Liver-specific tropism of adeno-associated viruses

Abstract

To provide a composition and a method for changing or varying tissue tropism of adeno-associated virus (AAV) such as liver tropism.SOLUTION: An adeno-associated virus (AAV) containing a capsid protein selected from the group consisting of the followings: a) AAV9 capsid protein including a modification including AAV3B-VR1(SQSGASNDN) in a liver toggle region thereof; b) AAV80 L65 capsid protein including a modification including AAV3B-VR1(SQSGASNDN) in a liver toggle region thereof; c) AAV80 L65 capsid protein including a modification including AAV9-VR1(NSTSGGSSNDN) in a liver toggle region thereof; and d) AAV3B capsid protein including S268T modification .SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 841,179, filed April 30, 2019, and and priority to U.S. Provisional Patent Application No. 62 / 670,543, filed May 11, 2018 claims.

[0002] The present disclosure relates generally to altering the tissue tropism of adeno-associated viruses (AAVs), and specifically to , regarding the regulation of AAV liver tropism. [Background technology]

[0003] Adeno-associated viruses (AAV) are viruses that belong to the family Parvoviridae. It is a small virus belonging to the genus Dependoparvovirus. The virus is a replication-deficient, non-enveloped virus that infects but not in humans and AAV is not known to cause disease in any particular primate species. In natural viruses, the host genome contains the genes that the virus carries. Although integration into the host cell may occur, it may remain extrachromosomal without being integrated into the genome of the host cell. These characteristics make AAV a valuable viral vector. However, certain AAV serotypes are candidates for use in gene therapy. They exhibit hepatotropism which may or may not be desirable depending on the disease being treated.

[0004] We will understand how AAV liver tropism is determined and develop strategies to target AAVs based on this information. Operationalizing gender is beneficial. Summary of the Invention

[0005] The tissue specificity, i.e., tissue tropism, of AAV is determined by the capsid serotype. The methods and compositions described herein modify the tissue tropism of a particular AAV with specificity to This allows for improved therapies delivered by such modified AAVs. Altering or changing the liver tropism of AAV can be useful, for example, when the liver is a desired target. For example, by enhancing the natural liver tropism, and in cases where the liver is not the desired target, In such cases, for example, it may be beneficial to reduce the natural liver tropism. More effective delivery of the virus to liver (or non-liver) cells and more efficient transduction When transfecting a human AAV, a lower dose of a given AAV can be administered to the subject.

[0006] In one aspect, the present disclosure provides a method for modifying the tissue tropism of an adeno-associated virus (AAV) vector. Such methods include the step of converting the capsid protein of Anc80 (SEQ ID NO: 1) to a The amino acid position in the AAV capsid protein corresponding to position 266 in the protein was determined. and replacing the naturally occurring amino acid at the designated position with glycine (G) amino acid. acid residues to improve the liver tropism of the resulting AAV vector, or Alanine (A) amino acid residues are replaced to reduce the liver tropism of the resulting AAV, i.e. i.e., a step that results in detargeting to the liver.

[0007] In some embodiments, such methods involve the use of naturally occurring amino acids at the mapped positions. amino acid with a G amino acid residue to improve the liver tropism of the resulting AAV vector, e.g. For example, the method includes a step of providing enrichment in the liver. is obtained by replacing the naturally occurring amino acid at the designated position with an A amino acid residue. This includes reducing the liver tropism of the AAV produced, for example, resulting in liver detargeting.

[0008] In another aspect, the present disclosure provides methods for controlling the tissue tropism of adeno-associated viral (AAV) vectors. Such methods include modifying the capsid protein of Anc80 (SEQ ID NO: 1). The amino acid position in the AAV capsid protein corresponding to position 168 in the protein was determined. and replacing the naturally occurring amino acid at the positioned position with arginine ( R) amino acid residues to improve the liver tropism of the resulting AAV vector, e.g., or by replacing lysine (K) amino acid residues with the resulting AA and reducing the liver tropism of V, e.g., resulting in liver detargeting.

[0009] In some embodiments, such methods involve the use of naturally occurring amino acids at the mapped positions. Replacing the amino acid with an R amino acid residue results in liver enrichment of the resulting AAV vector. In some embodiments, such a method includes the step of: Replacing naturally occurring amino acids with K amino acid residues improves liver targeting of the resulting AAV. The method may include a step of causing a decrease.

[0010] In another embodiment, a method for altering the tissue tropism of an adeno-associated viral (AAV) vector Such methods include incorporating the liver toggle region defined in Figure 14 into the AAV capsid protein. and positioning a naturally occurring liver toggle region within a protein derived from a heterologous serotype. The resulting AAV vectors are then replaced with sequences derived from the liver toggle region or de novo. and modifying the liver tropism of the compound, e.g., resulting in improved or reduced liver targeting. Contains type.

[0011] In some embodiments, the heterologous or de novo derived liver toggle region is AAV capsid protein corresponding to position 266 in the capsid protein of SEQ ID NO: 80 (SEQ ID NO: 1) When the protein contains a G amino acid residue, the resulting AAV vector is enriched in the liver. In some embodiments, a heterologous or de novo derived liver toggle is produced. A region corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) If the position in the AV capsid protein contains an A amino acid residue, the resulting AAV vector This results in reduced liver targeting of the drug.

[0012] In some embodiments, the replacing step involves replacing an existing or de novo compound. site-directed mutagenesis of the synthesized DNA, restriction digestion and ligation, existing or De novo synthesized DNA, homology-based assembly, or a combination of these. In some embodiments, the step of locating is performed by sequencing. This will be implemented.

[0013] In another aspect, the present disclosure provides a method for enhancing or reducing liver transfection. Such methods include screening for AAV capsid proteins. sequencing a nucleic acid encoding a protein; and Amino acid in the AAV capsid protein corresponding to position 266 in the capsid protein a step of locating a position, and inserting a G amino acid residue or a G amino acid residue at the located position; identifying AAV capsid proteins having an A amino acid residue at the selected position; Generally, G amino acid residues at the designated positions are used for transfection into the liver. The A amino acid residue at the designated position exhibited enhanced AAV activity, whereas the A amino acid residue at the designated position exhibited enhanced activity in the liver. 1 shows AAVs with reduced transfection into the mouse.

[0014] In another aspect, the present disclosure provides a method for enhancing or reducing liver transfection. Such methods include screening for AAV capsid proteins. sequencing a nucleic acid encoding a protein; and Amino acid in the AAV capsid protein corresponding to position 168 in the capsid protein a step of positioning a position, and placing an R amino acid residue or a identifying AAV capsid proteins having a K amino acid residue at a specific position; Generally, R amino acid residues at the designated positions are used for transfection into the liver. The AAV showed enhanced expression, while the K amino acid residue at the designated position was found to be involved in the liver 1 shows AAVs with reduced transfection into the mouse.

[0015] In yet another embodiment, the present disclosure provides an amino acid sequence in which X3 at position 266 is selected from G or A. An AAV having the sequence shown in SEQ ID NO: 1 [Anc80] is provided.

[0016] In another embodiment, the present disclosure provides the sequence An AAV having the sequence shown in number 1 [Anc80] is provided.

[0017] In another embodiment, the present disclosure provides a nucleic acid sequence having the sequence shown in SEQ ID NO: 2 [Anc80L65]. Provide AAVs.

[0018] In yet another embodiment, the present disclosure provides SEQ ID NO: 3 [Anc80L65 G266A] An AAV having the sequence shown in

[0019] In another embodiment, the present disclosure provides a method for the production of a recombinant AAV comprising the sequence set forth in SEQ ID NO: 4 [AAV9 G267A]. We provide AAVs that can

[0020] In another embodiment, the present disclosure provides a method for the preparation of a human AAV comprising administering to a mammalian subject the ... An AAV having the sequence shown is provided.

[0021] In yet another embodiment, the present disclosure provides SEQ ID NO: 6 [AAV9 Anc80L65-VR I].

[0022] In another embodiment, the present disclosure provides SEQ ID NO: 7 [AAV9 Anc80L65 G266 AAV having the sequence shown in A-VRI is provided.

[0023] In another aspect, the present disclosure provides a method for the preparation of a human avian medicament comprising the step of: The present invention provides an AAV having the gene encoding the HIV-1 virus.

[0024] In yet another embodiment, the present disclosure provides SEQ ID NO: 9 [AAV3B A266G S26 7 N268T].

