WO2024186275A1

WO2024186275A1 - Novel compositions of adeno-associated viruses engineered for enhanced tissue transduction and specificity

Info

Publication number: WO2024186275A1
Application number: PCT/SG2024/050144
Authority: WO
Inventors: Mohamed Amine MELIANI; Wei Leong CHEW
Original assignee: Agency For Science, Technology And Research
Priority date: 2023-03-09
Filing date: 2024-03-11
Publication date: 2024-09-12

Abstract

The invention relates to the production of engineered adeno-associated viruses. More specifically, the invention provides for modified adeno-associated viruses comprising a nucleotide sequence encoding a 7-mer peptide inserted in the capsid. Further provided is the use of engineered adeno-associated viruses for enhanced tissue transduction and specificity, and compositions thereof.

Description

NOVEL COMPOSITIONS OF ADENO-ASSOCIATED VIRUSES ENGINEERED

FOR ENHANCED TISSUE TRANSDUCTION AND SPECIFICITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore application No. 10202300637W, filed 9 March 2023, the contents of it being hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] The invention is in the field of biotechnology. In particular, the invention relates to production of engineered adeno-associated viruses More specifically, the invention provides the use of engineered adeno-associated viruses for enhanced tissue transduction and specificity and compositions thereof.

BACKGROUND OF THE INVENTION

[0003] Adeno-associated virus (AAV) vector-based therapeutics are rapidly advancing to the clinic. Systemic vector infusion represents the ideal delivery route in many gene therapy applications, especially when local administration is not feasible Currently, most AAV capsids are based on naturally occurring serotypes, which are sequestrated in the liver and have reduced therapeutic efficacy in organs such as in the central nervous system (CNS) or in skeletal muscles. Consequently, higher vector doses are required, resulting in off-target toxicities. Thus, there is a need for AAV capsids with organ-specific tropism at low vector doses.

SUMMARY

[0004] Tn one aspect, provided herein is a modified adeno-associated virus serotype DJ (AAV- DJ), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1767th and 1768th nucleotides of SEQ ID NO: 399, wherein the modified AAV-DJ has a 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% sequence identity with the sequence of SEQ ID NO: 399 outside the nucleotide sequence encoding the 7-mer peptide

[0005] In another aspect, provided herein is a modified adeno-associated virus serotype 2 (AAV2), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 401, wherein the modified AAV2 has a 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% sequence identity with the sequence of SEQ ID NO:

401 outside the nucleotide sequence encoding the 7-mer peptide.

[0006] In another aspect, provided herein is a modified adeno-associated virus serotype 5 (AAV5), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1722th and 1723th nucleotides of SEQ ID NO: 402, wherein the modified AAV5 has a 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% sequence identity with the sequence of SEQ ID NO:

402 outside the nucleotide sequence encoding 7-mer peptide.

[0007] In another aspect, provided herein is a modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1770th and 1771th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO:

403 outside the nucleotide sequence encoding 7-mer peptide.

[0008] In another aspect, provided herein is a modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO:

403 outside the nucleotide sequence encoding the 7-mer peptide.

[0009] In yet another aspect, provided herein is a modified adeno-associated virus serotype 9 (AAV9), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 404, wherein the modified AAV9 has a 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% sequence identity with the sequence of SEQ ID NO:

404 outside the nucleotide sequence encoding the 7-mer peptide.

[0010] In another aspect, provided herein is a composition comprising any modified AAV as described herein, and a pharmaceutically acceptable excipient.

[0011] In another aspect, provided herein is a nucleic acid encoding any modified AAV as described herein.

DEFINITIONS

[0012] As used herein, the term “AAV” or “adeno-associated virus” refers to a non-pathogenic, small viruses of the Parvovirus family which infect humans. The term “AAV” includes, but is not limited to, naturally occurring and engineered AAV subtypes or serotypes, for example, 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

[0013] As used herein, the term “capsid” refers to the protein shell of a virus particle. The capsid of a virus is composed of one or more capsid units or subunits. For example, the capsid of an AAV is composed of VP1, VP2 and/or VP3 subunits. The capsid of a virus may have several functional purposes. For example, the capsid of a virus may protect the viral genome from degradation, assist the virus in evading host immune response, and/or determine viral tropism to different cell or tissue types.

