodiment of a recombinant nucleic acid molecule comprising a nucleic acid (e.g., cap gene) encoding a capsid protein variant

[0460] F01. A recombinant nucleic acid molecule comprising a cap gene having a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs 02 to 31.

[0461] F02. In F01, the recombinant nucleic acid molecule further comprises the rep gene.

[0462] F03. A recombinant nucleic acid molecule, wherein in any one of F01 to F02, the recombinant nucleic acid molecule is a plasmid.

[0463] F04. A recombinant nucleic acid molecule in any one of F01 to F03, wherein the recombinant nucleic acid molecule further comprises a promoter operably linked to a cap gene.

[0464]

[0465] Exemplary embodiment of engineered host cells for AAV variant production

[0466] G01. An engineered host cell comprising any one of the recombinant nucleic acid molecules F01 to F04.

[0467] G02. In G01, the engineered host cell further comprises a transport plasmid containing a nucleic acid encoding a product of interest.

[0468] G03. In G02, the engineered host cell further comprises a helper plasmid containing a helper gene.

[0469] G04. In any one of G01 to G03, the host cell is a engineered host cell of mammalian origin.

[0470] G05. In any one of G01 to G04, the host cell is a engineered host cell that is HEK293, HEK293T, Huh-7, HeLa, HepG2, Hep1A, SV40, CHO, COS, MeWo, NIH3T3, A549, PERC6, HT1180, 293 AAV cell, monocyte, or dendritic cell.

[0471]

[0472] Exemplary embodiment of a method for producing AAV variants

[0473] H01. A method for producing an AAV variant comprising the following:

[0474] A cap gene having a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs 02 to 31, and a rep-cap plasmid comprising a rep gene,

[0475] A transport plasmid containing a nucleic acid encoding the product of interest, and

[0476] Helper plasmid containing a helper gene

[0477] Introducing into a host cell; and

[0478] AAV variants were obtained from host cells into which the above plasmids were introduced.

[0479]

[0480] The invention provided by the present disclosure will be described in more detail below through experimental examples and embodiments. These embodiments are intended to illustrate the contents disclosed by the present disclosure, and the scope of the contents disclosed by the present disclosure is not limited by these embodiments.

[0481]

[0482] Examples

[0483] To develop superior AAV2 variants, the inventors of the present disclosure developed a machine learning model and, through this, obtained a plasmid library capable of producing AAV2 variants and an AAV library obtained therefrom, and finally developed AAV2 variants with superior performance from the AAV library. The embodiments of the present disclosure are disclosed in detail below.

[0484]

[0485] Example 1. Development of a manipulated AAV library with improved diversity performance through a machine learning model

[0486] 1-1. Method

[0487] The region designed by the inventors of the present disclosure to introduce mutations for the development of AAV2 variants is positions 561–588 (28 amino acids) of AAV2 VP1. All mutations that may exist in the amino acid sequence consisting of the above 28 amino acids are 20 28For personal reasons, the inventors of the present disclosure first constructed an appropriate machine learning model and used it to select candidate sequences. The inventors of the present disclosure constructed an AAV plasmid library using the selected candidate sequences and constructed an AAV library.

[0488] 1-1-1. Machine Learning Model Development and Candidate Sequence Derivation

[0489] A functional prediction model was constructed using viability (packaging) data from a random mutation library targeting the 561-588 position (28 amino acids) of AAV2 VP1. The data was divided into training, validation, and test sets in an 8:1:1 ratio for model construction and evaluation. One-hot embedding and transformer-based ESM-2 were used as protein sequence fetishization methods. After one-hot encoding embedding, training was performed using a 1D-CNN model, while after ESM-2-based embedding, training was conducted using Linear Regression, kNN, SVM, Random Forest, and ANN models. The structure of each model was optimized using the validation data. In this process, RMSE and R² were used as model performance evaluation metrics. 2 used