[0025] In another embodiment, the present disclosure provides SEQ ID NO: 10 [AAV3B G265 A266A] An AAV having the sequence shown in

[0026] In yet another embodiment, the present disclosure provides a method for the preparation of a nucleic acid sequence encoding SEQ ID NO: 11 [AAV3B G265 A266 G].

[0027] In another embodiment, the present disclosure provides SEQ ID NO: 12 [AAV3B G265 A266A S268T].

[0028] In another embodiment, the present disclosure provides SEQ ID NO: 13 [AAV3B G265 A266G S268T].

[0029] In yet another aspect, the present disclosure provides SEQ ID NO: 14 [AAV3B AAV9-VRI] An AAV having the sequence shown in

[0030] In another embodiment, the present disclosure provides SEQ ID NO: 15 [AAV3B Anc80L65-VR I].

[0031] In yet another embodiment, the present disclosure provides SEQ ID NO: 16 [AAV3B Anc80L65 G266A-VRI].

[0032] In another aspect, the present disclosure provides a method for the preparation of a polypeptide as set forth in SEQ ID NO: 17 [Anc80L65 R168K]. The present invention provides an AAV having a sequence similar to that described above.

[0033] As used herein, "tissue tropism" refers to the infection and proliferation of cells by a particular AAV. refers to the natural tissue specificity for transfection and / or transcription. For example, many AAs V exhibits hepatotropism, which means that such AAVs are selective for hepatocytes rather than cells of other tissue types. Tissue tropism refers to the ability to selectively infect and / or transfect a particular tissue. specific surface proteins found on the surface of tissue cells and / or on the surface of specific AAVs; For example, they are often based on receptor proteins.

[0034] As used herein, "toggle" refers to the AA associated with the tissue tropism of that AAV. V refers to a specific location or region within the capsid protein. When a naturally occurring amino acid at a toggle position or region is replaced with a different amino acid, this The tissue tropism of the AAV is altered. Thus, a "liver toggle," e.g. "Lead 1" indicates that transfection is predominantly or essentially completely induced in hepatocytes. or "switched" to hepatocytes so that they do not occur predominantly or essentially completely. This refers to a residue that can be "removed."

[0035] Also, as used herein, "liver toggle region" refers to the region of the liver that is located between the liver and the spleen, as defined in FIG. This refers to the 20 amino acid residues located between the two beta strands where the "liver toggle" resides. Therefore, the "liver toggle region" allows transfection to be preferentially delivered to hepatocytes. or occurring essentially entirely, or predominantly or essentially entirely in hepatocytes To avoid this, transfection from heterologous AAV or de novo derivatives may be used to "switch" The liver toggle region is a contiguous series of residues that can be "switched" to other regions. Because of the overlap with , all toggle region swaps use this term as shorthand.

[0036] As used herein, "Toggle 2" refers to a switch that is independent of "Toggle 1." Therefore, "Liver Toggle 2" is different from the residue in question in Liver Toggle 1, Transfection occurs predominantly or essentially completely in hepatocytes, or or hepatocytes, "switching" them to occur predominantly over others or essentially completely refers to another possible residue.

[0037] Unless otherwise defined, all technical and scientific terms used herein are The methods and compositions of the present invention are the same as those commonly understood by those skilled in the art. Methods and materials similar or equivalent to those described herein are intended to be illustrative and not restrictive. Suitable methods and materials may be used in the practice or testing of the methods and compositions. In addition, the materials, methods, and examples are illustrative only and should not be construed as limiting. All publications, patent applications, patents, and other references incorporated by reference in their entireties.

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication are available upon request and payment of the necessary fee. The Agency will provide the necessary information. [Brief explanation of the drawings]

[0039] [Figure 1] 1 is an Anc80 scaffold sequence (SEQ ID NO: 1) showing the 11 positions that were altered to generate a 211 (2048) variant Anc80 library for in vivo screening. The highlighted positions X1 and X3 were determined to be liver toggle positions and correspond to residues 168 and 266 of the Anc80 capsid sequence. The X3 residue is a critical position that defines whether the vector will efficiently deliver genes to hepatocytes, and is therefore referred to as the "liver toggle" or "toggle." Position X1 is referred to herein as "toggle 2." [Figure 2]Figures 2A and 2B show the amino acid sequences of Anc80L65 (SEQ ID NO: 2) and Anc80L65 G266A (SEQ ID NO: 3), respectively. Both sequences are identical except for the liver toggle. The glycine (G) / alanine (A) liver toggle in both the nucleic acid and protein sequences is shown in bold and underlined. [Figure 3] Figures 3A and 3B show the amino acid sequences of AAV9 G267A (SEQ ID NO: 4) and AAV9 G267A S269T (SEQ ID NO: 5), respectively. These sequences represent the identification and modification of the liver toggle in AAV9, and further modification in the latter to match the corresponding position in Anc80L65. The residues that have been modified from AAV9 are bold and underlined. [Figure 4] Figures 4A and 4B show the amino acid sequences of AAV9 Anc80L65-VRI (SEQ ID NO: 6) and AAV9 Anc80L65 G266A-VRI (SEQ ID NO: 7), respectively. These sequences represent the transfer of the liver toggle region from Anc80L65 to AAV9, which provides both liver enrichment and liver detargeting. The transferred sequence is shown in bold and underlined. [Figure 5] Figures 5A and 5B show the sequences of AAV3B A266G (SEQ ID NO: 8) and AAV3B A266G S267_N268T (SEQ ID NO: 9), respectively. Although truncated compared to the Anc80 liver toggle region, making its identity uncertain, AAV3B A266 may represent the liver toggle in this serotype. AAV3B transduces mouse liver poorly, suggesting that the A266G modification may improve this function. Inserting a T generates a liver toggle region with even higher identity to Anc80. Residues changed or inserted from AAV3B are bold and underlined. [Figure 6]6A and 6B show the sequences of AAV3B G265_A266A (SEQ ID NO: 10) and AAV3B G265_A266G (SEQ ID NO: 11), respectively. The liver toggle region of AAV3B is shortened compared to the Anc80 liver toggle region. This function can be transferred by inserting a liver-enriched G or a liver-detargeted A from the corresponding position in Anc80. The residues changed or inserted from AAV3B are bold and underlined. [Figure 7] 7A and 7B are the sequences of AAV3B G265_A266A S268T (SEQ ID NO: 12) and AAV3B G265_A266G S268T (SEQ ID NO: 13), respectively. The liver toggle region of AAV3B is shortened compared to the Anc80 liver toggle region. Insertion of a liver-enriched G or a liver-detargeted A from the corresponding position in Anc80 can transfer this function. Changing the S at position 268 to a T generates a region with even higher identity to Anc80. Residues changed or inserted from AAV3B are bold and underlined. [Figure 8] Figures 8A-8C show the sequences of AAV3B AAV9-VRI (SEQ ID NO: 14), AAV3B Anc80L65-VRI (SEQ ID NO: 15), and AAV3B Anc80L65 G266A-VRI (SEQ ID NO: 16), respectively. These sequences represent the transfer of the liver toggle region from AAV9, Anc80L65, and Anc80L65 G266A, which confers both liver enrichment and liver detargeting, to AAV3B. The transferred sequences are shown in bold and underlined. [Figure 9] Figure 10 is an MA plot showing the abundance of 2048 Anc80 library members in liver versus viral input in C57 / BL6J mice. On the y-axis, zero indicates no change in input, and positive and negative values indicate relative enrichment or detargeting, respectively. Of the 11 toggle positions, position X3 correlates with the observed bimodality. [Figure 10]A, MA plot showing the abundance of 2048 Anc80 library members in liver relative to viral input in the non-human primate rhesus macaque. On the y-axis, zero indicates no change from input, while positive and negative values indicate relative enrichment or detargeting, respectively. As in mouse liver, the X3 of the 11 toggle positions correlates with the observed bimodality. [Figure 11]

