Novel compositions of adeno-associated viruses engineered to enhance tissue transduction and specificity

Abstract

The invention relates to the production of engineered adeno-associated viruses. More specifically, the present invention provides a modified adeno-associated virus comprising a nucleotide sequence encoding a 7-mer peptide inserted into the capsid. Also provided are uses of the engineered adeno-associated viruses for enhancing tissue transduction and specificity, and compositions thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Singapore Application No. 10202300637W, filed on 9 March 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] This invention belongs to the field of biotechnology. Specifically, this invention relates to the generation of engineered adeno-associated viruses. More specifically, this invention provides the use of engineered adeno-associated viruses for enhancing tissue transduction and specificity, and compositions thereof. Background of the Invention

[0005] Adeno-associated virus (AAV) vector-based therapies are rapidly entering clinical practice. Systemic vector infusion represents an ideal delivery route for many gene therapy applications, especially when local administration is not feasible. Currently, most AAV capsids are based on naturally occurring serotypes that are isolated in the liver, resulting in reduced efficacy in organs such as the central nervous system (CNS) or skeletal muscle. Consequently, higher vector doses are required, leading to off-target toxicity. Therefore, there is a need for AAV capsids with organ-specific targeting at low vector doses.

[0006] Overview

[0007] In one aspect, this document provides a modified adeno-associated virus serotype DJ (AAV-DJ) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1767 and 1768 of SEQ ID NO:399, wherein the modified AAV-DJ has at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with the sequence of SEQ ID NO:399, in addition to the nucleotide sequence encoding the 7-mer peptide.

[0008] On the other hand, this document provides a modified adeno-associated virus serotype 2 (AAV2) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:401, wherein the modified AAV2, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:401 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0009] On the other hand, this document provides a modified adeno-associated virus serotype 5 (AAV5) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1722 and 1723 of SEQ ID NO:402, wherein the modified AAV5 has at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with the sequence of SEQ ID NO:402, except for the nucleotide sequence encoding the 7-mer peptide.

[0010] On the other hand, this document provides a modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1770 and 1771 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0011] On the other hand, this document provides a modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0012] On another front, this document provides a modified adeno-associated virus serotype 9 (AAV9) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:404, wherein the modified AAV9, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:404 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0013] On the other hand, this document provides compositions comprising any modified AAV as described herein and pharmaceutically acceptable excipients.

[0014] On the other hand, this paper provides nucleic acids that encode AAVs with any modifications as described herein.

[0015] definition

[0016] As used herein, the term “AAV” or “adeno-associated virus” refers to a nonpathogenic small virus of the Parvoviridae family that infects humans. The term “AAV” includes, but is not limited to, naturally occurring and engineered AAV subtypes or serotypes, such as AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), and AAV-DJ.

[0017] As used herein, the term "capsid" refers to the protein shell of a viral particle. A viral capsid contains one or more capsid units or subunits. For example, the capsid of AAV contains VP1, VP2, and / or VP3 subunits. The viral capsid can serve multiple functional purposes. For example, it can protect the viral genome from degradation, help the virus evade the host's immune response, and / or determine the virus's tropism towards different cell or tissue types.

[0018] As used herein, the term "identity" in the context of polynucleotide or polypeptide sequences refers to the overall relevance or homology between polymer molecules (e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules). For example, the percentage of identity between two polynucleotide sequences can be calculated by aligning the two sequences for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and dissimilar sequences can be ignored for comparison purposes). The nucleotides at corresponding nucleotide positions are then compared. The molecules are identical at that position when a position in the first sequence is occupied by the same nucleotide as a corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared positions, taking into account the number of vacancies and the length of each vacancy that needs to be introduced for optimal alignment. Mathematical algorithms can be used to perform sequence comparisons and determine the percentage of identity between two sequences.

[0019] As used herein, the term "library" refers to a mixture of heterogeneous peptides or polynucleotides. A library contains members with similar peptide or polynucleotide sequences. Sequence differences between library members are the cause of library diversity. Libraries can be in the form of a mixture of peptides or polynucleotides, or they can be in the form of an organism or cell transformed with a polynucleotide library, such as yeast cells.