[0490] A Genetic Algorithm (GA) was applied based on the model constructed for deriving candidate sequences. To reduce false positive predictions, an ensemble strategy utilizing an ESM-2-based ANN model and a one-hot encoding-based 1D-CNN model was employed for prediction during the genetic algorithm process. Additionally, a Position-Specific Scoring Matrix (PSSM) was used to restrict the generation of unlikely sequences and to secure diverse candidate sequences. The PSSM was calculated from the training data. During the execution of the genetic algorithm, constraints on the PSSM score (scores of 60 or higher, or 90 or higher) were applied to ensure diversity while maintaining similarity to the sequence distribution of the training data. The genetic algorithm was performed for 100 generations, combining various mutation rates (0.1–0.3) and crossover rates (0.2–0.7) to generate sequences under diverse conditions. To filter the sequences generated by the genetic algorithm, the following two filtering strategies were used to finally select 70,000 sequences of 28aa (AAV2 VP1 positions 561-588) (i.e., 70,000 sequences based on machine learning models): 1) top sequences that received high scores in both the 1D-CNN model and the ESM-based ANN model, and 2) top sequences with high 1D-CNN scores for each PSSM score interval.

[0491]

[0492] 1-1-2. Creating a Plasmid Library

[0493] Pooled DNA oligos were synthesized for 100 wild-type (WT) sequences, 100 stop codon sequences, 27,000 random mutant sequences, and 70,000 machine learning model-based sequences (Total 97,200) corresponding to the VP1 561-588 position of AAV2. As a screening vector, a vector capable of expressing a portion of the C-terminal region of the AAV2 REP and the entire VP1 sequence of the AAV2 CAP under a CAG promoter between inverted terminal repeats (ITRs) was used. The VP1 561-588 corresponding portion of the screening vector was cloned using the synthesized pooled DNA oligos via Gibson assembly to construct each plasmid in the plasmid library.

[0494] That is, the plasmid library consisted of 100 plasmids for wild-type sequences designed to produce wild-type AAV2 capsid, 100 plasmids for stop codon sequences, 27,000 random mutation-based variant sequence plasmids designed to produce AAV2 capsid variants containing VP1 variants with VP1 positions mutated at amino acid sequence sites VP1 561-588 (random library), and 70,000 machine learning-based variant sequence plasmids designed to produce AAV2 capsid variants containing VP1 variants with VP1 positions mutated at amino acid sequence sites VP1 561-588 (machine learning library).

[0495] The key elements of each plasmid in the plasmid library and the structure of each plasmid based on them are illustrated as follows:

[0496] Plasmid for wild-type sequence: ITR-CAG promoter-wild-type AAV2 cap gene-bGH poly A-ITR

[0497] Random mutation-based variant sequence plasmid: ITR-CAG promoter-AAV2 variant cap gene-bGH poly A-ITR

[0498] Machine learning-based variant sequence plasmid: ITR-CAG promoter-AAV2 variant cap gene-bGH poly A-ITR

[0499]

[0500] 1-1-3. Next Generation Sequencing (NGS) for Plasmid Libraries

[0501] The region containing VP1 561-588 of the constructed plasmid library was amplified using 10 ng of the plasmid library, Phusion High-Fidelity DNA Polymerase (NEB), and primers (Table 01), after which the samples were purified using a Fragment DNA purification kit (intronbio). The purified samples were prepared for NGS through Truseq Nano DNA library construction, and sequencing reads were produced via paired-end sequencing using the Illumina Novaseq platform 150PE. For NGS on the plasmid library, two technical replicates were performed, and the criterion was set as an average of 100 reads across two technical replicates per variant. Out of a total of 99,000 sequences, 96,400 sequences that passed the read > 100 criterion were selected (100 WT sequences, 100 Stop sequences, 27,000 random mutation sequences, and 69,200 machine learning-based sequences). The 96,400 selected sequences were used in subsequent experiments, such as frequency analysis and diversity analysis through AAV library packaging. Consequently, the coverage of the constructed plasmid library was confirmed to be approximately 99% (100 x 96,400 / 97,200). Frequency was defined as the relative read count within the same NGS and calculated using the formula "frequency = read count of the corresponding variant / total read count of 96,400." Additionally, the correlation between the frequencies of each variant between two technical replicates was confirmed to be high, with a Pearson R value of 0.99.

[0502] [Table 01] Information on primers used

[0503]

[0504]

[0505] 1-1-4. AAV Library Manufacturing (AAV Capsid Library Packaging)

[0506] Expi293F™ Suspension Cells (A14527, Gibco) were maintained by subculture twice a week in Expi293™ Expression medium (Thermo) with 0.5X penicillin / streptomycin (Thermo).