[0023] Figure 1 is a bar graph illustrating clonal validation of Anc80 liver toggle in mouse liver. Both Anc80L65 (SEQ ID NO: 2) and Anc80L65 G266A (SEQ ID NO: 3) were generated by triple transfection with the eGFP-expressing genome CB7.CI.eGFP.FF2A.hA1AT.RGB. Five mice were injected with these vectors at a dose of 1.25e12 gc / kg, and the mice were sacrificed three days later. Biodistribution of harvested livers revealed a 100x enrichment of the eGFP-encoding genome per cell in the Anc80L65 toggle "on" vector relative to the Anc80L65 G266A toggle "off" vector. [Figure 12] Figures 12A-12K are a series of 11 microscopic images illustrating the results of eGFP staining in the livers of 10 mice plus the one uninjected control mouse from Figure 11. There is significantly higher eGFP staining in the livers of mice injected with the Anc80L65 toggle "on" vector versus the Anc80L65 G266A toggle "off" vector. [Figure 13]Figure 13A is a schematic representation of the crystal structure of the adeno-associated virus 2 (AAV2) VP3 capsid monomer. The location of the liver toggle ("LT") is indicated by an arrow, and the structure of the region encompassing the LT is boxed. Figure 13B is a schematic representation of the crystal structure showing an overlay of the LT regions of AAV2, 3, 6, 8, and 9, highlighting the local secondary structures that define the toggle position. The toggle region is defined as residues located between and encompassing the β-ascending (βa) and β-descending (βd) secondary structures, with the primary functional group encoded two to three residues N-terminal to the α-toggle (αt). The LT residues are located within VR1, a beta-sheet-binding loop, N-terminal to the three-residue alpha helix. [Figure 14] Sequence alignments of AAV clade and clonal prototypes (e.g., lines 1, 4, 8-10, and 13), clinically relevant AAV serotypes (both naturally occurring and engineered serotypes; e.g., lines 2, 3, 5-7, 11, and 12), and Anc variants (e.g., lines 14-22). Lines 1-22 correspond to SEQ ID NOs: 18-39. The positions of the b upper (ba), b lower (bd), a toggle (at), the toggle region, and the toggle position itself are shown. The b upper (ba) begins at a conserved tyrosine, and the b lower (bd) ends at a conserved serine. The toggles represent residue 266 in Anc80L65, residue 267 in AAV9, and residue 257 in AAV5; it is emphasized that the position numbers are relative when defining the toggle. [Figure 15]

[0039] Figure 1 is a table of liver toggle region sequences (SEQ ID NOS: 40-55, top to bottom) directed at the a-toggle (NDN) at the C-terminus of the Anc80L65, AAV3B, and AAV9 serotypes, as well as variants constructed to test the liver toggle hypothesis. The last two columns list the known "on" or "off" transduction efficiencies of Anc80L65, AAV3B, and AAV9 in mouse and primate livers; in vivo results for variants are described herein in bold. Hypothesized predicted efficiencies for untested variants are in italics. [Figure 16]Schematic diagram showing how the liver toggle region can be transplanted into heterologous AAV Caps to further test the findings of single-residue toggle. The region is flanked by two conserved domains suitable for homology-specific association of the "swap" region. SEQ ID NO: 86. [Figure 17] 15A-15C are tables (similar to FIG. 15A) showing predicted liver transduction efficiencies of variants with "swapped" liver toggle regions (SEQ ID NOS: 56-63, top to bottom). The last two columns list in bold the "on" or "off" transduction efficiencies determined in vivo in mouse liver for such variants, as described herein. Hypothetically predicted efficiencies for untested variants are in italics. [Figure 18] Figures 18A and 18B are bar graphs illustrating the in vitro transduction efficiency of AAV9 and AAV9-based liver toggle variants. The variants were generated by triple transfection of 293 cells and packaged into a genome encoding CMV luciferase. The variants were titrated and then added to Huh7 cells at a multiplicity of infection (MOI) of 100,000. Huh7 cells are a liver cancer cell line, and transduction efficiency, as measured by normalized RLU, can be interpreted as a rough indication of whether a variant was liver "on" or "off." Changing the native AAV9 G267 to A (SEQ ID NO: 4) significantly reduced transduction efficiency of Huh7 cells, as did the double mutant G267A S269T (SEQ ID NO: 5). AAV9 does not appear to tolerate liver toggle region swaps well, but toggling may be evident by the relative efficiencies of the Anc80L65 (SEQ ID NO: 6) and Anc80L65 G266A (SEQ ID NO: 7) variants. [Figure 19]Figures 19A and 19B are bar graphs illustrating the in vitro transduction efficiency of AAV3B and AAV3B-based liver toggle variants. The variants were generated by triple transfection of 293 cells and packaged into a genome encoding CMV luciferase. The variants were titrated and then added to Huh7 cells at an MOI of 100,000. Huh7 cells are a liver cancer cell line, and transduction efficiency, as measured by normalized RLU, can be interpreted as a rough indication of whether a variant was liver "on" or "off." Changing native AAV3B A266 to G (SEQ ID NO: 8) unexpectedly significantly reduced the transduction efficiency of Huh7 cells, but inserting a T at the corresponding position of Anc80L65 (SEQ ID NO: 9) rescued this variant, outperforming AAV3B. Similarly, inserting A (SEQ ID NO: 10) or G (SEQ ID NO: 11) before A266 with or without altering S268T (SEQ ID NO: 12 and SEQ ID NO: 13) results in a liver toggle-like efficiency pattern with both +G "on" variants outperforming AAV3B. AAV3B tolerates the liver toggle region swap well, with toggling evident by the relative efficiency of the liver "on" AAV9 variant (SEQ ID NO: 14), and the Anc80L65 (SEQ ID NO: 15) and Anc80L65 G266A (SEQ ID NO: 16) variants. [Figure 20] Figures 20A-20C are graphs showing the in vivo kinetic expression of luciferase in mice injected with AAV9 and the AAV9-based variants G267A and G267A S269T. Mice were followed for 57 days and imaged during their lifetime. Regions of interest were defined as the whole body, liver, and gluteal region (the major muscle groups of the thigh and buttocks). Both liver-off AAV9 variants exhibited very low radiance in the liver region, whereas AAV9 G267A S269T had an overall total radiance comparable to AAV9. This double mutant may generate the majority of this total radiance from the gluteal region. [Figure 21-1]Figures 21A-21F are bar graphs supporting the data observed in in vivo luciferase experiments. Mice injected with the GFP-expressing vectors AAV9, AAV9 G267A, or AAV9 G267A S269T were sacrificed 28 days post-injection to examine the biodistribution of both the eGFP-containing genome (DNA) and eGFP expression (RNA). While both hepatocyte "off" mutants had DNA and RNA levels three orders of magnitude lower in the liver (Figures 21A-21B), AAV9 G267A and AAV9 G267A S269T were comparable to AAV9 in cardiac cells (Figures 21C-21D). Significantly, in quadriceps cells, AAV9 G267A S269T surpassed AAV9 in both gene delivery and gene expression levels (Figures 21E-21F). [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22] Figures 22A and 22B are graphs showing the in vivo kinetic expression of luciferase in mice injected with AAV3B or AAV3B-based variants G265_A266A, G265_A266G, Anc80L65-VRI, Anc80L65 G266A-VRI, or AAV9. Mice were followed for 29 days and imaged throughout their lives. Regions of interest were defined as the whole body and liver. Both liver "on" variants of AAV3B emitted light with higher radiance than their liver "off" counterparts, supporting the toggling hypothesis. Indeed, both liver "off" AAV3B variants exhibited very low radiance in the liver region. Interestingly, the Anc80L65-VRI mutant was as efficient as wild-type AAV3B, but significantly, a simple glycine insertion produced a vector with a liver-domain signal equivalent to AAV9. The total signal of the variant also matched that of AAV9, suggesting that the majority of the AAV3B G265 A266G signal originates from the liver. [Figure 23] The sequence is Anc80L65 R266K (SEQ ID NO: 17). The sequence is identical to Anc80L65 except for liver toggle 2. The arginine (R) / lysine (K) liver toggle is highlighted. [Figure 24] Sequence alignments of AAV clade and clonal prototypes (e.g., lines 1, 4, 8-10, and 13), clinically relevant AAV serotypes (both naturally occurring and engineered serotypes; e.g., lines 2, 3, 5-7, 11, and 12), and Anc variants (e.g., lines 14-22). Lines 1-22 correspond to SEQ ID NOs: 64-85. The location of liver toggle 2, the orientation of the conserved proline and lysine, and the non-canonical start codon for VP2 are shown. Liver toggle 2 represents residue 168 of Anc80L65 and AAV9, and residue 151 of AAV5. It is emphasized that position numbers are relative when defining toggles. [Figure 25] Figures 25A and 25B are graphs and heatmaps. Figure 25A shows, on the left, an MA plot showing the abundance of 2048 Anc80 library members in C57BL / 6 mouse hepatocytes, and Figure 25B shows, on the left, an MA plot of the xenograft FRG mouse model on day 28 of viral input. Variants enriched in these cells are boxed, and the identities of each of the 11 toggle positions are shown in a "heatmap" to the right of both figures. Of the 11 toggle positions, the liver toggle X3 position correlates with enrichment. X1 position toggle 2, state 1, also correlates with enrichment, particularly in the most enriched variants. [Figure 26-1] Figures 26A-26C show three MA plots on the left showing the abundance of 2048 Anc80 library members in animal and in vitro models of day 28 NHP (Figure 26A), day 28 xenograft FRG mouse model (Figure 26B), and day 3 human hepatocytes (Figure 26C) relative to viral input. Variants enriched in these cells are boxed, and the identity of each of the 11 toggle positions is shown in a "heat map" to the right of each figure. Of the 11 toggle positions, the liver toggle X3 position correlates with enrichment. The X1 position, toggle 2, state 1, also correlates with enrichment, particularly in the most enriched variants. [Figure 26-2] This is a continuation of Figure 26-1. DETAILED DESCRIPTION OF THE INVENTION