[0014] As used herein, the term “identity” in the context of a polynucleotide or polypeptide sequence refers to the overall relatedness or homology between polymeric molecules, e.g., between polynucleotide molecules (e g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e g , gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes) The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0015] As used herein, the term “library” refers to a mixture of heterogeneous polypeptides or polynucleotides. A library is composed of members that have similar polypeptide or polynucleotide sequences. Sequence differences between library members are responsible for the diversity of the library. The library can take the form of a mixture of polypeptides or polynucleotides, or can be in the form of organisms or cells, for example yeast cells and the like, that are transformed with a library of polynucleotides.

[0016] As used herein, “modified virus” and “virus variant” are interchangeable terms referring to an engineered or recombinant virus comprising an alteration, that is, “modification”, in the sequence of a polypeptide or a polynucleotide encoding a polypeptide compared to that of a reference sequence. The reference sequence may be a genomic sequence or a subsection thereof of a virus, or the amino acid sequence encoded by the genomic sequence or a subsection thereof of a virus. The reference sequence may be a sequence from a wild-type or native virus, or it may be the sequence from a different modified virus. A wild-type virus refers to a virus with phenotype or genotype that predominates in nature. “Modified AAV” or “variant AAV” refers to an AAV that has a change in its genomic sequence or a subsection thereof, or in the polypeptide encoded by its genomic sequence or a subsection thereof, compared to a reference AAV. The alteration or modification of a sequence may refer to an 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 genomic sequence or a subsection thereof compared to a reference AAV or has one or more amino acids inserted into the polypeptide encoded by its genomic sequence or a subsection thereof.

[0017] As used herein, “peptide” refers to the condensation product of two or more amino acids, wherein the length of the polypeptide is less than or equal to 50 amino acids long, e.g., 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 long.

[0018] As used herein, “targeting” and “tropism” are interchangeable terms that refer to the ability of a virus to infect different cell types to produce a successful infection. A successful infection may be defined as when the virus gains entry to the host cell, when the virus has begun replication of its genome in the host cell, when intact progeny virus is detectable within the host cell, when the virus progeny exits the host cell, or any combination thereof. In one example, a virus with tropism to the CNS or a CNS-targeting virus indicates that the virus is capable of infecting cells that are from or derived from the CNS and/or that the virus is capable of infecting cells that are from or derived from the CNS at a higher specificity than other cell types. In another example, a virus with tropism to the muscle or a muscle-targeting virus refers to a virus that is capable of successfully infecting cells that are from or derived from skeletal muscle and/or that the virus is capable of infecting cells that are from or derived from muscle at a higher specificity than other cell types. Conversely, a virus that does not have tropism or has reduced tropism to a cell type would not be capable or have a reduced capability of infecting said cell type. For example, a virus with reduced liver tropism or that is liver de-targeting indicates that the virus is less capable or unable to successfully infect cells that are from or derived from the liver. It would be understood by those skilled in the art that “tropism” may also mean “specificity to”. For example, virus tropism to the CNS means that a virus has specificity for the CNS. Therefore, as used herein, the terms “tropism” and “specificity” are interchangeable and mean the ability of a virus to successfully infect a selected cell type. Further, a virus may also be defined as “specific targeting” or having “specific tropism”, to indicate that it is specific for a selected tissue type. For example, a virus with specific tropism to the brain is one that specifically infects the brain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0020] Figure 1 shows a schematic map of the construction of the AAV-DJ library. The top diagram shows the AAV-DJ library plasmid, where the grey lines represent the insertion site of the peptide library (between N589 and R590), and the bottom diagram shows the full-length Rep2 plasmid. Abbreviations: ITR, inverted terminal repeat.

[0021] Figure 2 shows the next-gen sequencing (NGS)-driven evolution of the AAV-DJ peptide library. C57BL/6 mice (n=5) were injected intravenously with 2.5 x 10⁹ vg/mouse of AAV-DJ peptide library. 28 days later, organs were collected, rnRNA were enriched and capsid DNA variants were amplified and analyzed by NGS. Figures 2A, 2B and 2C show distribution of the length of the inserted sequences in the AAV-DJ capsid DNA sequenced from the liver, brain, and muscle respectively. The X-axis represents the length of the nucleotide sequence inserted, and the Y-axis represents the probability density of each length of nucleotide sequences.