[0507] Transfection was performed on Expi293F cells (2.5E+6 cells / ml) with a total volume of 500ml and N=3 (3 biological replicates) using the constructed plasmid library (pAAV2-Library), pREP (rep plasmid), and pHelper (helper plasmid) in a mass ratio of 1:10:15 and PEI in a ratio of 2:1 (PEI ul : DNA ug). Approximately 18 hours after transfection, 0.1M (final concentration) sucrose was added, and approximately 4 days after transfection, 0.5M (final concentration) NaCl was added, followed by incubation at 37℃ for 3 hours. Subsequently, the culture medium was transferred to 250ml tubes and centrifuged at 3300g / 30min / 4℃, and the cell supernatant was filtered through a 0.45μm PES filter. After adding an amount of 40% PEG8000 solution equivalent to 1 / 4 of the cell supernatant volume, incubation was carried out sequentially in a stirrer at 125 rpm / 1 hr / 4℃ and 0 rpm / 16 hrs / 4℃. Subsequently, the same amount was transferred to a 250 ml tube and centrifuged at 3300 g / 30 min / 4℃. Afterward, the pellet was dissolved in resuspension buffer and collected in a 50 ml conical tube; 2 mM MgCl2 and 50 units / ml Benzonase were added, followed by incubation at 37℃ / 1 hr and centrifugation at 2000 g / 30 min / 4℃ in a water bath, and the supernatant was subjected to iodixanol gradient purification.AAVs were extracted from a 40% iodixanol fraction, and only high-purity AAVs were collected via silver staining. The final purified AAV library was prepared by replacing the buffer with formulation buffer (PBS-0.001% PF-68) using a spin concentrator (Satorius, Cat no. VS2042). The structure of the viral genome of each AAV in the prepared library, based on key elements, is as follows: ITR-CAG promoter-wild-type AAV2 or AAV2 variant cap gene-bGH poly A-ITR.

[0508]

[0509] 1-1-5. NGS for AAV Library

[0510] After treating the constructed AAV library with Denase1 and Proteinase K, the viral genome was extracted using a DNA purification kit. Subsequently, the capsid region of the viral genome was amplified using approximately 10 ng of viral DNA, Phusion High-Fidelity DNA Polymerase (NEB), and primers (Table 01), followed by sample purification using a Fragment DNA purification kit (intronbio). The purified samples were prepared for NGS through Truseq Nano DNA library construction, and sequencing reads were generated via paired-end sequencing using the Illumina Novaseq platform 150PE. For NGS on the AAV library, two technical replicates were performed for each of the three biological replicates; the correlation between variant frequencies among these technical replicates was confirmed to be high, with a Pearson R value of >0.98.

[0511]

[0512] 1-1-6. AAV Library Correlation and Diversity Performance Analysis

[0513] The frequencies of 96,400 selected sequences for each of the three AAV library biological replicates were calculated, and the Pearson R correlation between biological replicates was analyzed using these values.

[0514] The diversity performance of the AAV library was analyzed by determining viability based on the number of mutations. Specifically, the frequency ratio (Frequency_V / Frequency_P) was calculated by normalizing the frequency of the packaged AAV library for each sequence to the frequency value of the plasmid library used for virus production. Subsequently, 1.4, which is 5% of the average frequency ratio (Frequency_V / Frequency_P) value (27.9) of the WT, was set as the viable cutoff, and sequences with a value of 1.4 or higher were classified as viable sequences. Considering that the proportion of viable sequences decreases as the number of mutations increases, the ratio of viable sequences based on the number of mutations was calculated for the random mutation library and the machine learning-based library, respectively, and the diversity performance of these two libraries was compared.

[0515]

[0516] 1-2. Results

[0517] A machine learning model was developed by training on viability (packaging function) data from a random mutation library created by targeting the VP1 561-588 position (28 amino acids) of the AAV2 VP1 amino acid sequence. Variant sequences predicted to have viable function were obtained through the developed machine learning model. Subsequently, to compare under identical conditions, plasmid libraries for the random mutation sequence (random mutation library or random library) and the machine learning-based variant sequence (machine learning library) were created in the same pool, and AAV libraries corresponding to three biological replicates were produced.