[0040] Adeno-associated viruses (AAV) are naturally hepatotropic. This tropism has been implicated in liver pathogenesis. This is advantageous for gene therapy treatment of diseases that involve the organ being efficiently transduced. The number of genome-containing viral particles required to deliver a vaccine places a burden on both patients and donors. In contrast, relevant treatments for diseases with non-hepatic etiology are not related to AAV. The liver acts as a sink for most therapeutic substances delivered via the liver, resulting in reduced efficacy. In addition, promising AAV serotypes may be able to infect the mouse liver. does not transduce cells efficiently, limiting the use of mouse models for clinical relevance and drug testing. severely restrict.

[0041] Previous methods to identify AAV sequences that correlate with tropism have not identified motifs that define liver tropism. For example, comparison with the well-defined characteristics of existing highly related serotypes, unrelated These methods rely on random domain swaps between different serotypes or on structural considerations, e.g. Mapping of AAV tropism determinants was performed by comparing highly related serotypes. One such example is the mutation of a single amino acid (E) between AAV1 and AAV6. 531K), which improves AAV1-mediated transduction of mouse liver (Wu et al. ., 2006, J. Virol., 80(22):11393-7). Another example is the domain between AAV2 and AAV8. This is a reciprocal swap of genes, which alters tropism but does not affect any stable specific tissue. The target motif could not be defined either (Raupp et al., 2012, J. Virol., 86(17): Furthermore, overall structural considerations may provide a basis for determining the overall relationship between good and poor liver transducing agents. It emphasizes only practical differences and is more observational than practically useful. (Nam et al., 2007, J. Virol., 81(22):12260-71).

[0042] Identification of the liver toggle in AAV capsid proteins In the present disclosure, amino acids at a single site (e.g., " Rationally designed AAV capsidrives to identify mutations in the liver toggle The present disclosure also describes screening of rallies. or, if necessary, detargeting to the liver, thereby lowering the effective dose. b) improves mouse liver transduction while retaining other favorable properties. With minimal modification of the sex, such serotypes can be used in mouse disease models. The specific residues in the AAV capsid that result in

[0043] As described herein, the liver-specific tropism of AAV capsid proteins is due to the Anc8 0 (SEQ ID NO: 1) by mutating a single residue at position 266. For example, a non-glycine (G) amino acid residue at this position can be changed to a glycine (G) amino acid residue. The difference results in improved or increased tropism or targeting of AAV to the liver. Alternatively, the non-alanine (A) amino acid residue at this position may be mutated to an alanine (A) amino acid residue. This results in a decrease in the tropism or targeting of AAV to the liver ("detargeting"). Therefore, the propensity of AAV to infect and / or transfect the liver is Mutating the non-G residue at position 266 in the AAV capsid protein of SEQ ID NO: 1) to a G residue On the other hand, AAV can infect and / or translocate the liver. The propensity to infect is shown to be related to position 266 in the AAV capsid protein of Anc80 (SEQ ID NO: 1). can be reduced by mutating non-A residues to A residues.

[0044] In some embodiments, for example, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or 11, which are known to be derived from other capsid proteins, correspond to position 266 of Anc80. The corresponding amino acid position may be a G amino acid residue or an A amino acid residue, depending on the desired liver-specific tropism. In some embodiments, the amino acid residue can be mutated to any of the G amino acid residues. Insertion of either A or B amino acid residues can alter liver-specific tropism as desired. In some embodiments, further modifications, insertions, or Deletions can improve liver-specific tropism, if desired. The entire native liver toggle region, including the toggle residues, is then transferred to a heterologous capsid or de novo. o Replace the liver toggle region of the synthesized sequence to achieve the desired increased or decreased liver tropism It is possible.

[0045] In some embodiments, the capsid protein comprises amino acid sequence 266 of Anc80 (SEQ ID NO: 1). The target amino acid sequence is genetically modified or designed to have the desired amino acid residue corresponding to the target position. When the virus components are mixed with the AAV virus particles and the AAV virus particles are assembled, It can be enriched in the liver or detargeted to the liver (e.g., capsid sequence, or the corresponding capsid without G or without A at the appropriate position. or the corresponding nucleotide sequence having A instead of G or G instead of A at the appropriate position. compared to the capsid sequence).

[0046] Also, as described herein, the liver tropism of AAV capsid proteins is This can be changed by mutating a single residue at position 168 of c80 (SEQ ID NO: 1). For example, a non-arginine amino acid residue at this position can be replaced with an arginine (R) amino acid residue. Mutations at this position may result in improved or increased liver tropism of AAV or may result in the non- Mutation of lysine amino acid residues to lysine (K) amino acid residues reduces the liver tropism of AAV. Therefore, AAV is unable to infect and / or transfect the liver. The propensity to infect is due to the 168 can be increased or improved by mutating non-R residues at positions 1 and 2 to R residues, while The propensity of AAV to infect and / or transfect the liver is mediated by the expression of Anc80 (SEQ ID NO: 1). 1) By mutating the non-K residue at position 168 in the AAV capsid protein to a K residue It can be reduced by

[0047] In some embodiments, for example, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or 11, which are known to be derived from other capsid proteins, correspond to position 168 of Anc80. The corresponding amino acid positions may be R amino acid residues or K amino acid residues depending on the desired liver-specific tropism. The acid residues can be mutated to either:

[0048] Similar properties (e.g., polarity, acidity / basicity) to the amino acids that confer the tropisms described herein Amino acids having different affinity, hydrophobicity, charge, and / or size can be used (e.g., For example, instead of G or A at position 266 for Anc80, or It is understood that the nucleotide sequence of ...

[0049] As described herein, the liver-specific tropism of AAV capsid proteins is shown (Figure 14 can be altered by mutating additional residues within the liver toggle region (defined as In some embodiments, a naturally occurring liver toggle region can confer the desired liver tropism. (e.g., to enhance or reduce the liver tropism of the resulting AAV vector) The liver toggle region can be replaced with that from a different serotype to reduce the In embodiments, the naturally occurring liver toggle region has the desired liver tropism (e.g., To improve or decrease the liver tropism of the resulting AAV vector As described herein, heterologous or de novo synthesis may be used. The novo-derived liver toggle region is located in the capsid protein of Anc80 (SEQ ID NO: 1). contains a G amino acid residue at the position in the AAV capsid protein corresponding to position 266 in The liver tropism of the resulting AAV vector is improved, and heterologous or de novo derived liver vectors are used. The ribonucleic acid toggle region corresponds to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1). If the corresponding AAV capsid protein contains an A amino acid residue at that position, the resulting AAV The liver tropism of the vector is reduced.

[0050] The discoveries described herein allow for the identification of proteins enriched in or derived from hepatocytes. For detargeted AAVs, the capsid protein sequences and, in detail, The amino acid residues corresponding to positions 266 and / or 168 in c80 (SEQ ID NO: 1) It is possible to screen AAVs based on the sequence of the nucleic acid they encode. Methods for sequencing nucleic acids are well known in the art and include chain termination. PCR amplification (e.g., Sanger sequencing) or chemical degradation (e.g., Maxam-Gill Many variations and improvements have been made, including but not limited to the bart sequencing method. Forms of automated sequencing and high-throughput sequencing have been developed and are well known in the art. It has been used in sequencing, including put sequencing methods.

[0051] As used herein, enriched or enriched means that the capsid protein is In Anc80 (SEQ ID NO: 1), a G amino acid residue at the amino acid position corresponding to position 266 and / or does not have an R amino acid position corresponding to position 168 (e.g., the original AAV genome count in hepatocytes (null or wild-type sequence, or sequence before mutation) As used herein, the term "hepatocyte proliferation" refers to an increase in the number of AAV genomes in hepatocytes compared to normal hepatocytes. In this case, detargeted, or detargeting, is when the capsid protein is Anc80 (SEQ ID NO: 1), an A amino acid residue at the amino acid position corresponding to position 266, and / or If the K amino acid position corresponding to position 68 is not present (e.g., the original or wild-type sequence), The number of AAV genomes in hepatocytes was compared with that in hepatocytes containing the AAV genome sequence (or the sequence before mutation). This refers to a reduction in the number of AAV genomes in the infected area.