[0022] Figure 3 shows a table of values of the fold-change difference detected by NGS of selected AAV-DJ variants normalized to wild-type AAV-DJ, leading to identification of AAV- DJ variants with enhanced brain, muscle transduction efficiency with complete liver detargeting at low vector doses. The labels on the left denote the 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 scale bar on the right represents the fold-change of the modified AAV genome sequenced from the respective organ versus the wild-type. Lighter shades denote higher fold-change, darker shades denote lower fold-change, with black denoting no change (baseline). Low or absence of a fold-change increase in an organ indicates a decrease or absence of infectivity or tropism in the organ, and high or presence of a fold-change increase in an organ indicates an increase or presence of infectivity or tropism in the organ. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides for modified AAV s with altered tropism to various cell types, tissue types and/or organs compared to a reference AAV.

[0024] The reference AAV may be a wild-type AAV or unmodified AAV, and it may be of the same or different serotype as the modified AAV.

[0025] A modified virus or virus variant may be modified so that its tropism or targeting to one or more selected cell types, tissues and/or organs is altered. For example, a wild-type virus may be modified to possess enhanced tropism, reduced tropism, or no change in tropism towards one or more selected organs, tissues and/or cell types. In some examples, the reduction in tropism is to the extent that tropism is abolished, that is, the virus is no longer capable of infecting the one or more selected organs, tissues and/or cell types. The organ may be any organ from a human or an animal. For example, the organ may be the CNS, ear, eye, heart, intestine, kidney, joint/synovium, lung, liver, pancreas, or skeletal muscle. The tissues may be primary or clinical isolates from an organ, or it may be a cultured tissue originating from an organ. The cells may be primary or clinical isolates from an organ or tissue, or it may be a cultured cells originating from an organ or tissue.

[0026] An AAV with altered tropism may be obtained for example by introducing one or more mutations into the viral genome or sections thereof, or within a specific gene. For example, an AAV may be modified by introducing mutations into one or more genes encoding the capsid protein. Mutations may be introduced by any method known by those skilled in the art, for example, site-directed mutagenesis or site-saturation mutagenesis, and may include insertion or deletion of nucleotides (frame-shift mutations), replacement/substitution of nucleotides, or combinations thereof. For example, site-saturation mutagenesis may be used to substitute a selected site in a polypeptide with all possible amino acids through the use of degenerate codons in the nucleotide sequence encoding the polypeptide. In an example, the degenerate codon is NNK, where K stands for keto nucleotides such as guanine and thymine, and N is for any nucleotide. It would be understood by those skilled in the art that a modification of the polynucleotide sequence encoding a gene may result in the modification of the polypeptide encoded by the polynucleotide sequence. It would also be understood that mutations in the context of the present invention may refer to mutations of a viral polynucleotide and/or viral polypeptide sequence.

[0027] In one example, a wild-type or reference AAV is modified by insertion of a polynucleotide sequence encoding a peptide to a desired site in the nucleotide sequence of the reference or wild-type AAV. The reference or wild-type AAV may include but is not limited to the serotypes AAV-2, AAV-5, AAV-6, AAV-9, and AAV-DJ (Table 4). It would be understood by those skilled in the art that the resulting AAV, that is, “modified AAV”, will have a nucleotide sequence identical to that of the reference or wild-type AAV (i.e., the unmodified AAV) apart from, that is, “outside”, the nucleotide sequence encoding the inserted peptide

[0028] The desired site at which the polynucleotide sequence is inserted may be located within a coding or non-coding region of the viral genome. In one example, the wild-type or reference AAV may be modified by insertion of one or more amino acids or a peptide into a polypeptide sequence that encodes for a protein or proteins forming the capsid or part of the virus.

[0029] The peptide that is inserted to alter tropism of a wild-type or reference virus may vary in length. For example, peptides may be 5-15 amino acids in length. As non-limiting examples, the peptide may be 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in length. For example, the inserted peptide may 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 the 7-mer peptide may comprise any permutation of 7 amino acids. In some examples, the amino acids are the 20 standard amino acids. Therefore, provided herein are modified AAVs comprising a 7-mer peptide sequence, wherein the peptide sequence is any permutation of the 20 standard amino acids. It would be understood by those skilled in the art which amino acids are the 20 standard amino acids.