[0518] After performing Illumina Nova-seq NGS (amplicon sequencing) on ​​these three AAV libraries, the frequency (relative read count) for each variant was calculated to analyze the correlation between the AAV libraries. The analysis results confirmed a high positive correlation between the libraries (Pearson R=0.94 between Library 1 and Library 2; Pearson R=0.91 between Library 1 and Library 3; Pearson R=0.95 between Library 2 and Library 3) (Fig. 01). Specifically, Fig. 01 presents the results of the correlation analysis of three biological replicates of the AAV libraries. Two NGS technical replicates were produced for each biological replicate, and the average frequency value of the two technical replicates was used for the correlation analysis.

[0519] Through correlation analysis of biological replicates in the AAV library, we were able to confirm the high reliability of overall AAV library production and NGS data.

[0520] Subsequently, as described above, the diversity capabilities of the random library and the machine learning library were compared by analyzing the viability of each variant according to the number of mutations. For the viability analysis, the value (Frequency_V / Frequency_P) was calculated by normalizing the frequency within the AAV library (Frequency_V) and the frequency within the plasmid library used for AAV production (Frequency_P) for each variant. The average frequency ratio (Frequency_V / Frequency_P) of 100 WT sequences was set as 5% of the viability cutoff (1.4), and variant sequences (or AAV variants) with a value greater than this were classified as viable sequences for analysis. The results of the diversity performance analysis between the random library and the machine learning library are disclosed in Fig. 02. The x-axis of the graph in Fig. 02 represents the number of mutations (number of mutations when compared to WT AAV2), which is indicated as distance to WT AAV2 in the graph. For example, if the amino acid sequence of the region corresponding to 561–588 of wild-type AAV2 VP1 of an AAV2 variant has a difference of two amino acids (mutation) when compared to the 28-amino acid sequence of 561–588 of wild-type AAV2 VP1, it belongs to the group with a Distance to WT AAV2 of 2. As another example, an AAV2 variant with a Distance to WT AAV2 of 4 has a difference of 4 amino acids (i.e., mutation) when compared to the 561–588 amino acid sequence of wild-type AAV2 VP1. The viability % in Fig. 02 represents the proportion of AAV variants having a value greater than or equal to the aforementioned viability cutoff. For example, among AAV variants with a Distance to WT AAV2 of 2, those with a Frequency_V / Frequency_P value of 1.Approximately 90% of AAV variants were identified as having a mutation level of 4 or higher. Frequency_V utilized the average frequency value of the AAV library (average over a total of 6 AAV library replicates: 3 biological replicates, 2 technical replicates per biological replicate), while Frequency_P utilized the average frequency value of the plasmid library (average over 2 technical replicates). The diversity performance analysis results demonstrate that the machine learning library exhibits significantly higher viability compared to the random library across all mutation count conditions. In the case of the random library, partially viable sequences existed under conditions involving only 2 to 3 minor amino acid mutations, but almost no viable sequences were identified under conditions with 5 or more mutations. In contrast, the machine learning library demonstrated over 30% viability under the 5-mutation condition (Distance to WT AAV2 = 5) and over 10% viability even under the 10-mutation condition. Accordingly, for example, it was confirmed that more than 30% of AAV variants among 5 mutation conditions had a packaging function, and more than 10% of AAV variants among 10 mutation conditions had a packaging function. The functions of sequences predicted to have a packaging function based on machine learning models could be experimentally verified, and the utility of machine learning was clearly confirmed, particularly in multiple mutation conditions required to enhance the delivery function of WT.

[0521]

[0522] Example 2. Securing an AAV variant (capsid variant sequence) with excellent RPE delivery function

[0523] 2-1. Method

[0524] The inventors of the present disclosure injected an AAV library into the eyes of mice and analyzed the RPE delivery function of each variant of the AAV library to finally select AAV variants with superior function from the AAV library. The process of selecting superior AAV variants is described in detail below.

[0525]

[0526] 2-1-1. In vivo Mouse Injection and RPE Tissue Separation

[0527] A total of 1E+9 vg (virus genome) of an AAV library (1E+12 vg / ml) was administered via subretinal injection at a volume of 1 μL / eye to adult C57BL / 6J mice (8 weeks old, 22-30g, N=6), and the injection was performed unilaterally into only the left eye (OS). To determine the success of the subretinal injection, retinal bleb formation was confirmed via FP / OCT imaging, and only mice with confirmed blebs were used for subsequent analysis. Additionally, the same dose of the AAV library was intravitreally injected into adult C57BL / 6J mice (8 weeks old, 22-30g, N=6) at a volume of 1 μL / eye.