[0020] As used herein, “modified virus” and “viral variant” are interchangeable terms referring to an engineered or recombinant virus that contains alterations (i.e., “modifications”) in the sequence of a polypeptide or polynucleotide encoding a polypeptide compared to a reference sequence. The reference sequence can be a viral genome sequence or a subsection thereof, or an amino acid sequence encoded by a viral genome sequence or a subsection thereof. The reference sequence can be a sequence derived from a wild-type or natural virus, or it can be a sequence derived from a virus with different modifications. A wild-type virus is a virus whose phenotype or genotype is dominant in nature. A “modified AAV” or “variant AAV” refers to an AAV that has changes in its genome sequence or a subsection thereof, or in the polypeptide encoded by its genome sequence or a subsection thereof, compared to a reference AAV. Sequence alterations or modifications can refer to the insertion, deletion, or substitution of one or more residues in the sequence. For example, a modified AAV may be an AAV that has one or more nucleotides inserted into its genome sequence or a sub-part of its genome sequence compared to a reference AAV, or an AAV that has one or more amino acids inserted into a polypeptide encoded by its genome sequence or a sub-part of its genome sequence.

[0021] As used herein, “peptide” refers to a condensation product of two or more amino acids, wherein the length of the polypeptide is less than or equal to 50 amino acids, for example, 5, 6, 7, 8, 9, 10, 11-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 or 45-50 amino acids.

[0022] As used herein, “targeting” and “tropism” are interchangeable terms referring to the ability of a virus to infect different cell types to produce a successful infection. Successful infection can be defined as when a virus enters a host cell, when a virus begins to replicate its genome within a host cell, when intact progeny viruses are detectable within a host cell, when viral progeny leave a host cell, or any combination thereof. In one instance, a CNS-tropy virus or a CNS-targeting virus indicates that the virus is capable of infecting cells originating from or derived from the CNS and / or that the virus is capable of infecting cells originating from or derived from the CNS with higher specificity than other cell types. In another instance, a muscle-tropy virus or a muscle-targeting virus refers to a virus capable of successfully infecting cells originating from or derived from skeletal muscle and / or that is capable of infecting cells originating from or derived from muscle with higher specificity than other cell types. Conversely, a virus with no tropism or reduced tropism for a particular cell type cannot infect that cell type, or its ability to infect that cell type is reduced. For example, a liver-reduced tropism or liver-detargeting virus indicates that the virus is less capable or unable to successfully infect cells originating from or derived from the liver. Those skilled in the art will understand that “tropism” can also mean “specificity.” For example, CNS-oriented viruses mean that the virus is specific to the CNS. Therefore, as used herein, the terms "oriented" and "specific" are interchangeable and refer to the ability of a virus to successfully infect a selected cell type. Furthermore, a virus can also be defined as "specifically targeted" or "specifically oriented" to indicate its specificity to a selected tissue type. For example, a brain-specific oriented virus is a virus that specifically infects the brain. Brief description of the attached diagram

[0024] The invention will be better understood when considered in conjunction with the detailed description in the accompanying drawings, which are non-limiting examples:

[0025] Figure 1 The diagram illustrates the construction of the AAV-DJ library. The top image shows the AAV-DJ library plasmid, where gray lines represent insertion sites for the peptide library (between N589 and R590), and the bottom image shows the full-length Rep2 plasmid. Abbreviation: ITR, inverted terminal repeat sequence.

[0026] Figure 2 The evolution of the AAV-DJ peptide library driven by next-generation sequencing (NGS) was demonstrated. 2.5 × 10⁻⁶ peptides were injected intravenously into C57BL / 6 mice (n=5). 9 A Vg / mouse AAV-DJ peptide library was obtained. After 28 days, organs were collected, mRNA was enriched, and capsid DNA variants were amplified and analyzed by NGS. Figure 2A, 2B, and 2C show the length distribution of inserted sequences in AAV-DJ capsid DNA sequenced from liver, brain, and muscle, respectively. The X-axis represents the length of the inserted nucleotide sequence, and the Y-axis represents the probability density of each nucleotide sequence length.

[0027] Figure 3 A table showing the fold change variance values ​​of selected AAV-DJ variants detected by NGS, normalized against wild-type AAV-DJ, resulted in the identification of AAV-DJ variants with enhanced brain and muscle transduction efficiency at low vector doses and complete liver detargeting. The labels on the left indicate AAV-DJ variants, corresponding to the peptides listed in Table 3. The values ​​in the table represent the fold change of each variant in the liver, brain, and muscle compared to wild-type AAV-DJ. The bar on the right represents the fold change of the modified AAV genome sequenced from each organ relative to wild-type. Lighter hues indicate higher fold changes, darker hues indicate lower fold changes, and black indicates no change (baseline). Lower or absent increases in fold change in an organ indicate reduced or absent infectivity or tropism in that organ, while higher or absent increases in fold change in an organ indicate increased or present infectivity or tropism in that organ. Invention Details

[0029] This disclosure provides modified AAVs that alter the orientation of multiple cell types, tissue types, and / or organs compared to reference AAVs.

[0030] The reference AAV can be wild-type AAV or unmodified AAV, and it can have the same or different serotypes as modified AAV.