[0052] Methods for screening AAV genome numbers in the liver are known in the art. Immortalized and / or transduced primary hepatocytes in vitro, as well as wild-type This typically involves in vivo systemic injection into both humanized and humanized mice (e.g., Grimm, et al., 2008, J. Virol., 82:5887-911; Lisowski et al., 2014, Nature, 382:doi:10. 1038 / nature12875).

[0053] Efficient (or increased) or inefficient (or decreased) liver tropism Representative capsid proteins that confer efficient liver tropism are provided herein. Representative capsid proteins that contribute to the enrichment of AAV genomes in hepatocytes, for example The sequences are SEQ ID NO: 2 [AAV9 Anc80+266G], SEQ ID NO: 6 [AAV9 A nc80+266G VRI], SEQ ID NO: 9 [AAV3B A266G S267 N2 68T], SEQ ID NO: 11 [AAV3B G265 A266G], SEQ ID NO: 13 [AAV 3B G265 A266G S268T], SEQ ID NO: 14 [AAV3B AAV9+2 67G-VRI], SEQ ID NO: 15 [AAV3B Anc80+266G-VRI] , while conferring reduced liver tropism (e.g., resulting in liver detargeting by AAV particles) ) Representative capsid protein sequences are SEQ ID NO: 3 [Anc80+266A], SEQ ID NO: 4 [AAV9 G267A], SEQ ID NO: 5 [AAV9 G267A S269T], SEQ ID NO: No. 7 [AAV9 Anc80+266A-VRI], SEQ ID NO: 10 [AAV3B G2 65 A266A], SEQ ID NO: 12 [AAV3B G265 A266A S268T] , SEQ ID NO: 16 [AAV3B Anc80+266A-VRI], SEQ ID NO: 17 [Anc 80L65 R168K].

[0054] As described in detail below, two AAV capsules exhibited a bimodal pattern of liver transduction. The sequences of the cytosine proteins, SEQ ID NO: 2 and SEQ ID NO: 3, have the sequence shown in SEQ ID NO: 1. In this case, position 266, designated X3 in FIG. 1, is G or A. Similarly, two AAVs exhibiting a bimodal pattern of liver transduction The capsid protein sequences SEQ ID NO: 16 and SEQ ID NO: 17 are identical to the sequence shown in SEQ ID NO: 1. The Anc80 scaffold sequence has the sequence: in this case, position 168, designated X1, is R or is one of K.

[0055] Nucleic acids encoding AAV capsid proteins, including liver toggle As described herein, the amino acid residues in the AAV capsid protein are 266 Mutation or insertion of the G and A amino acid residues in the toggle position results in The liver tropism of the resulting AAV switches between enrichment and detargeting. The amino acid residues in the protein are switched between R and K amino acid residues at position 168. Mutation between the sequences switches the liver tropism of the resulting AAV between enrichment and detargeting. The mutations are made at the nucleic acid level, and the mutations are not present in the encoded amino acid sequence (e.g., As used herein, a nucleic acid refers to a single molecule that is typically translated into a single protein. or may include DNA and RNA, including those containing multiple nucleic acid analogs or backbone modifications. Nucleic acids can be single-stranded or double-stranded, generally depending on the intended use.

[0056] Mutations can be introduced into nucleic acids using a variety of methods, many of which are well known in the art. For example, mutations can be induced by mutagenesis (e.g., site-directed mutagenesis, PCR) or chemically synthesizing a nucleic acid molecule containing the desired mutation(s). For example, Sambrook, Fritsch & Maniatis ( Molecular Cloning: a laboratory manual, 1989, Ed. 2) and Dieffenbach & Dveksle Please refer to (PCR primer: a laboratory manual, 2003, Ed. 2).

[0057] Nucleic acids can be obtained (e.g., isolated) using techniques routine in the art. For example, the nucleic acid can be prepared by, but not limited to, recombinant nucleic acid techniques and / or polynucleotides. The vectors can be isolated using any method, including polymerase chain reaction (PCR). The PCR technique is described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, E ds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid technology is For example, restriction enzyme digestion and ligation can be used to isolate nucleic acids. Isolated nucleic acids are chemically synthesized either as single nucleic acid molecules or as a series of oligonucleotides. can be synthesized into

[0058] An "isolated" nucleic acid molecule is a nucleic acid molecule that is isolated from at least one end of the genome of the organism from which the isolated nucleic acid molecule is derived. or nucleic acid molecules that do not contain naturally occurring flanking sequences at either end (e.g., PCR or restriction enzyme (cDNA or genomic DNA fragments generated by proteolysis). The fragment is generally chosen for ease of manipulation or to generate fusion nucleic acid molecules, which are discussed in more detail below. In order to In addition, isolated nucleic acid molecules include modified nucleic acid molecules, such as recombinant or synthetic nucleic acid molecules. obtain.

[0059] Polypeptides were purified by DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. obtained from natural sources (e.g., biological samples) by known methods such as chromatography (e.g., The polypeptide can also be expressed, for example, by the nucleic acid expression of an expression vector. In addition, the purified polypeptide can be purified by expressing it. The degree of purity of the polypeptide can be determined by any suitable method, e.g. , column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis can be measured using

[0060] As used herein, a "purified" polypeptide is one that is purified from the polypeptide in which it is naturally associated. A polypeptide is a polypeptide that has been separated or purified from cellular components that contain it. Typically, the polypeptide is At least 70% by dry weight (e.g., at least 75%, 80%, 85%, 90%, 9 5% or 99%) that is free from naturally associated proteins and naturally occurring molecules. A chemically synthesized polypeptide is considered "purified" if it is The synthetic polypeptides are "purified" because they are separated from the components that naturally accompany them.

[0061] The nucleic acid can be propagated in a vector. The vector can be a viral vector or The vector may include a viral or non-viral vector, and may include an expression vector. Many vectors are commercially available and vectors can be used in recombinant DNA applications conventional in the art. In some cases, vectors containing nucleic acids can be readily produced using techniques such as The vector may have an expression element that can be operably linked to a nucleic acid such as The present invention also includes sequences such as sequences encoding selectable markers (e.g., antibiotic resistance genes). The vector containing the nucleic acid may further comprise a chimeric or fusion polypeptide (i.e., a polynucleotide The peptide is operably linked to a heterologous polypeptide, which may be at either the N-terminus or C-terminus of the peptide. A typical heterologous polypeptide can be encoded by the encoded These can be used in the purification of polypeptides containing 6xHis tags, thione S-transferase (GST)).

[0062] Expression elements are known in the art and are used to direct and control the expression of coding sequences. An example of an expression element is a promoter sequence. Expression elements include introns, enhancer sequences, and response elements that regulate the expression of nucleic acids. The expression elements may be of viral origin or may contain inducible elements. or by non-viral molecular biology techniques (e.g., simple propagation of plasmid vectors) ) or the expression element may be derived from, but is not limited to, bacteria, yeast, insects, or The expression elements may be of various origins, such as mammalian origin, or may be a combination of elements of various origins. As used herein, operably linked means a promoter. or other expression element(s) to direct or control expression of the nucleic acid. In some instances, operably linked refers to being located within a vector relative to a nucleic acid. means that the two sequences are in frame.

[0063] Methods for introducing viral vectors into host cells are known in the art. These methods typically utilize the natural infectivity of the virus. Methods for the production of microbial cells are known in the art. As used herein, a "host cell" refers to a refers to the specific cells into which a viral or non-viral vector is introduced, and The host cell may be a prokaryotic or eukaryotic cell, as appropriate. For example, nucleic acids can be obtained in bacterial cells such as E. coli or in insect cells. , yeast or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or The vector can be expressed in COS cells. Other suitable host cells are known to those skilled in the art. Non-viral nucleic acids can be prepared by, but are not limited to, electroporation, phosphate phosphatase, and the like. Calcium precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofuscin known methods such as transfection, microinjection, and viral transfection The method can be used to transduce host cells both in vivo and in vitro. It can be introduced.