[0030] It would be understood by those skilled in the art that certain methods of modifying a polypeptide sequence, for example, site saturation mutagenesis, allows for the screening of every of the 20 standard amino acids for a selected site in a polypeptide sequence. Therefore, the peptides listed in the present disclosure are non-limiting examples of 7-mer peptides with the property of conferring altered tropism to an AAV when inserted to the viral capsid.

[0031] Provided herein are modified AAVs comprising a nucleotide sequence encoding a 7- mer peptide inserted between two nucleotides of a reference or wild-type AAV capsid gene. The two nucleotides may be within the sequence of a VP1, VP2 and/or VP3 gene. In one example, the two nucleotides are within the sequence of a VP3 gene. It would be understood by those skilled in the art that the VP1 gene encodes for the full-length capsid, while VP2 and VP3 genes encode for subunits of the capsid. Those skilled in the art will also recognize that the nucleotide sequences of the VP2 and VP3 genes are subsumed within the VP1 nucleotide sequence.

[0032] In one aspect, provided herein is a modified adeno-associated virus serotype DJ (AAV- DJ), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1767th and 1768th nucleotides of SEQ ID NO: 399, wherein the modified AAV-DJ has a 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% sequence identity with the sequence of SEQ ID NO: 399 outside the nucleotide sequence encoding the 7-mer peptide.

[0033] In another aspect, provided herein is a modified adeno-associated virus serotype 2 (AAV2), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 401, wherein the modified AAV2 has a 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% sequence identity with the sequence of SEQ ID NO:

401 outside the nucleotide sequence encoding the 7-mer peptide.

[0034] In another aspect, provided herein is a modified adeno-associated virus serotype 5 (AAV5), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1722th and 1723th nucleotides of SEQ ID NO: 402, wherein the modified AAV5 has a 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% sequence identity with the sequence of SEQ ID NO:

402 outside the nucleotide sequence encoding 7-mer peptide.

[0035] In another aspect, provided herein is a modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1770th and 1771th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO:

403 outside the nucleotide sequence encoding 7-mer peptide.

[0036] In another aspect, provided herein is a modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO:

403 outside the nucleotide sequence encoding the 7-mer peptide.

[0037] In another aspect, provided herein is a modified adeno-associated virus serotype 9 (AAV9), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 404, wherein the modified AAV9 has a 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% sequence identity with the sequence of SEQ ID NO:

404 outside the nucleotide sequence encoding the 7-mer peptide. [0038] It would generally be understood that there may be some variation in the sequence identity of a virus, for example a modified virus, compared to a reference virus, for example a wild-type virus. 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 to a reference virus. The comparison may be between the AAV of the same or different serotypes. For example, an AAV-DJ may have at least 50% sequence identity with a reference AAV-2, AAV-5, AAV-6, AAV-9, or AAV-DJ virus. In one example, an AAV-DJ has at least 57% sequence identity with a reference AAV-5.

[0039] In one example, provided herein is 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.

[0040] In another example, provided herein is 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.

[0041] In another example, provided herein is 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.

[0042] In another example, provided herein is a modified AAV6 comprising a sequence corresponding to a 7-mer peptide inserted between the 1770th and 1771th nucleotides 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.

[0043] In yet another example, provided herein is a modified AAV6 comprising a sequence corresponding to a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 403, wherein the modified AAV 6 has at least 57% sequence identity with the sequence of SEQ ID NO: 403 outside the 7-mer peptide sequence.

[0044] In yet another example, provided herein is 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.