[0031] Viruses or viral variants can be modified to alter their tropism or targeting toward one or more selected cell types, tissues, and / or organs. For example, wild-type viruses can be modified to have enhanced, reduced, or no tropism toward one or more selected organs, tissues, and / or cell types. In some instances, the reduction in tropism reaches the point of elimination, meaning the virus is no longer able to infect one or more selected organs, tissues, and / or cell types. Organs can be any organ from humans or animals. For example, organs can be the CNS, ear, eye, heart, intestine, kidney, joint / synovium, lung, liver, pancreas, or skeletal muscle. Tissues can be primary or clinical isolates from organs, or can be cultured tissues derived from organs. Cells can be primary or clinical isolates from organs or tissues, or can be cultured cells derived from organs or tissues.

[0032] AAVs with altered tropism can be obtained, for example, by introducing one or more mutations into the viral genome or a portion thereof or into a specific gene. For example, an AAV can be modified by introducing a mutation into one or more genes encoding a capsid protein. Mutations can be introduced by any method known to those skilled in the art (e.g., site-directed mutagenesis or site-saturation mutagenesis) and can include nucleotide insertions or deletions (frameshift mutations), nucleotide substitutions / replacements, or combinations thereof. For example, site-saturation mutagenesis can be used to replace selected sites in a polypeptide with all possible amino acids by using degenerate codons in the nucleotide sequence encoding the polypeptide. In an example, the degenerate codon is NNK, where K represents a keto nucleotide, such as guanine and thymine; and N represents any nucleotide. Those skilled in the art will understand that modification of a polynucleotide sequence encoding a gene can result in modification of the polypeptide encoded by that polynucleotide sequence. It should also be understood that mutation in the context of this invention can refer to mutations in viral polynucleotide and / or viral polypeptide sequences.

[0033] In one example, a wild-type or reference AAV is modified by inserting a polynucleotide sequence encoding a peptide into a desired site in the nucleotide sequence of a reference or wild-type AAV. The reference or wild-type AAV may include, but is not limited to, serotypes AAV-2, AAV-5, AAV-6, AAV-9, and AAV-DJ (Table 4). Those skilled in the art will understand that the resulting AAV, i.e., the “modified AAV,” has the same nucleotide sequence as the reference or wild-type AAV (i.e., the unmodified AAV), except for the nucleotide sequence encoding the inserted peptide.

[0034] The desired site for inserting the polynucleotide sequence can be located within a coding or non-coding region of the viral genome. In one instance, wild-type or reference AAV can be modified by inserting one or more amino acids or peptides into a polypeptide sequence encoding one or more proteins that form part of the capsid or virus.

[0035] The length of the peptide inserted to alter the tropism of the wild-type or reference virus can vary. For example, the peptide length can be 5-15 amino acids. As a non-limiting example, the peptide length can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. For example, the inserted peptide can be a 5-mer, 6-mer, 7-mer, 8-mer, 9-mer, or 10-mer peptide. In one example, the peptide is a 7-mer peptide. The amino acid sequence of a 7-mer peptide can contain any arrangement of 7 amino acids. In some instances, the amino acids are the 20 standard amino acids. Therefore, this document provides AAVs modified with a 7-mer peptide sequence, wherein the peptide sequence is any arrangement of the 20 standard amino acids. Those skilled in the art will understand which amino acids are the 20 standard amino acids.

[0036] Those skilled in the art will understand that certain methods of modifying polypeptide sequences, such as site saturation mutagenesis, allow screening for each of 20 standard amino acids at selected sites in the polypeptide sequence. Therefore, the peptides listed in this disclosure are non-limiting examples of 7-mer peptides possessing the property of conferring AAV alterations upon insertion into a viral capsid.

[0037] This document provides modified AAVs comprising a nucleotide sequence encoding a 7-mer peptide inserted between two nucleotides of a reference or wild-type AAV capsid gene. These two nucleotides may be within the VP1, VP2, and / or VP3 gene sequences. In one example, the two nucleotides are within the VP3 gene sequence. Those skilled in the art will understand that the VP1 gene encodes the full-length capsid, while the VP2 and VP3 genes encode capsid subunits. Those skilled in the art will also recognize that the nucleotide sequences of the VP2 and VP3 genes are contained within the VP1 nucleotide sequence.

[0038] In one aspect, this document provides a modified adeno-associated virus serotype DJ (AAV-DJ) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1767 and 1768 of SEQ ID NO:399, wherein the modified AAV-DJ has at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with the sequence of SEQ ID NO:399, in addition to the nucleotide sequence encoding the 7-mer peptide.