[0528] On day 28 after administration, an electroretinogram (ERG) was performed on the animals that received the injection to confirm that there were no abnormalities in overall retinal function, and on day 29 after administration, the eyes of all mice that received the injection were enucleated to separate the neural retina and RPE tissues.

[0529]

[0530] 2-1-2. NGS for Mouse RPE

[0531] Genomic DNA and RNA were extracted from isolated RPE tissues using homogenization and the Quick DNA / RNA Miniprep Plus kit (Zymo Research). The extracted RNA was synthesized using the cDNA Reverse-transcription kit (ThermoFisher), and the capsid library region was amplified using Phusion High-Fidelity DNA Polymerase (NEB) and primers (Table 01). Subsequently, the samples were purified using the Fragment DNA purification kit (intronbio). The purified samples were prepared for NGS-ready through Truseq Nano DNA library construction, and sequencing reads were produced using paired-end sequencing on the Illumina Novaseq platform 150PE.

[0532]

[0533] 2-1-3. Analysis of RPE Delivery Function of AAV Libraries

[0534] The analysis of delivery efficiency was performed by calculating the frequency (Frequency_RPE) for each sequence (i.e., each AAV variant) from NGS data on RPE tissue cDNA and comparing this value with the frequency ratio (Frequency_RPE / Frequency_V) value, which was normalized by the frequency (Frequency_V) for each sequence (i.e., each AAV variant) within the AAV library used for injection. For each injection route (subretinal, intravitreal), the frequency ratio (Frequency_RPE / Frequency_V) values ​​for each variant analyzed for 6 individuals were compared with the frequency ratio (Frequency_RPE / Frequency_V) values ​​of the reference WT AAV2, and statistical significance was analyzed using a Student's t-test. At this time, the frequency ratio (Frequency_RPE / Frequency_V) of WT AAV2 per individual was the average frequency ratio (Frequency_RPE / Frequency_V) of the 100 injected sequences.

[0535] Statistical analysis was performed using the Student t-test, and a significance criterion of p<0.05 was used.

[0536]

[0537] 2-2. Results

[0538] AAV libraries were injected into mouse eyes using subretinal injection and intravitreal injection, the two most commonly used Route of Administration (ROA) in gene therapy clinical practice, and sequences with superior delivery function compared to WT AAV2 for each ROA were analyzed. For the delivery function analysis, the frequency value (Frequency_RPE) of the NGS for RNA samples expressed on the CAG promoter after delivery to RPE (Retinal Pigment Epithelium cells) was analyzed, and the frequency ratio (Frequency_RPE / Frequency_V), normalized by the frequency (Frequency_V) of the AAV library used for injection, was calculated for each capsid sequence and compared. For each ROA, for the 6 individuals used, capsid sequences (AAV variants) with superior delivery capabilities compared to WT AAV2 were analyzed by comparing them with the frequency ratio (Frequency_RPE / Frequency_V) value of WT AAV2 used as a benchmark.

[0539] For subretinal injection, out of a total of 96,400 AAVs in the AAV library, 85,164 AAV variants were identified as having lower delivery efficiency compared to wild-type AAV2, and 11,236 (approximately 11%) were identified as having higher delivery efficiency compared to wild-type AAV2 [AVs with a (Frequency_RPE / Frequency_V) value greater than 1 times that of wild-type AAV]. Furthermore, 28 AAV variants with statistically significantly superior RPE delivery function in subretinal injection were identified, and the results of comparing these 28 AAV variants with WT AAV2 are shown in Fig. 03. Specifically, Fig. 03 shows the fold change (AAV frequency ratio / WT AAV2 frequency ratio) value calculated through the frequency ratio of the 28 AAV variants and the WT AAV2 frequency ratio. All 28 AAV variants showed RPE delivery more than 10 times higher than WT AAV2, and among them, 8 AAVs demonstrated delivery function more than 50 times higher. The 3 AAV variants with the highest delivery function showed delivery superiority of 125 times, 115 times, and 89 times, respectively, compared to WT AAV2. Statistical analysis was performed using the Student's t-test, and sequences corresponding to p<0.05 were classified as significant sequences. In Figure 03, SR refers to subretinal injection, and RPE-SR-1 to RPE-SR-28 represent each of the 28 AAV variants.