[0045] In one example, the inserted peptides of the present invention may comprise a sequence as set forth in Table 1 and confers enhanced liver, brain, and muscle tropism to a modified AAV compared to a reference or wild-type AAV. In another example, the targeting peptide may comprise a sequence as set forth in Table 2 and confers enhanced brain and muscle tropism with reduced liver tropism to a modified AAV compared to a wild-type AAV. In yet another example, the targeting peptide may comprise a sequence as set forth in Table 3 and confers enhanced brain and muscle tropism with liver de-targeting to a modified AAV compared to a wild-type AAV

[0046] The modified virus or virus variant of the present invention may have increased or decreased specificity to one or more selected cell types compared to a reference virus. The increase or decrease in specificity may refer to the increased ability of the virus to bind to and/or infect said cell type. The increase or decrease in specificity of a virus to a cell type may be compared to that of a reference cell type may be measured by quantitative methods known to those skilled in the art. The degree of enhancement or reduction in tropism of a modified AAV relative to a reference AAV, such as a wild-type AAV, may be calculated by comparing the infectivity of the variant relative to the infectivity of the reference virus in a selected tissue sample. Those skilled in the art will be aware of the methods of measuring infectivity of viruses. For example, infectivity of a virus may be measured by plaque assay, enzyme-linked immunosorbent assay (ELSA), transduction assay using reporter genes, polymerase chain reaction (PCR), high-throughput sequencing or any combination thereof. In one example, the infectivity or tropism of an AAV is assessed by next-generation sequencing (NGS).

[0047] The comparing of the infectivity or tropism of an AAV variant relative to the infectivity of a reference or original AAV, wherein infectivity is measured by NGS, may be done by dividing the number of reads of the AAV variant in a selected tissue sample, for example the brain, muscle, or liver, by the number of reads of the reference or original virus in the tissue sample to obtain the degree fold-change in infectivity or tropism of the AAV variant relative to the reference or original virus. When the AAV variant has fold-change of > 1 relative to the reference or original AAV in a tissue type, it has increased or improved tropism for that tissue type compared to the reference or original virus. When the AAV variant has fold-change of < 1 relative to the reference or original AAV in a tissue type, it has decreased or reduced tropism forthat tissue type compared to the reference or original virus. When the AAV variant has foldchange = 1 relative to the reference or original AAV in a tissue type, it has same or similar tropism for that tissue type compared to the reference or original virus. When the AAV variant has fold-change = 0 relative to the reference or original AAV in a tissue type, it is not detectable in that tissue type or de-targeting towards that tissue type compared to the reference or original virus.

Table 1: AAV-DJ variants with enhanced brain, muscle, and liver tropism

Table 2: AAV-DJ Variants with Enhanced Brain and Muscle Tropism and Reduced Liver

Tropism

Table 3: AAV-DJ Variants with Superior Brain and Muscle Tropism and Liver Detargeting

[0048] In one example, the modified AAV comprises a 7-mer peptide sequence selected from the sequences set forth in Table 1 , and the insertion increases brain, muscle, and liver tropism compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0049] Tn another example, the modified AAV comprises a 7-mer peptide sequence selected from the sequences set forth in Table 2, and the insertion increases brain, and muscle tropism, and decreases liver tropism compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0050] In yet another example, the modified AAV comprises a 7-mer peptide sequence selected from the sequences set forth in Table 3, and wherein the insertion increases brain, and muscle tropism, and is liver de-targeting. [0051] In yet another example, the modified AAV comprises a 7-mer peptide sequence selected from the group consisting of SEQ ID NOs: 22, 24, 32, 85, 106, 198, 216, 232, 251, 293, 312, 320, 321, 224, 346 or 376.

Table 4: AAV nucleotide sequences

[0052] In any of the DNA sequences referenced and/or described herein, the single letter symbol has the following description: A for adenine; C for cytosine; G for guanine; T for thymine; K for keto nucleotides such as guanine and thymine; and N is for any nucleotide (which is not a gap). The use of the NNK codon in site-saturation mutagenesis allows for coverage of codons for all 20 standard amino acids, including degenerate codons. “Degenerate codons” refers to codons differing in one or two nucleotides but coding for the same amino acid. For example, the amino acid Alanine may be encoded by the codons GCT, GCC, GCA or GCG and the amino acid Leucine may be encoded by the codons CTT, CTC, CTA, CTG, TTA and TTG. [0053] In any of the amino acid sequences referenced and/or described herein, the single letter symbol has the following description: G (Gly) for Glycine; A (Ala) for Alanine; L (Leu) for Leucine; M (Met) for Methionine; F (Phe) for Phenylalanine; W (Trp) for Tryptophan; K (Lys) for Lysine; Q (Gin) for Glutamine; E (Glu) for Glutamic Acid; S (Ser) for Serine; P (Pro) for Proline, V (Vai) for Valine, I (He) for Isoleucine; C (Cys) for Cysteine; Y (Tyr) for Tyrosine; H (His) for Histidine; R (Arg) for Arginine; N (Asn) for Asparagine; D (Asp) for Aspartic Acid; and T (Thr) for Threonine.