[0064] Methods using AAV capsid proteins containing liver toggle AAV viruses deliver transgenes (in cis or trans, along with other viral sequences) The transgene may be delivered to the cell containing, for example, a reporter gene (e.g., a base pair). ta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, Thymidine kinase, green fluorescent polypeptide (GFP), chloramphenicol acetylcholine transferase (CAT), or luciferase, or hemagglutinin or M yc) or a therapeutic gene (e.g., hormones or their receptors, growth factors or their receptors, differentiation factors or their receptors , immune system regulators (e.g., cytokines and interleukins) or their receptors , enzymes, RNA (e.g., inhibitory or catalytic RNA) or target antigens (e.g., oncogenic The genes may be genes encoding immune antigens (antigens, autoimmune antigens).

[0065] The particular therapeutic gene will depend, at least in part, on the particular disease or deficiency being treated. By way of example only, gene transfer or gene therapy may be used to treat diseases such as hemophilia, retinitis pigmentosa, cystic leukemia, and fibrosis, Leber's congenital amaurosis, lysosomal storage disorders, inborn errors of metabolism (e.g., phenylalanine, Inborn errors of amino acid metabolism, including ketonuria; inborn errors of organic acid metabolism, including propionic acidemia Usually, inborn errors of fatty acid metabolism, including medium-chain acyl-CoA dehydrogenase deficiency (MCAD) ), cancer, color blindness, cone-rod dystrophy, macular degeneration (e.g., age-related macular degeneration) ), lipopolypeptide lipase deficiency, familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, Alzheimer's disease, Parkinson's disease, obesity, inflammatory bowel disease Disability, diabetes, congestive heart failure, hypercholesterolemia, hearing loss, coronary heart disease, familial renal Amyloidosis, Marfan syndrome, fatal familial insomnia, Creutzfeldt-Jakob syndrome disease, sickle cell disease, Huntington's disease, frontotemporal lobar degeneration, Usher syndrome, lactose Intolerance, lipid storage disorders (e.g., Niemann-Pick disease type C), Batten disease, choroideremia A, glycogen storage disease type II (Pompe disease), ataxia-telangiectasia (Louis-Bar syndrome), congenital Hypothyroidism, severe combined immunodeficiency (SCID), and / or amyotrophic lateral sclerosis The present invention can be applied to the treatment of amyotrophic lateral sclerosis (ALS). Therapy can be applied to treat retinal dystrophies (e.g., biallelic RP). One-time gene therapy indicated for treatment of patients with confirmed E65 mutation-associated retinal dystrophy The product (LUXTURNA™) (boretigene neparvovec-rzyl (vo retigene neparvovec-rzyl))(Spark Therapeutics Inc., Philadelphia, PA).

[0066] The therapeutic gene can also be used to infect, for example, a subject (e.g., a human, an animal (e.g., a pet, livestock, For example, the immunogen can be an immunogen useful for immunizing an organism (e.g., an endangered animal). pathogen) or an immunogenic portion or component thereof (e.g., a toxin polypeptide or For example, immunogenic polypeptides can be obtained from The active ingredient is a virus (e.g., picornavirus, enterovirus, orthomyxovirus). viruses, reoviruses, retroviruses), prokaryotes (e.g., Streptococcus pneumoniae, Staphylococci, Listeria, Pseudomonas )), and eukaryotes (e.g., amoeba, malaria, leishmania, nematode). The methods described herein and compositions produced by such methods are not intended to be limiting of any particular It is understood that there is no limitation on the transgene.

[0067] Typically, AAV virus suspended in a physiologically compatible carrier is transfected into the target tissue using standard techniques. The compound can be administered to a mammal (e.g., a human or non-human mammal). Suitable buffers (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, Saline solutions that can be formulated using cereals, gelatin, dextran, agar, pectin, and water The AAV virus transduces or infects the appropriate cells and produces sufficient levels of the gene. Administered in amounts sufficient to provide gene transfer and expression and to provide therapeutic benefit without undue adverse effects. Common pharmaceutically acceptable routes of administration include oral, intranasal, intratracheal, inhalation, intravenous, By intramuscular, intraocular, subcutaneous, intradermal, transmucosal, or other route of administration, e.g., into the liver or lung The route of administration may be, but is not limited to, direct delivery to an organ such as , can be combined.

[0068] The dose of AAV virus administered to a subject will depend primarily on the condition being treated, as well as the subject's age, weight, and For example, the therapeutic agent administered to a human subject may vary depending on factors such as the type of treatment and the health of the subject. The effective dose of AAV virus is generally about 1 x 10 1 ~1×10 12 Genome copies (GC) Concentration of virus (e.g., approximately 1 x 10 3 ~1×10 9 About 0.1 ml to about 0.1 ml of a solution containing GC Transduction and / or expression of transgenes can be performed using DNA, RNA, or at various time points after administration by protein assay. In this case, the expression level of the transgene can be monitored to determine the frequency and / or amount of administration. Dosage regimens similar to those described for therapeutic purposes are also available for immunization. It can be used.

[0069] In accordance with the present invention, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques are employed. Such techniques are well explained in the literature. The present invention does not limit the scope of the methods and compositions of the present invention, as set forth in the claims. These are further described in the Examples below. [Example]

[0070] [Example 1] Materials and methods for identifying AAV liver toggles 2. AAV Sequence 11 (2048) Variant Anc80 libraries (e.g., U.S. Pat. No. 6,412,139) 9,695,220) into the Anc80 scaffold sequence (SEQ ID NO: 1; Figure 1). One position (X1 to X in Figure 1) 11 ) was generated by modifying each variant. Cloned into mammalian expression plasmids, pRep and pAd helper accessors Transfect HEK293 cells with the lysed plasmid to express the viral vector. This library was then used to generate a liver-localized in vivo used for screening in vo (e.g., enrichment vs. detargeting). Briefly, one experiment In this study, three mice were transfected with the Anc80 vector library, with a total of 2.7311 (approximately Three days after injection, the mice were sacrificed and the livers were collected and frozen. In a separate experiment, two rhesus monkeys were transfected with 1.6 e of the Anc80 vector library. The test was terminated 28 days after injection, and the livers were harvested. NA was extracted from the liver, and the population of viral variants present in the tissue was analyzed by next-generation sequencing. As described below, the Anc80 scaffold sequence shown in Figure 1 The X3 position, highlighted by the nucleotide sequence X3, was determined to be the liver toggle position. The X3 position is located in the Anc80 scaffold sequence. This corresponds to residue 266 of

[0071] The results of the in vivo screening experiments are shown in Figures 9 and 10. 2048 variant Anc80 library versus viral input (x-axis) A plot showing the abundance of members (y-axis) is shown. On the y-axis, zero indicates a change in input. indicates the absence of targeting, and positive and negative values indicate relative enrichment or detargeting, respectively. Of the 11 toggle positions within the c80 scaffold sequence, position X3 correlated with the observed hepatic bimodality. .

[0072] Inferior, but nevertheless associated with liver enrichment or detargeting, is the X1 position. The amino acid residue identity corresponds to residue 168 of the Anc80 scaffold sequence. The consideration of the identity of all toggle positions is visualized by a method called "smartmap." Furthermore, by selecting a group of variants according to the desired properties, The heat map can reveal multiple affected toggle locations, if any. Using the same data from Figures 9 and 10, wild-type C57BL / 6 mice (Figure 25) and rhesus macaques (Figure 26) both had the most liver-enriched Anc80 variant. The X3=1 (G) and X1=1 (R) are present in the For both mouse and human hepatocytes recovered from the FRG human liver xenograft mouse model, and primary human hepatocytes ( A similar pattern was observed in micropatterned co-cultures (MPCCs).

[0073] [Example 2] Generation and testing of AAV liver toggles Specific sequences from the 2048 variant Anc80 library, including liver toggles (e.g. See, for example, U.S. Pat. No. 9,719,070, which is incorporated herein by reference in its entirety. Anc80L65 (SEQ ID NO: 2) contains a G at position 266. Anc80L65 G266A (SEQ ID NO: 3) shows liver enrichment and contains an A at position 266. The G / A liver toggle is highlighted and color-coded to match the color coding shown in Figure 2. The colors are coded in Figure 3 as shown below.

[0074] Each individual variant was identified by site-directed mutagenesis or gene synthesis with PCR-generated fragments. These fragments are derived from either isothermal (Gibson) cloning or by mixing the fragments with the original fragments. The variants are cloned into standard rep / cap "trans" plasmids to Then, vectors expressing GFP and alpha-1-antitrypsin are generated. The variant was identified at the Grousbeck Gene Therapy Center It is generated by the Gene Transfer Vector Core.