[0540] The VP1 561-588 amino acid sequences (amino acid sequences of the variant region) of the capsids of these 28 AAV variants (RPE-SR 1 to RPE-SR-28) and the DNA sequences encoding them are shown in Table 02, and the frequency ratio (Frequency_RPE / Frequency_V), t-test results for comparison of WT AAV2 delivery function, and fold change comparison results with WT AAV2 are shown in Table 03.

[0541] [Table 02] VP1 561-588 amino acid sequences of WT AAV2 and 28 AAV variant capsids and the DNA sequences encoding them

[0542]

[0543]

[0544]

[0545] [Table 03] Results of comparison of frequency ratios of 28 AAV variants in each mouse and delivery efficiency with WT AAV2

[0546]

[0547]

[0548] For intravitreal injection, out of a total of 96,400 AAVs in the library, 88,773 AAV variants were identified as having lower delivery efficiency compared to wild-type AAV2, and 7,627 AAV variants (approximately 7%) were identified as having higher delivery efficiency compared to wild-type AAV2 [AVs with a (Frequency_RPE / Frequency_V) value greater than 1 times that of wild-type AAV]. Furthermore, five AAV variants with statistically significantly superior RPE delivery function in intravitreal injection were identified, and the results of comparing these five AAV variants with WT AAV2 are shown in Fig. 04. Specifically, Fig. 04 shows the fold change (AAV frequency ratio / WT AAV2 frequency ratio) value calculated through the frequency ratio of the five AAV variants and the WT AAV2 frequency ratio. All five AAV variants showed a delivery function more than 9 times higher than WT AAV2, and among them, three AAV variants showed high delivery functions of 34 times, 19 times, and 19 times, respectively, compared to WT AAV2. In Fig. 04, IVT refers to intravitreal injection, and RPE-IVT-1 to RPE-IVT-5 represent each of the five AAV variants.

[0549] The VP1 561-588 amino acid sequences (amino acid sequences of the variant region) of the capsids of these five AAV variants (RPE-IVT-1 to RPE-IVT-5) and the DNA sequences encoding them are shown in Table 04, and the frequency ratio (Frequency_RPE / Frequency_V), t-test results for comparing WT AAV2 delivery function, and fold change comparison results with WT AAV2 are shown in Table 05.

[0550] Meanwhile, RPE-SR-20 (variant 9), RPE-SR-19 (variant 10), and RPE-SR-15 (variant 14) were confirmed to have excellent delivery capabilities even in intravitreal injection (RPE-SR-20 is identical to RPE-IVT-1, RPE-SR-19 is identical to RPE-IVT-2, and RPE-SR-15 is identical to RPE-IVT-3).

[0551]

[0552] [Table 04] VP1 561-588 amino acid sequences of the capsids of WT AAV2 and 5 AAV variants and the DNA sequences encoding them

[0553]

[0554]

[0555] [Table 05] Results of comparison of frequency ratios of 5 AAV variants and delivery efficiency with WT AAV2

[0556]

[0557]

[0558] The full amino acid sequences of VP1, VP2, and VP3 of the capsids of variants 1 to 30 are disclosed in Table 06.

[0559] [Table 06] Total amino acid sequences of VP1, VP2, and VP3 of variants 1 to 30

[0560]

[0561]

[0562] Meanwhile, the AAV2 variant developed through this experimental example holds additional significance in that, in addition to possessing excellent RPE delivery capabilities in mice, it serves as an important foundation for developing superior AAVs in human or non-human primates. The literature [Dalkara, D., Byrne, LC, Klimczak, RR, Visel, M., Yin, L., Merigan, WH, ... & Schaffer, DV (2013). In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Science translational medicine, 5(189), 189ra76-189ra76.] discloses that an AAV2 variant library was constructed, and after injecting this library via intravitreal injection in mice, a 7m8 capsid was developed through screening. This 7m8 was confirmed to possess excellent delivery capabilities in the primate retina as well. Furthermore, the safety and efficacy of this 7m8 have been verified, and a phase 2 clinical program is currently underway (see reference [Khanani, AM, Boyer, DS, Wykoff, CC, Regillo, CD, Busbee, BG, Pieramici, D., ... & Kiss, S. (2024). Safety and efficacy of ixoberogene soroparvovec in neovascular age-related macular degeneration in the United States (OPTIC): a prospective, two-year, multicentre phase 1 study. EClinicalMedicine, 67.]). As such, the AAV developed through mouse experiments can serve as an important foundation for developing superior AAVs in primates in the future.