[0054] The modified AAVs provided herein may possess advantageous characteristics when used for research and/or therapeutic applications as a result of their enhanced tropism to an organ, tissue and/or cell type. For example, modified AAVs with enhanced tropism may be used at lower vector doses resulting reduced off-target toxicities. Modified AAVs with enhanced tropism may also allow for the regulation of anti-capsid immune responses, and may be used for therapeutic purposes, such as CRISPR delivery and gene augmentation therapy. Such targeting may enhance the specificity of AAV s to the desired cell types and improve the overall therapeutic effect. In yet another example, in the case of liver targeting, liver sinusoidal endothelial cells (LSECs) and hepatocytes and other cell types in the liver could be targeted, whereas in the case of brain targeting, neurons and astrocytes and other cell types in the brain could be targeted. Additionally, the LSEC targeting can reduce the induction of the immune responses to AAV capsid. The modified AAVs provided herein may also possess advantageous characteristics when used for research and/or therapeutic applications as a result of their reduced tropism to an organ, tissue and/or cell type. For example, the modified AAVs with reduced tropism could be used in therapy at lower vector doses because they are not sequestrated in the liver, leading to reduced off-target toxicities. AAV dosage in therapy may be represented as the amount of vector genomes or viral genomes administered to a subject. For example, the dose may be specified as the amount of vector genomes per body weight (vg/kg) or amount of vector genomes per volume (vg/ml or vg/L) or vector genomes per animal (vg/animal). The therapeutically effective vector dose may be a range. For example, the effective dose per animal may be as low as 1 x 10⁹ to as high as 1 x IO¹⁷ vg, depending on the route and site of administration.

[0055] In one example, provided herein is the modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 1 or Table 7, and wherein the insertion increases specificity to cells selected from the group consisting of: liver sinusoidal endothelial cells, or hepatocytes, compared to a wild-type AAV without a 7-mer peptide sequence insertion. [0056] In another example, provided herein is the modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 1, Table 2 or Table 3, and wherein the insertion increases specificity to cells selected from the group consisting of: neurons, or astrocytes, compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0057] In another example, provided herein is the modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 7, and wherein the insertion increases production of antigen-specific regulatory T cells compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0059] In some examples, insertion of the 7-mer peptide sequence may reduce the induction of immune responses to the modified AAV capsid compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0060] In other examples, insertion of the 7-mer peptide sequence may regulate anti-capsid immune response compared to a wild-type AAV without a 7-mer peptide sequence insertion.

[0058] In yet another example, provided herein is the modified AAV as described herein, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 1, Table 2 or Table 3, and wherein the insertion improves therapeutic effect compared to a wild-type AAV without a 7-mer peptide sequence insertion. In some examples, the improvement in therapeutic effect may be reduction of off-target effects or the reduction of vector dose required for therapy. Off-target effects refer to effects in organs or tissues other than in the intended organ or tissue when an AAV is used in therapy.

[0059] In one aspect, there is provided herein a composition comprising one or more of the modified AAV described herein. In one example, the composition is a pharmaceutical composition comprising one or more of the modified AAV described herein and a pharmaceutically acceptable excipient.

[0060] In another aspect, there is provided a nucleic acid encoding the modified AAV as described herein.

[0061] The modified AAV described herein may be produced using several methods known in the art. For example, the modified AAV may be produced via the use of a virus packaging cell, such as the HEK-293T cell line It would be understood by those skilled in the art that one or more nucleic acid molecules comprising a nucleic acid sequence encoding the virus would be introduced into the packaging cell, which would then produce intact, infectious virus particles. [0062] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0063] The invention has been described broadly and generically herein Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0064] Other embodiments are within the following claims and non- limiting examples In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[0065] Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[0066] Example 1: Construction of the NNK-based 7-mer peptide insertion library and vector production.