[0075] Three 8-week-old male mice per variant and parental controls were transfected with a total genome copy of 1e11 ( Approximately 5 e 12 gc / kg) was injected, and liver tissue was collected on the third day. The peaks were determined by qPCR and are expressed as absolute values and as a percentage of the parental control. Anc80L65 (SEQ ID NO: 2), which contains G at position 266, shows liver enrichment; Anc80L65 G266A (SEQ ID NO: 3), which contains A, exhibits liver detargeting. Furthermore, Figure 12 shows that the expression from the genome is observed by eGFP staining of liver tissue. When using Anc80L65 (SEQ ID NO: 2) containing G at position 266, A at position 266 is used. It is shown that the nucleotide sequence is more pronounced for Anc80L65 G266A (SEQ ID NO: 3) containing .

[0076] [Example 3] Identifying the corresponding liver toggle residues in other serotypes Figure 13A shows the crystal structure of the AAV2 VP3 capsid monomer. The location of the LT is indicated by an arrow and the structure of the region encompassing the LT is boxed. The residues are 2–3 residues N-terminal to the α-toggle at position 266 of Anc80. Figure 13B shows the overlap of the LT region of AAV2, 3, 6, 8 and 9VP3. The crystal structure of AAV2 VP3 shows the ribosomal domain and highlights the local secondary structures that define the toggle position. The toggle region is located between the β-ascending (βa) and β-descending (βd) secondary structures. These secondary structures are encoded by the α-toggle (αt) at the N-terminal end of the α-toggle (αt) domain. The LT residue is defined as a residue with the main functional group that is It is located within VR1, a beta-sheet connecting loop, N-terminal to the ribosomal region.

[0077] Figure 14 shows the primary VR from the original and clinically relevant serotype, encompassing the liver toggle. I Amino acid sequence alignment, Anc80L65 is located on line 15. The positions of ascending (βa), descending (βd), α toggle (αt), the toggle region, and the toggle itself. The β-ascending (βa) sequence begins at a conserved tyrosine, and the β-descending (βd) sequence begins at a conserved tyrosine. terminates at a conserved serine, equivalent to Anc80 266 in most serotypes The location of the cleavage site can be easily inferred from the close identity. The rhesus monkey B viruses AAV2 and AAV3, and the related viruses AAV4 and Rh In 32 and 33 the corresponding position is more vague or less clear. Nevertheless, the primary sequence was prepared as a guide to identify liver toggles in other serotypes. As shown in Figure 5, the toggle is located at residue 266 in Anc80L65, the AAV 9 and residue 267 in AAV5, and define the toggle as , it is emphasized that the position numbers are relative.

[0078] [Example 4] Generation and testing of AAV liver toggles in other serotypes Similarly, Figure 15 shows the Anc80L65, AAV3B, and AAV9 serotypes, as well as The α-toggle (ND) variant was constructed to test the liver toggling hypothesis. Sequence alignment of the liver toggle region, oriented in the N-direction. AAV3B, AAV9, and other variants can be transfected into mice (M) or primates (primates). The in vivo assay was performed in the liver of mice with P. Transduction efficiency is indicated by "on" or "off" in the last two columns. Phenotypes for experiments still in progress are in bold, and phenotypes for experiments still in italics. Show it with your body.

[0079] In addition to the single residue liver toggles identified herein, the entire liver toggle region The liver toggle function is transferred to the heterologous serotype while retaining other desirable characteristics of the liver toggle source serotype. In Figure 16, the conserved domains adjacent to such residues are used to We describe a method to quickly and easily test the transplantability of the liver toggle region. Homology-specific association to heterologous serotypes by amplification or de novo synthesis of a 0-bp region This becomes possible.

[0080] FIG. 17 shows a portion of this study and the associated liver toggle regions as illustrated in FIG. The last two columns list the range swaps in mice and primates determined by this study. In vivo liver toggling functions are shown in bold or predicted liver toggling functions are shown in bold. It is written in italics.

[0081] In vitro transduction of Huh7 hepatoma cells resulted in the expression of all liver tether variants. To test the viability of these variants, we investigated the in vivo liver toggle activity of these variants. This is useful in both in vitro and in suggesting an "on" or "off" phenotype. and titers as determined by vector-delivered luciferase expression as illustrated in FIG. When normalized for , most of the altered variants were surprisingly not stable. Furthermore, the amount of luciferase activity observed showed that such variants with a liver-on “G” at the position equivalent to Anc80 are involved in the liver The expected liver function was higher in the off-A group than in their off-A siblings in that they had higher RLU values. This was consistent with the liver toggle phenotype.

[0082] Importantly, the toggle is located in vi at a position equivalent to position 266 of Anc80. This is demonstrated in vo, which is position 267 in AAV9. Changing 7 to A resulted in the production of live luciferase synthases, as illustrated in Figures 20 and 21. Signal, genome copies per cell and delivered marker expression were 100% in the liver. Furthermore, the need for alteration of other toggle region residues is reduced by the difference in the Toggle single mutant AA Superior potency of the double mutant AAV9 G267A S269T over AAV9 G267A This result is further substantiated by the fact that the double mutant not only exhibits a liver "off" phenotype, but also This suggests advantages for gene delivery to liver and skeletal muscle.

[0083] Human liver xenotransplantation models in NHP and FRG mice, and in vitro human liver xenotransplantation models Further studies in a mouse liver model will reveal a second factor affecting hepatic gene delivery by AAV. The location was identified and is referred to herein as "liver toggle 2." This toggle is located in the Anc8 It is encoded by residue X1 of the 0 scaffold and corresponds to position 168 of Anc80L65.

[0084] Figure 22 shows the putative liver toggle "off" virus AAV3B with A266 changed to G. Insertion of T two residues C-terminal (SEQ ID NO: 9) or change of residue at position 268 from S to T or by leaving it unchanged and inserting a G before A266 (SEQ ID NO: 11) The liver can be "turned on" by the IL-1 receptor, as demonstrated by luciferase expression during life. The liver "off" prediction is compatible with the A "off" insertion in these latter two variants. (SEQ ID NO: 10). Furthermore, the native AAV3B liver toggle "off" region , liver toggle from AAV9 (SEQ ID NO: 14) and Anc80L65 (SEQ ID NO: 15) Swapping the "on" region also improves mouse liver transduction. Liver Toggle "Off" A Swapping nc80L65 to G266A reduces luciferase activity in vivo Ze expression is reduced compared to its "on" counterpart Anc80L65 (SEQ ID NO: 16).

[0085] Figure 24 shows the location and localization of X1 in clinically relevant serotypes and AncAAV. This was confirmed by sequence alignment of all AAVs except AAV5. The VP2 N-terminal region is located in a positively charged motif that is easily identifiable by the VP2 N-terminal region. The end is 17–20 residues shorter. Nevertheless, all serotypes share this conserved region. Related to adjacent residues: two residues N-terminal proline and one residue C-terminal lysine Liver Toggle 2 can be defined by gender.

[0086] Figures 25 and 26 illustrate the suitability of Liver Toggle 2 for liver enrichment. In this study, we performed experiments using wild-type C57BL / 6 mice and a human liver xenograft mouse model. We searched for enrichment of Anc80 variants in both mouse hepatocyte components of the FRG. Figure 2 In 6, experiments were performed using rhesus monkey liver, reciprocal human hepatocytes from FRG experiments, and myocytes. They were cultured in vitro using a technique called multipattern coculture (MPCC). We searched for the enrichment of Anc80 variants in human hepatocytes. Once the identified variants are selected from the accompanying MA plot, the fitness of Liver Toggle X3 is evaluated. In addition, the suitability of X1 is clear, and the ranking order is from lowest to highest. As enrichment increases, the variant switches from toggle 0 = K to toggle 1 = R. This pattern indicates that liver toggle 2 at position X1 is independent of liver toggle at position X3, but The X3 toggle suggests a dominant position.

[0087] AAV3B requires the human hepatocyte growth factor receptor (HuHG) for efficient transduction into hepatocytes. Since the discovery that FR is dependent on the FR (Ling et al., 2010, Hum. Gen. Ther., 21(12): 1741- 1747), which has been well-received as a clinically relevant serotype. Prior to this discovery, this serotype was However, this insufficient liver delivery ability in mice has led to its rejection in gene therapy. Nevertheless, most preclinical disease models are in mice, and Mouse models are useful, for example, in determining the efficacy, safety, and dosage of gene therapy drugs. AAV3B plays an important role in the development of HIV-1-associated HIV-1-mediated HIV-1-associated ... Modifying AAV3B to improve liver delivery capacity in humans to the same level as in primates is It is very useful for industry. AAV3B has a shortened VR compared to Anc80. I and liver toggle region, which corresponds to position 266 of Anc80. This makes it difficult to assign residues (see Figures 14 and 15).