[0039] On the other hand, this document provides a modified adeno-associated virus serotype 2 (AAV2) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:401, wherein the modified AAV2, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:401 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0040] On the other hand, this document provides a modified adeno-associated virus serotype 5 (AAV5) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1722 and 1723 of SEQ ID NO:402, wherein the modified AAV5 has at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with the sequence of SEQ ID NO:402, except for the nucleotide sequence encoding the 7-mer peptide.

[0041] On the other hand, this document provides a modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1770 and 1771 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0042] On the other hand, this document provides a modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0043] On the other hand, this document provides a modified adeno-associated virus serotype 9 (AAV9) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:404, wherein the modified AAV9, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:404 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

[0044] It is generally understood that the sequence identity of a virus (e.g., a modified virus) compared to a reference virus (e.g., a wild-type virus) can vary. For example, a virus may have at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with a reference virus. Comparisons can be made between AAVs of the same or different serotypes. For example, AAV-DJ may have at least 50% sequence identity with reference AAV-2, AAV-5, AAV-6, AAV-9, or AAV-DJ viruses. In one instance, AAV-DJ has at least 57% sequence identity with reference AAV-5.

[0045] In one instance, this document provides a modified AAV-DJ as described herein, wherein the modified AAV-DJ has at least 57% sequence identity with the sequence of SEQ ID NO:399 outside the 7-mer peptide sequence.

[0046] In another instance, this document provides a modified AAV2 as described herein, wherein the modified AAV2 has at least 57% sequence identity with the sequence of SEQ ID NO:401 outside the 7-mer peptide sequence.

[0047] In another instance, this document provides a modified AAV5 as described herein, wherein the modified AAV5 has at least 57% sequence identity with the sequence of SEQ ID NO:402 outside the 7-mer peptide sequence.

[0048] In another example, this document provides a modified AAV6 comprising a sequence corresponding to a 7-mer peptide inserted between nucleotides 1770 and 1771 of SEQ ID NO:403, wherein the modified AAV6 has at least 57% sequence identity with the sequence of SEQ ID NO:403 outside the 7-mer peptide sequence.

[0049] In yet another example, this document provides a modified AAV6 comprising a sequence corresponding to a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:403, wherein the modified AAV6 has at least 57% sequence identity with the sequence of SEQ ID NO:403 outside the 7-mer peptide sequence.

[0050] In yet another instance, this document provides a modified AAV9 as described herein, wherein the modified AAV9 has at least 57% sequence identity with the sequence of SEQ ID NO:404 outside the 7-mer peptide sequence.

[0051] In one example, the insert peptide of the present invention may comprise the sequence shown in Table 1, and imparts enhanced liver, brain, and muscle targeting to the modified AAV compared to a reference or wild-type AAV. In another example, the targeting peptide may comprise the sequence shown in Table 2, and imparts enhanced brain and muscle targeting, and reduced liver targeting, to the modified AAV compared to wild-type AAV. In yet another example, the targeting peptide may comprise the sequence shown in Table 3, and imparts enhanced brain and muscle targeting, and deliverable targeting, to the modified AAV compared to wild-type AAV.

[0052] Compared to a reference virus, the modified virus or viral variant of the present invention may have increased or decreased specificity for one or more selected cell types. Increased or decreased specificity may refer to an enhanced ability of the virus to bind to and / or infect said cell type. The increase or decrease in viral specificity for a particular cell type can be compared to the specificity of a reference cell type and can be measured using quantitative methods known to those skilled in the art. The degree of enhancement or reduction in tropism of the modified AAV relative to a reference AAV (e.g., wild-type AAV) can be calculated by comparing the infectivity of the variant in selected tissue samples with that of the reference virus. Those skilled in the art will understand methods for measuring viral infectivity. For example, viral infectivity can be measured by plaque assay, enzyme-linked immunosorbent assay (ELSA), transduction assay using a reporter gene, polymerase chain reaction (PCR), high-throughput sequencing, or any combination thereof. In one example, the infectivity or tropism of AAV is assessed by next-generation sequencing (NGS).

[0053] The infectivity or tropism of an AAV variant relative to a reference or original AAV can be compared by dividing the number of reads of the AAV variant in a selected tissue sample (e.g., brain, muscle, or liver) by the number of reads of the reference or original virus in the tissue sample, to obtain the fold change in infectivity or tropism of the AAV variant relative to the reference or original virus, where infectivity is measured by NGS. When an AAV variant has a fold change >1 relative to the reference or original AAV in a tissue type, its tropism to that tissue type is increased or improved compared to the reference or original virus. When an AAV variant has a fold change <1 relative to the reference or original AAV in a tissue type, its tropism to that tissue type is decreased or weakened compared to the reference or original virus. When an AAV variant has a fold change =1 relative to the reference or original AAV in a tissue type, it has the same or similar tropism to that tissue type compared to the reference or original virus. When an AAV variant has a multiple of 0 relative to the reference or original AAV in a tissue type, it is either undetectable in that tissue type compared to the reference or original virus, or detargeted to that tissue type.