Claims

1. AAV variants including the following: AAV capsid variant, At this time, the above AAV capsid variant includes a VP1 variant having the amino acid sequences of SEQ ID NOs 02 to 31.

2. In Paragraph 1, The above AAV capsid variant is an AAV variant selected from any one of the following: An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 02, a VP2 variant having the amino acid sequence of SEQ ID NO. 33, and a VP3 variant having the amino acid sequence of SEQ ID NO. 64; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 03, a VP2 variant having the amino acid sequence of SEQ ID NO. 34, and a VP3 variant having the amino acid sequence of SEQ ID NO. 65; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 04, a VP2 variant having the amino acid sequence of SEQ ID NO. 35, and a VP3 variant having the amino acid sequence of SEQ ID NO. 66; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 05, a VP2 variant having the amino acid sequence of SEQ ID NO. 36, and a VP3 variant having the amino acid sequence of SEQ ID NO. 67; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 06, a VP2 variant having the amino acid sequence of SEQ ID NO. 37, and a VP3 variant having the amino acid sequence of SEQ ID NO. 68; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 07, a VP2 variant having the amino acid sequence of SEQ ID NO. 38, and a VP3 variant having the amino acid sequence of SEQ ID NO. 69; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 08, a VP2 variant having the amino acid sequence of SEQ ID NO. 39, and a VP3 variant having the amino acid sequence of SEQ ID NO. 70; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 09, a VP2 variant having the amino acid sequence of SEQ ID NO. 40, and a VP3 variant having the amino acid sequence of SEQ ID NO. 71; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 10, a VP2 variant having the amino acid sequence of SEQ ID NO. 41, and a VP3 variant having the amino acid sequence of SEQ ID NO. 72; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 11, a VP2 variant having the amino acid sequence of SEQ ID NO. 42, and a VP3 variant having the amino acid sequence of SEQ ID NO. 73; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 12, a VP2 variant having the amino acid sequence of SEQ ID NO. 43, and a VP3 variant having the amino acid sequence of SEQ ID NO. 74; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 13, a VP2 variant having the amino acid sequence of SEQ ID NO. 44, and a VP3 variant having the amino acid sequence of SEQ ID NO. 75; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 14, a VP2 variant having the amino acid sequence of SEQ ID NO. 45, and a VP3 variant having the amino acid sequence of SEQ ID NO. 76; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 15, a VP2 variant having the amino acid sequence of SEQ ID NO. 46, and a VP3 variant having the amino acid sequence of SEQ ID NO. 77; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 16, a VP2 variant having the amino acid sequence of SEQ ID NO. 47, and a VP3 variant having the amino acid sequence of SEQ ID NO. 78; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 17, a VP2 variant having the amino acid sequence of SEQ ID NO. 48, and a VP3 variant having the amino acid sequence of SEQ ID NO. 79; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 18, a VP2 variant having the amino acid sequence of SEQ ID NO. 49, and a VP3 variant having the amino acid sequence of SEQ ID NO. 80; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 19, a VP2 variant having the amino acid sequence of SEQ ID NO. 50, and a VP3 variant having the amino acid sequence of SEQ ID NO. 81; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 20, a VP2 variant having the amino acid sequence of SEQ ID NO. 51, and a VP3 variant having the amino acid sequence of SEQ ID NO. 82; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 21, a VP2 variant having the amino acid sequence of SEQ ID NO. 52, and a VP3 variant having the amino acid sequence of SEQ ID NO. 83; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 22, a VP2 variant having the amino acid sequence of SEQ ID NO. 53, and a VP3 variant having the amino acid sequence of SEQ ID NO. 84; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 23, a VP2 variant having the amino acid sequence of SEQ ID NO. 54, and a VP3 variant having the amino acid sequence of SEQ ID NO. 85; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 24, a VP2 variant having the amino acid sequence of SEQ ID NO. 55, and a VP3 variant having the amino acid sequence of SEQ ID NO. 86; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 25, a VP2 variant having the amino acid sequence of SEQ ID NO. 56, and a VP3 variant having the amino acid sequence of SEQ ID NO. 87; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 26, a VP2 variant having the amino acid sequence of SEQ ID NO. 57, and a VP3 variant having the amino acid sequence of SEQ ID NO. 88; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 27, a VP2 variant having the amino acid sequence of SEQ ID NO. 58, and a VP3 variant having the amino acid sequence of SEQ ID NO. 89; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 28, a VP2 variant having the amino acid sequence of SEQ ID NO. 59, and a VP3 variant having the amino acid sequence of SEQ ID NO. 90; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 29, a VP2 variant having the amino acid sequence of SEQ ID NO. 60, and a VP3 variant having the amino acid sequence of SEQ ID NO. 91; An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 30, a VP2 variant having the amino acid sequence of SEQ ID NO. 61, and a VP3 variant having the amino acid sequence of SEQ ID NO. 92; and An AAV capsid variant comprising a VP1 variant having the amino acid sequence of SEQ ID NO. 31, a VP2 variant having the amino acid sequence of SEQ ID NO. 62, and a VP3 variant having the amino acid sequence of SEQ ID NO.