[0088] Changing A to G at position 266 did not improve gene delivery into Huh7 cells; in fact, The modifications can generate loss-of-function phenotypes in AAV3B. Reinforcing the need to consider the entire liver toggle region, the T Insertion of A266 into Anc80 with concomitant change to G at A266, or the corresponding Insertion of either A or G at the position where it would be inserted resulted in the formation of a chromosome in Huh7 cells and in In vivo, variants that exhibit both liver "on" and "off" phenotypes are generated. Furthermore, AAV3B is a good candidate for liver toggle region swapping. Appears to be well tolerated and produces a liver "on" liver toggle from AAV9 and Anc80L65 When the region was transferred, the Huh7 variant was expressed in the mouse liver in vivo. Cellular gene delivery is improved relative to AAV3B alone. Liver "off" Anc80L65 Transfection with the G266A liver toggle region reduced this activity to near background levels. (Figure 19 and Figure 22). Importantly, a single G is inserted between G265 and A266. Upon introduction, Aβ was detected in mice as determined by luciferase signal in the liver. A 3B-based serotype with potency comparable to AV9 is generated (Fig. 22).

[0089] Other embodiments The methods and compositions of the present invention are described herein in several different aspects. Although the foregoing description of the various embodiments is illustrative, it is to be understood that the present invention is not limited to the methods and compositions of the present invention. It should be understood that no limitation of the scope of the invention is intended. and modifications are within the scope of the following claims.

[0090] Disclosed are methods and compositions that can be used, can be used together, can be used in preparation for, or products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are also disclosed. That is, specific reference to various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, but each is specifically contemplated and described herein. For example, if a particular composition or a particular method is disclosed and discussed, and several compositions or methods are discussed, any and all combinations and permutations of the compositions and methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is specifically contemplated and disclosed. Various embodiments of the present invention are described below. 1. A method for altering the tissue tropism of an adeno-associated viral (AAV) vector, comprising: Mapping an amino acid position within an AAV capsid protein that corresponds to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) or that corresponds to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1); replacing the naturally occurring amino acid at a mapped position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) with a glycine (G) amino acid residue to improve liver tropism of the resulting AAV vector, or with an alanine (A) amino acid residue to decrease liver tropism of the resulting AAV; or replacing the naturally occurring amino acid at a mapped position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1) with an arginine (R) amino acid residue to improve liver tropism of the resulting AAV vector, or with a lysine (K) amino acid residue to decrease liver tropism of the resulting AAV vector; A method comprising: 2. The method described in 1 above, comprising a step of replacing the naturally occurring amino acid at a positioned corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) with a G amino acid residue to improve the liver tropism of the resulting AAV vector. 3. The method described in 1 above, comprising a step of replacing the naturally occurring amino acid at a positioned corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) with an A amino acid residue to reduce the liver tropism of the resulting AAV vector. 4. The method described in 1 above, comprising a step of replacing the naturally occurring amino acid at a positioned corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1) with an R amino acid residue to improve the liver tropism of the resulting AAV vector. 5. The method described in 1 above, comprising replacing the naturally occurring amino acid at a positioned corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1) with a K amino acid residue to reduce the liver tropism of the resulting AAV. 6. The method according to any one of 1 to 5 above, wherein the replacing step is carried out using site-directed mutagenesis. 7. A method according to any one of 1 to 6 above, wherein the replacing step is carried out by restriction digestion and ligation of existing or de novo synthesized DNA. 8. A method according to any one of 1 to 7 above, wherein the replacing step is carried out by homologous association of existing or de novo synthesized DNA. 9. A method according to any one of 1 to 8 above, wherein the step of locating is carried out by sequencing. 10. A method for screening the level of liver tropism of an adeno-associated virus (AAV), comprising: sequencing nucleic acids encoding AAV capsid proteins; mapping an amino acid position within the AAV capsid protein corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) or an amino acid position within the AAV capsid protein corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1); identifying an AAV capsid protein having a G or A amino acid residue at a position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1), or having an R or K amino acid residue at a position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1); wherein a G amino acid residue at a positioned position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) or an R amino acid residue at a positioned position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1) indicates an AAV that exhibits liver tropism, and an A amino acid residue at a positioned position corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1) or a K amino acid residue at a positioned position corresponding to position 168 in the capsid protein of Anc80 (SEQ ID NO: 1) indicates an AAV that exhibits little or no liver tropism. 11.266th place X 3 is selected from G or A, and X at position 168 1 is selected from R or K. An adeno-associated virus (AAV) having the sequence set forth in SEQ ID NO: 1 [Anc80]. 12. SEQ ID NO:2 [Anc80L65], SEQ ID NO:3 [Anc80L65 G266A], SEQ ID NO:4 [AAV9 G267A], SEQ ID NO:5 [AAV9 G267A S269T], SEQ ID NO:6 [AAV9 Anc80L65-VRI], SEQ ID NO:7 [AAV9 Anc80L65 G266A-VRI], SEQ ID NO:8 [AAV3B A266G], SEQ ID NO:9 [AAV3B A266G S267 N268T], SEQ ID NO:10 [AAV3B G265 A266A], SEQ ID NO:11 [AAV3B G265 A266G], SEQ ID NO:12 [AAV3B G265 A266A S268T], SEQ ID NO:13 [AAV3B G265 A266G S268T], SEQ ID NO:14 [AAV3B An adeno-associated virus (AAV) having a sequence selected from the group consisting of SEQ ID NO: 15 [AAV3B Anc80L65-VRI], SEQ ID NO: 16 [AAV3B Anc80L65 G266A-VRI], and SEQ ID NO: 17 [Anc80L65 R168K]. 13. A method for altering the tissue tropism of an adeno-associated viral (AAV) vector, comprising: positioning the liver toggle region defined in FIG. 14 within an AAV capsid protein; Replacing the naturally occurring liver toggle region with a liver toggle region from a heterologous serotype or a de novo-derived sequence to improve or decrease the liver tropism of the resulting AAV vector. A method comprising: 14. The method described in claim 13, wherein the liver tropism of the resulting AAV vector is improved when the heterologous or de novo-derived liver toggle region contains a G amino acid residue at a position in the AAV capsid protein corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1). 15. The method described in claim 13, wherein when the heterologous or de novo-derived liver toggle region contains an A amino acid residue at a position in the AAV capsid protein corresponding to position 266 in the capsid protein of Anc80 (SEQ ID NO: 1), the liver tropism of the resulting AAV vector is reduced.

Claims

1. An adeno-associated virus (AAV) comprising a capsid protein selected from the group consisting of: a) an AAV9 capsid protein containing a modification in its liver toggle region that includes AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 58); b) Anc80L65 capsid protein containing a modification in its liver toggle region that includes AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63); c) an Anc80L65 capsid protein comprising a modification in its liver toggle region comprising AAV9-VR1 (NSTSGGSSNDN, SEQ ID NO: 62); and d) AAV3B capsid protein containing the S268T modification.

2. 2. The adeno-associated virus (AAV) of claim 1, wherein the capsid protein is an AAV9 capsid protein that contains a modification in its liver toggle region that includes AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 58).

3. 2. The adeno-associated virus (AAV) of claim 1, wherein the capsid protein is an Anc80L65 capsid protein that comprises a modification in its liver toggle region that includes AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63).

4. 2. The adeno-associated virus (AAV) of claim 1, wherein the capsid protein is an Anc80L65 capsid protein that comprises a modification in its liver toggle region that includes AAV9-VR1 (NSTSGGSSNDN, SEQ ID NO: 62).

5. 2. The adeno-associated virus (AAV) of claim 1, wherein the capsid protein is an AAV3B capsid protein containing the S268T modification.

6. 1. A method for altering the tissue tropism of an adeno-associated virus (AAV), comprising: a) modifying the liver toggle region of the AAV9 capsid protein to include AAV3B-VR1 (SQSGASNDN, SEQ ID NO:58); b) modifying the liver toggle region of the Anc80L65 capsid protein to include AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63); c) modifying the liver toggle region of the Anc80L65 capsid protein to include AAV9-VR1 (NSTSGGSSNDN, SEQ ID NO: 62); or d) modifying the liver toggle region of the AAV3B capsid protein to include the S268T modification; A method comprising:

7. An adeno-associated virus (AAV) produced by the method of claim 6.

Images