[0054] Table 1: AAV-DJ variants with enhanced brain, muscle, and liver orientation

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Table 2: AAV-DJ variants with enhanced brain and muscle tropism and reduced liver tropism

[0064]

[0065]

[0066]

[0067] Table 3: AAV-DJ variants with excellent brain and muscle orientation and liver detargeting

[0068]

[0069] In one instance, the modified AAV contained a 7-mer peptide sequence selected from the sequences shown in Table 1, and the insertion increased brain, muscle, and liver tropism compared to wild-type AAV without the inserted 7-mer peptide sequence.

[0070] In another example, the modified AAV contained a 7-mer peptide sequence selected from the sequences shown in Table 2, and compared with wild-type AAV without the inserted 7-mer peptide sequence, the insertion increased brain and muscle orientation and decreased liver orientation.

[0071] In yet another example, the modified AAV contains a 7-mer peptide sequence selected from the sequences shown in Table 3, and the insertion therein increases brain and muscle affinity and is deliverable to the liver.

[0072] In yet another instance, the modified AAV comprises a 7-mer peptide sequence selected from the group consisting of SEQ ID NO: 22, 24, 32, 85, 106, 198, 216, 232, 251, 293, 312, 320, 321, 224, 346, or 376.

[0073] Table 4: AAV nucleotide sequences

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] In any DNA sequence mentioned and / or described herein, single-letter symbols have the following descriptions: A represents adenine; C represents cytosine; G represents guanine; T represents thymine; K represents a ketone nucleotide, such as guanine and thymine; and N represents any nucleotide (which is not a vacancy). The use of the NNK codon in site-saturated mutagenesis allows for codons covering all 20 standard amino acids, including degenerate codons. A “degenerate codon” is a codon that differs in one or two nucleotides but encodes the same amino acid. For example, the amino acid alanine can be encoded by codons GCT, GCC, GCA, or GCG, and the amino acid leucine can be encoded by codons CTT, CTC, CTA, CTG, TTA, and TTG.

[0090] In any amino acid sequence mentioned and / or described herein, the single-letter symbols have the following descriptions: G (Gly) represents glycine; A (Ala) represents alanine; L (Leu) represents leucine; M (Met) represents methionine; F (Phe) represents phenylalanine; W (Trp) represents tryptophan; K (Lys) represents lysine; Q (Gln) represents glutamine; E (Glu) represents glutamic acid; S (Ser) represents serine; P (Pro) represents proline; V (Val) represents valine; I (Ile) represents isoleucine; C (Cys) represents cysteine; Y (Tyr) represents tyrosine; H (His) represents histidine; R (Arg) represents arginine; N (Asn) represents asparagine; D (Asp) represents aspartic acid; and T (Thr) represents threonine.

[0091] The modified AAVs described herein may possess advantageous properties for research and / or therapeutic applications due to their enhanced tropism towards organ, tissue, and / or cell types. For example, modified AAVs with enhanced tropism can be used at lower carrier doses, resulting in reduced off-target toxicity. Modified AAVs with enhanced tropism can also allow for the modulation of anti-capsid immune responses and can be used for therapeutic purposes, such as CRISPR delivery and gene enhancement therapy. This targeting can enhance the specificity of AAVs for desired cell types and improve overall therapeutic efficacy. In yet another example, in the case of liver targeting, hepatic sinusoidal endothelial cells (LSECs) and hepatocytes, as well as other cell types in the liver, can be targeted, while in the case of brain targeting, neurons and astrocytes, as well as other cell types in the brain, can be targeted. Furthermore, LSEC targeting can reduce the induction of immune responses to the AAV capsid. The modified AAVs described herein may also possess advantageous properties for research and / or therapeutic applications due to their reduced tropism towards organ, tissue, and / or cell types. For example, AAVs with reduced tropism modifications can be used for treatment at lower vector doses because they are not isolated in the liver, thus reducing off-target toxicity. The AAV dose in treatment can be expressed as the amount of vector genome or viral genome administered to the subject. For example, the dose can be specified as the amount of vector genome per body weight (vg / kg), or the amount of vector genome per volume (vg / ml or vg / L), or the amount of vector genome per animal (vg / animal). The therapeutically effective vector dose can be a range. For example, depending on the route and site of administration, the effective dose per animal can be as low as 1 × 10⁻⁶. 9 Up to 1×10 17 vg.