93.

3. In any one of paragraphs 1 to 2, The above AAV variant further comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid encoding a product of interest, and wherein the nucleic acid molecule is an AAV variant packaged in the AAV capsid variant.

4. In Paragraph 3, The nucleic acid molecule further comprises a promoter, wherein the promoter is operably linked to the nucleic acid encoding the product of interest, an AAV variant.

5. In Paragraph 4, The nucleic acid molecule further comprises two ITRs, wherein the nucleic acid encoding the promoter and the product of interest is an AAV variant located between the two ITRs.

6. In any one of paragraphs 3 through 5, The above nucleic acid molecule is an AAV variant that is single-stranded DNA.

7. In any one of paragraphs 1 through 6, The above AAV variant is an AAV variant having enhanced delivery ability to RPE (retinal pigment epithelium) tissue or RPE cells.

8. In any one of paragraphs 1 through 7, At this time, the AAV capsid variant of the above AAV variant is an AAV capsid variant comprising a VP1 variant having an amino acid sequence selected from any one of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31, and At this time, the above AAV variant is an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject via intravitreal injection.

9. In any one of paragraphs 1 through 7, At this time, the AAV capsid variant of the above AAV variant is an AAV capsid variant comprising a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29, and At this time, the above AAV variant is an AAV variant having enhanced RPE tissue or RPE cell delivery ability when administered to a subject via subretinal injection.

10. AAV variants including the following: AAV capsid variant, At this time, the above AAV capsid variant comprises a VP1 variant having an amino acid sequence selected from SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 15, SEQ ID NO. 30, and SEQ ID NO. 31, and At this time, the above AAV variant is an AAV variant having enhanced ability to deliver to RPE (retinal pigment epithelium) tissue or RPE cells when administered to a subject via intravitreal injection.

11. AAV variants including the following: AAV capsid variant, At this time, the above AAV capsid variant comprises a VP1 variant having an amino acid sequence selected from any one of SEQ ID NOs 02 to 29, and At this time, the above AAV variant is an AAV variant having enhanced retinal pigment epithelium (RPE) tissue or RPE cell delivery ability when administered to a subject via subretinal injection.

12. A method for producing AAV variants including the following: A cap gene having a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs 02 to 31, and a rep-cap plasmid comprising a rep gene, A transport plasmid containing a nucleic acid encoding the product of interest, and Helper plasmid containing a helper gene Introducing into a host cell; and AAV variants were obtained from host cells into which the above plasmids were introduced.

13. In Paragraph 12, The above host cell is of mammalian origin, method.

14. Engineered host cells for the production of AAV variants, including the following: The above-described engineered host cell is a rep-cap plasmid comprising a cap gene having a nucleic acid sequence encoding any one amino acid sequence selected from SEQ ID NOs 02 to 31, and a rep gene.

15. In paragraph 14, the engineered host cell further comprises a transport plasmid containing a nucleic acid encoding a product of interest, and a helper plasmid.

16. In any one of paragraphs 14 to 15, the host cell is a engineered host cell of mammalian origin.

Images