[0067] AAV-DJ-based capsid libraries were generated by the insertion of random 7-mer peptide between amino acids 589 and 590. In order to generate a transcription-competent library for mRNA-driven directed evolution, the capsid gene is placed under the control of a ubiquitous promoter (cytomegalovirus “CMV” promoter) in tandem with Rep2 P40 promoter (starting at nucleotide #1700). The P40 sequence is required for capsid mRNA expression and splicing during AAV production. The sequence is itself flanked by inverted terminal repeats (ITR) so each variant will encapsulate its own capsid sequence (Figure 1). [0068] For cloning of capsid libraries, a 453-bp gBlock fragment of the wtAAV-DJ capsid was synthesized and used as a template to introduce the random 7-mer peptide library and overhangs for subcloning. PCR was performed for 15 cycles using Neb Q5 polymerase. The CMV promoter was amplified by PCR to introduce the Swal restriction site and overhangs. The resulting amplicons were purified via gel-extraction. The remaining fragments were synthesized as gBlocks. The different fragments were then assembled by Gibson assembly following manufacturer’s instructions into the recipient ITR plasmid previously digested with SphI and Mfel. The gBlocks were synthesized by IDT.

[0069] The full-length and unmodified Rep2 (Rep2-ACap, Figure 1) was provided in trans by a separate plasmid during vector production. The VP1 (MIL), VP2 (T138A), and VP3 (M205L, M21 IL, and M235L) were silenced and the plasmid was assembled by Gibson assembly using pRep2-Cap-DJ_MlL as a template and gBlock containing the AAV-DJ fragment with VP2/VP3 start codon mutations.

[0070] The diversity of the plasmid library was determined by the number of clones growing from a representative aliquot of transformed chemically competent DH5a bacteria. Library plasmids were harvested and purified using a maxi prep plasmid purification kit.

[0071] Virus production was performed by triple transfection in HEK293T cells using 15 pg of helper plasmid, 10 pg of Rep2-Acap plasmid and 1 pg of library DNA plasmid per T175 flask. 24 hours later, the medium was changed to a fresh culture medium containing 2%FBS. Three days after transfection, cells were collected and resuspended in lysis buffer (Tris HC1 pH

7.5 + 2 mM MgC12 + 150 mM NaCl) and lysed by three cycles of freezing/thawing. The supernatant was collected and treated with 50 U/ml of benzonase and 1 U/ml of Rnase cocktail for 30 min at 37°C to remove unpackaged nucleic acids. After incubation, the lysate was loaded on top of a discontinuous density gradient (15%, 25%, 40%, and 60%) and ultracentrifuged at 54,000 rpm, at 18 °C, for 1.5 hr, on a Type 70 Ti rotor. The 40% fraction was extracted and dialyzed with lx PBS (pH 7.2) with 0.001% pluronic acid, using Amicon® Ultra-15 (100 kDa MWCO) filters. Primers and probes localizing to within the AAV2 ITR were used for the quantification of the viral titer by digital droplet PCR (ddPCR).

[0072] Example 2: in vivo library screening in mice

[0073] AAV-DJ libraries were injected into C57BL/6 mice (male, N = 5) at a vector dose of

2.5 xlO⁹ vg/mouse into the lateral tail vein. Animals were euthanized 28 days later, and the organs were harvested and stored at -80°C. Total RNA was extracted from different organs using the Rneasy® Plus mini kit. The sample was then enriched for mRNA using Oligo(dT)25 magnetic beads, and cDNA synthesis was performed using SuperScript™ IV reverse transcriptase following the manufacturer’s instructions. Capsid DNA variants were amplified from cDNA for the generation of NGS amplicons The amplicons were quantified using a fluorometer, pooled at an equimolar ratio, and sequenced on an iSeq™ sequencer. Following in vivo selection, more than 10,000 unique capsid variants were identified, with some showing superior transduction efficiency in the liver, brain, and skeletal muscle (Figure 2, A-C). Moreover, potential variants with enhanced brain-tropic or myotropic with complete liver detargeting were also identified (Figure 3).