[0092] In one instance, this document provides a modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences shown in Table 1 or Table 7, and wherein the insertion increases specificity for cells selected from the group consisting of hepatic sinusoidal endothelial cells or hepatocytes compared to wild-type AAV without the inserted 7-mer peptide sequence.

[0093] In another instance, this document provides modified AAVs as described herein, wherein the 7-mer peptide sequence is selected from the sequences shown in Table 1, Table 2, or Table 3, and wherein the insertion increases the specificity for cells selected from groups composed of neurons or astrocytes compared to wild-type AAVs without the inserted 7-mer peptide sequence.

[0094] In another instance, this document provides a modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences shown in Table 7, and wherein the insertion increases the generation of antigen-specific regulatory T cells compared to wild-type AAV without the inserted 7-mer peptide sequence.

[0095] In some instances, inserting a 7-mer peptide sequence can reduce the induction of an immune response to the modified AAV capsid compared to wild-type AAV without the inserted 7-mer peptide sequence.

[0096] In other instances, the insertion of a 7-mer peptide sequence modulates the anti-capsid immune response compared to wild-type AAVs without the inserted 7-mer peptide sequence.

[0097] In yet another example, this document provides modified AAVs as described herein, wherein the 7-mer peptide sequence is selected from the sequences shown in Tables 1, 2, or 3, and wherein the insertion improves therapeutic efficacy compared to wild-type AAVs without the inserted 7-mer peptide sequence. In some instances, the improvement in therapeutic efficacy may be a reduction in off-target effects or a reduction in the dose of the vector required for treatment. Off-target effects refer to the effects produced in organs or tissues other than the target organ or tissue when AAVs are used for treatment.

[0098] In one respect, this document provides compositions comprising one or more modified AAVs as described herein. In one example, the composition is a pharmaceutical composition comprising one or more modified AAVs as described herein and a pharmaceutically acceptable excipient.

[0099] On the other hand, nucleic acids are provided that encode AAV modified as described herein.

[0100] The modified AAVs described herein can be generated using several methods known in the art. For example, modified AAVs can be generated using viral packaging cells (e.g., the HEK-293T cell line). Those skilled in the art will understand that introducing one or more nucleic acid molecules containing a nucleic acid sequence encoding a virus into packaging cells will cause the packaging cells to produce complete, infectious viral particles.

[0101] The invention exemplarily described herein may be suitably practiced in the absence of any element or more elements, or any limitation or limitation not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are used as descriptive rather than restrictive terms, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to recognize that various modifications may be made within the scope of the invention as claimed. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and alterations can be made to the specific invention disclosed herein by those skilled in the art, and such modifications and alterations are considered to be within the scope of the invention.

[0102] This invention has been described broadly and generally. Each narrower group of species and subgenerics falling within the scope of this general disclosure also constitutes part of the invention. This includes a general description of the invention, with incidental conditions or negative limitations removing any subject matter from that category, regardless of whether the removed material is specifically described herein.

[0103] Other embodiments are within the scope of the appended claims and non-limiting embodiments. Furthermore, where features or aspects of the invention are described according to the Markush group, those skilled in the art will recognize that the invention is also described according to any individual member or subgroup of the Markush group.

[0104] Experimental Section

[0105] Non-limiting examples and comparative examples of the present invention will be described in further detail with reference to specific embodiments, but these embodiments should not be construed as limiting the scope of the invention in any way.

[0106] Example 1: Construction and vector generation of 7-mer peptide insertion library based on NNK.

[0107] AAV-DJ-based capsid libraries were generated by inserting a random 7-mer peptide between amino acids 589 and 590. To generate a transcription-competent library for directed evolution driven by mRNA, the capsid gene was placed under the control of a ubiquitous promoter (cytomegalovirus “CMV” promoter) tandem with the Rep2 P40 promoter (starting from nucleotide #1700). The P40 sequence is essential for capsid mRNA expression and splicing during AAV production. This sequence itself is flanked by inverted terminal repeats (ITRs), thus each variant encapsulates its own capsid sequence (…). Figure 1 ).

[0108] To clone the capsid library, a 453-bp gBlock fragment of the wtAAV-DJ capsid was synthesized and used as a template to introduce a random 7-mer peptide library and overhangs for subcloning. PCR was performed using Neb Q5 polymerase for 15 cycles. The CMV promoter was amplified by PCR to introduce the SwaI restriction site and overhangs. The resulting amplicons were purified by gel extraction. The remaining fragments were synthesized as gBlocks. Then, following the manufacturer's instructions, the different fragments were assembled into a receptor ITR plasmid pre-digested with SphI and MfeI using Gibson assembly. The gBlocks were synthesized by IDT.

[0109] During the vector generation process, the full-length, unmodified Rep2 (Rep2-ΔCap, Figure 1 The plasmids were provided in trans form by individual plasmids. VP1 (M1L), VP2 (T138A), and VP3 (M205L, M211L, and M235L) were silenced, and the plasmids were assembled via Gibson assembly using pRep2-Cap-DJ_M1L as a template and gBlock containing an AAV-DJ fragment with VP2 / VP3 start codon mutations.

[0110] The diversity of the plasmid library was determined by the number of clones grown from representative aliquots of transformed chemocompetent DH5α bacteria. The library plasmids were harvested and purified using the Maxi Prep plasmid purification kit.

[0111] Virus generation was performed by triple transfection of HEK293T cells with 15 μg helper plasmid, 10 μg Rep2-Δcap plasmid, and 1 μg library DNA plasmid in each T175 flask. After 24 hours, the medium was replaced with fresh medium containing 2% FBS. Three days post-transfection, cells were collected and resuspended in lysis buffer (Tris HCl pH 7.5 + 2 mM MgCl2 + 150 mM NaCl) and lysed using three freeze-thaw cycles. The supernatant was collected and treated with a mixture of 50 U / ml benzonase and 1 U / ml RNase at 37°C for 30 min to remove unpackaged nucleic acids. After incubation, the lysates were loaded onto the top of a discontinuous density gradient (15%, 25%, 40%, and 60%) and ultracentrifuged at 54,000 rpm for 1.5 h at 18°C ​​on a Type 70 Ti rotor. A 40% fraction was extracted and used... The virus titer was determined by dialysis using an Ultra-15 (100 kDa MWCO) filter with 1×PBS (pH 7.2) containing 0.001% pluronic acid. Viral titers were quantified by digital droplet PCR (ddPCR) using primers and probes localized to the AAV2 ITR.

[0112] Example 2: Screening of mouse in vivo libraries

[0113] The AAV-DJ library was indexed at 2.5 × 10 9 A carrier dose of vg / mouse was injected into the lateral tail vein of male C57BL / 6 mice (N=5). Animals were euthanized after 28 days, and organs were harvested and stored at -80°C. The Plus Mini Kit was used to extract total RNA from different organs. Then, the mRNA in the samples was enriched using Oligo(dT)25 magnetic beads, and SuperScript was used according to the manufacturer's instructions. TM cDNA synthesis was performed using IV reverse transcriptase. Capsid DNA variants were amplified from the cDNA to generate NGS amplicones. The amplicones were quantified using a fluorometer, pooled in equimolar ratios, and analyzed on iSeq. TM Sequencing was performed on a sequencer. Following in vivo selection, over 10,000 unique capsid variants were identified, some of which showed excellent transduction efficiency in the liver, brain, and skeletal muscle. Figure 2 , AC). Furthermore, potential variants with enhanced brain or muscle targeting and complete deliverability were identified ( Figure 3 ).

[0114] The tropism change of AAV variants compared to a reference AAV (i.e., wild-type AAV) was assessed by calculating the fold change of the variant relative to wild-type virus in selected tissue types. First, a normalization factor (NF) was calculated by dividing the wild-type count in the original viral library (i.e., the library) by the wild-type count in the selected tissue (Normalization factor (NF) = AAV_WT count in the original viral library / AAV_WT count in the tissue). Next, the normalized variant count for each variant was obtained by multiplying the variant count in the selected tissue by the normalization factor for that tissue (Normalized AAV variant count = AAV variant count in the tissue × NF for tissue). Finally, the fold change of each variant was calculated by dividing the normalized variant count by the wild-type count in the original viral library (Fold change of conversion (FC) = Normalized AAV variant count / AAV_WT count in the original viral library). The counts, normalized counts, and fold changes (relative to wild-type AAV) for each variant in each selected tissue are shown in Tables 5 and 6. In the table, the "-" count indicates that no variant was detected in the tissue sample. The amino acids "N" and "R" at the beginning and end of each sequence represent N589 and R590 of the wild-type AAV-DJ capsid, respectively, with a 7-mer peptide inserted between them.

[0115] Table 5. Fold-change analysis of AAV variants in brain, muscle and liver

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Table 5 (continued). Fold-change analysis of AAV variants in brain, muscle and liver.

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] Table 6. Fold-change analysis of AAV variants in LSEC

[0138]

[0139]

[0140]

[0141] Example 3: Selection of peptides that confer tropism to hepatic sinusoidal endothelial cells (LSEC)

[0142] Animals received an injection of 2.5 × 10^9 vg / animal of AAV. Organs were harvested one month after AAV library injection. Specifically, for this round of selection, after liver collection, a portion of the liver was excised for cell suspension preparation. CD146+ hepatic sinusoidal endothelial cells (LSECs) were isolated from this suspension using fluorescence-activated cell sorting (FACS). This process led to the identification of AAV-DJ variants with enhanced LSEC tropism (Table 7).