[0074] The altered tropism of the AAV variants compared to that of the reference AAV, that is, a wild-type AAV, was assessed by calculating transduction fold-change of the variant relative to the wild-type virus for a selected tissue type. First, a normalization factor was calculated by factoring the wild-type count in the original virus pool (that is, the library) by the wild-type count in a selected tissue (Normalisation factor (NF)= AAV_WT count in original virus pool / AAV WT count in tissue). Next, a normalized variant count for each variant was obtained by multiplying each variant count in the selected tissue by the normalization factor of that tissue (Normalised AAV variant count = count AAV variant in tissue x NF Tissue). Finally, the transduction fold change of each variant was calculated by factoring the normalized variant count by the wild-type count in the original virus pool (Transduction fold-change (FC) = Normalised AAV variant count /AAV wt count in original virus pool). The counts, normalized counts and transduction fold change in each selected tissue for each variant relative to wildtype AAV can be found in Tables 5 and 6. In said tables, a count of indicates that the variant was not detected in that tissue sample. The amino acids “N” and “R” at the beginning and end of each sequence respectively denote N589 and R590 of the wild-type AAV-DJ capsid, between which the 7-mer peptide is inserted.

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

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

Table 6. Fold-change analysis of AAV variants in LSECs

[0075] Example 3: selection of peptides conferring tropism to liver sinusoidal endothelial cells (LSECs)

[0076] Animals received injections of 2.5 x 10^A9 vg/animal of AAV. One month after the injection of the AAV library, organs were harvested. Specifically, for this round of selection, after liver collection, a portion of the liver was sectioned off to prepare a cell suspension. From this suspension, CD146+ liver sinusoidal endothelial cells (LSECs) were isolated using fluorescence-activated cell sorting (FACS). This process led to the identification of AAV-DJ variants with improved tropism to LSECs (Table 7). Table 7. Peptides identified conferring tropism to liver sinusoidal endothelial cells (LSECs)

[0077] Equivalents

[0078] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.

Claims

1. A modified adeno-associated virus serotype DJ (AAV-DJ), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1767th and 1768th nucleotides of SEQ ID NO: 399, wherein the modified AAV-DJ has a 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% sequence identity with the sequence of SEQ ID NO: 399 outside 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 the 1764th and 1765th nucleotides of SEQ ID NO: 401, wherein the modified AAV2 has a 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% sequence identity with the sequence of SEQ ID NO: 401 outside the nucleotide sequence encoding the 7-mer peptide.

3. A modified adeno-associated virus serotype 5 (AAV5), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1722th and 1723th nucleotides of SEQ ID NO: 402, wherein the modified AAV5 has a 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% sequence identity with the sequence of SEQ ID NO: 402 outside the nucleotide sequence encoding the 7-mer peptide.

4. A modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1770th and 1771th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO: 403 outside the nucleotide sequence encoding the 7-mer peptide.

5. A modified adeno-associated virus serotype 6 (AAV6), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 403, wherein the modified AAV6 has a 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% sequence identity with the sequence of SEQ ID NO: 403 outside the nucleotide sequence encoding the 7-mer peptide.

6. A modified adeno-associated virus serotype 9 (AAV9), comprising a nucleotide sequence encoding a 7-mer peptide inserted between the 1764th and 1765th nucleotides of SEQ ID NO: 404, wherein the modified AAV9 has a 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% sequence identity with the sequence of SEQ ID NO: 404 outside the nucleotide sequence encoding the 7-mer peptide.

7. The modified A AV-DJ of claim 1 , wherein the modified A AV-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 of 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 of 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 of 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 of 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 of 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 of any one of claims 1-12, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 1 , and wherein the insertion increases brain, muscle, and liver tropism compared to a wild-type AAV without a 7-mer peptide sequence insertion.

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

15. The modified AAV of any one of claims 1 -12, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 3, and wherein the insertion increases brain, and muscle tropism, and is liver de-targeting.

16. The modified AAV-DJ of any one of claims 1-12, wherein the 7-mer peptide sequence is selected from the group consisting of SEQ ID NOs: 22, 24, 32, 85, 106, 198, 216, 232, 251, 293, 312, 320, 321, 224, 346 or 376.

17. The modified AAV of any one of claims 1-12, wherein the 7-mer peptide sequence is selected from the sequences set forth in Table 1 or Table 7, and wherein the insertion increases specificity to cells selected from the group consisting of: liver sinusoidal endothelial cells or hepatocytes, compared to a wild-type AAV without a 7-mer peptide sequence insertion.

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

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

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

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

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