[0143] Table 7. Identified peptides that confer tropism on hepatic sinusoidal endothelial cells (LSEC)

[0144]

[0145] Equivalent scheme

[0146] The foregoing embodiments are presented to illustrate the purpose of the present invention and should not be construed as limiting the scope of the invention in any way. It is evident that various modifications and changes can be made to the specific embodiments of the invention described above and illustrated in the embodiments without departing from the basic principles of the invention. All such modifications and changes are intended to be covered in this application.

Claims

1. A modified adeno-associated virus serotype DJ (AAV-DJ) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1767 and 1768 of SEQ ID NO:399, wherein the modified AAV-DJ has at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% sequence identity with the sequence of SEQ ID NO:399, excluding the nucleotide sequence encoding the 7-mer peptide.

2. A modified adeno-associated virus serotype 2 (AAV2) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:401, wherein the modified AAV2, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:401 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

3. A modified adeno-associated virus serotype 5 (AAV5) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1722 and 1723 of SEQ ID NO:402, wherein the modified AAV5, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:402 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

4. A modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1770 and 1771 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

5. A modified adeno-associated virus serotype 6 (AAV6) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:403, wherein the modified AAV6, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:403 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

6. A modified adeno-associated virus serotype 9 (AAV9) comprising a nucleotide sequence encoding a 7-mer peptide inserted between nucleotides 1764 and 1765 of SEQ ID NO:404, wherein the modified AAV9, apart from the nucleotide sequence encoding the 7-mer peptide, has sequence identity with the sequence of SEQ ID NO:404 in the range of at least 50% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99%.

7. The modified AAV-DJ according to claim 1, wherein the modified AAV-DJ has at least 57% sequence identity with the sequence of SEQ ID NO:399 outside the nucleotide sequence encoding the 7-mer peptide.

8. The modified AAV2 according to claim 2, wherein the modified AAV2 has at least 57% sequence identity with the sequence of SEQ ID NO:401 outside the nucleotide sequence encoding the 7-mer peptide.

9. The modified AAV5 according to claim 3, wherein the modified AAV5 has at least 57% sequence identity with the sequence of SEQ ID NO:402 outside the nucleotide sequence encoding the 7-mer peptide.

10. The modified AAV6 according to claim 4, wherein the modified AAV6 has at least 57% sequence identity with the sequence of SEQ ID NO:403 outside the nucleotide sequence encoding the 7-mer peptide.

11. The modified AAV6 according to claim 5, wherein the modified AAV6 has at least 57% sequence identity with the sequence of SEQ ID NO:403 outside the nucleotide sequence encoding the 7-mer peptide.

12. The modified AAV9 according to claim 6, wherein the modified AAV9 has at least 57% sequence identity with the sequence of SEQ ID NO:404 outside the nucleotide sequence encoding the 7-mer peptide.

13. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 1, and wherein the insertion increases brain, muscle, and liver orientation compared to wild-type AAV without the inserted 7-mer peptide sequence.

14. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 2, and wherein the insertion increases brain and muscle orientation and decreases liver orientation compared to wild-type AAV without the inserted 7-mer peptide sequence.

15. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 3, and wherein the insertion increases brain and muscle orientation and is liver-detargeting.

16. The modified AAV-DJ according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from SEQ ID NO: 22, 24, 32, 85, 106, 198, 216, 232, 251, 293, 312, 320, 321, 224, 346 or 376.

17. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 1 or Table 7, and wherein the insertion increases specificity for cells selected from hepatic sinusoidal endothelial cells or hepatocytes compared to wild-type AAV without the inserted 7-mer peptide sequence.

18. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 1, Table 2 or Table 3, and wherein the insertion increases specificity for cells selected from neurons or astrocytes compared to wild-type AAV without the inserted 7-mer peptide sequence.

19. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 7, and wherein the insertion increases the production of antigen-specific regulatory T cells compared to wild-type AAV without the inserted 7-mer peptide sequence.

20. The modified AAV according to any one of claims 1-12, wherein the sequence of the 7-mer peptide is selected from the sequences shown in Table 1, Table 2 or Table 3, and wherein the insertion improves therapeutic efficacy compared to wild-type AAV without the inserted 7-mer peptide sequence.

21. A composition comprising the modified AAV of any one of the preceding claims and a pharmaceutically acceptable excipient.

22. A nucleic acid encoding an AAV modified according to any one of the preceding claims.

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