Capsid proteins of an adeno-associated virus (AAV), nucleic acid molecule, recombinant AAV, adeno-associated viruses (AAV), and method for altering the tissue tropism of an adeno-associated virus (AAV)

Description

1 / 39 Capsid proteins of an adeno-associated virus (AAV), nucleic acid molecule, recombinant AAV, adeno-associated viruses (AAV), and method for altering the tissue tropism of an adeno-associated virus (AAV) Separated from BR112020022858-3, filed on 10 / 05 / 2019 CROSS-REFERENCE FOR RELATED ORDERS

[001] This application claims priority for U.S. Provisional Application Serial No. 62 / 841,179, filed April 30, 2019, and for U.S. Provisional Application Serial No. 62 / 670,543, filed May 11, 2018. TECHNICAL FIELD

[002] This description generally refers to the alteration of the tissue tropism of adeno-associated virus (AAV) and, specifically, to the control of the hepatic tropism of AAV. BACKGROUND

[003] Adeno-associated virus (AAV) is a small virus belonging to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. The virus is a non-enveloped, replication-defective virus that infects, but is not known to cause disease in, humans and certain primate species. AAV can infect dividing and quiescent cells and persist in an extrachromosomal state without integrating into the host cell genome, although in the native virus, some integration of virus-carrying genes into the host genome may occur. These characteristics make AAV a candidate for use as a viral vector in gene therapy. However, certain AAV serotypes exhibit hepatic tropism, which, depending on the disease being treated, may or may not be desirable.

[004] It would be beneficial to understand how the hepatic tropism of AAV is determined and, based on that information, be able to manipulate the Petition 870260051179, dated 05 / 28 / 2026, page 12 / 239 2 / 39 AAV tropism. SUMMARY

[005] The specificity of the tissue, that is, the tropism of the tissue, of AAV is determined by the capsid serotype, and the methods and compositions described herein allow for altering the tissue tropism of a particular AAV with specificity to improve therapies administered by these altered AAVs. For example, altering or changing the hepatic tropism of an AAV can be beneficial, for instance, when the liver is the desired target, e.g., by increasing a natural liver tropism, and similarly when the liver is not the desired target, e.g., by reducing a natural liver tropism. For example, a lower dose of a given AAV can be administered to an individual when that virus is more effectively released and more effectively transfects liver cells (or a non-hepatic organ).

[006] In one aspect, this description provides methods for altering the tissue tropism of adeno-associated virus (AAV) vectors. Such methods typically include locating an amino acid position within an AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No.: 1); and replacing a naturally occurring amino acid at the located position with a glycine (G) amino acid residue to provide enhanced hepatic tropism of the resulting AAV vector, or with an alanine (A) amino acid residue to reduce hepatic tropism, i.e., provide hepatic debleaching of the resulting AAV.

[007] In some embodiments, such methods include replacing a naturally occurring amino acid at the located position with a G amino acid residue to provide enhanced hepatic tropism, e.g., hepatic enrichment, of the resulting AAV vector. In some embodiments, such methods include Petition 870260051179, dated 05 / 28 / 2026, page 13 / 239 3 / 39 replacement of the naturally occurring amino acid at the localized position by an amino acid residue A to provide reduced hepatic tropism, e.g., hepatic bleaching by the resulting AAV.

[008] In another aspect, this description provides methods for altering the tissue tropism of adeno-associated virus (AAV) vectors. Such methods typically include localizing an amino acid position within an AAV capsid protein corresponding to position 168 in an Anc80 capsid protein (SEQ ID No: 1); and replacing a naturally occurring amino acid at the localized position with an arginine (R) amino acid residue to provide enhanced hepatic tropism, e.g., hepatic enrichment, of the resulting AAV vector, or with a lysine (K) amino acid residue to provide hepatic tropism, e.g., hepatic bleaching of the resulting AAV.

[009] In some embodiments, such methods include replacing the naturally occurring amino acid at the localized position with an R amino acid residue to provide hepatic enrichment of the resulting AAV vector. In some embodiments, such a method may include replacing the naturally occurring amino acid at the localized position with a K amino acid residue to provide reduced hepatic targeting of the resulting AAV.

[0010] In another aspect, methods for altering the tissue tropism of an adeno-associated virus (AAV) vector are provided. Such methods typically include locating a liver alternanance region, as defined in Figure 14, in an AAV capsid protein; and replacing a naturally occurring liver alternanance region with a liver alternanance region from a heterologous serotype or de novo derived sequence to alter liver tropism, for example, to provide increased or decreased liver targeting of the resulting AAV vector. Petition 870260051179, dated 05 / 28 / 2026, page 14 / 239 4 / 39

[0011] In some embodiments, when the heterologous or de novo derived hepatic alternation region comprises an amino acid residue G at a position within an AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No.: 1), hepatic enrichment of the resulting AAV vector is provided. In some embodiments, when the heterologous or de novo derived hepatic alternation region comprises an amino acid residue A at a position within an AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No.: 1), reduced hepatic targeting of the resulting AAV vector is provided.

[0012] In some embodiments, the replacement step is performed using site-directed mutagenesis, restriction digestion and ligation of existing or newly synthesized DNA, homology-mediated assembly of existing or newly synthesized DNA, or combinations thereof. In some embodiments, the localization step is performed by sequencing.

[0013] In another aspect, the description provides evaluation methods for an AAV whose liver transfection is enriched or reduced. Such methods typically include sequencing a nucleic acid encoding an AAV capsid protein; locating an amino acid position within the AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No: 1); and identifying an AAV capsid protein possessing a G amino acid residue at the located position or an A amino acid residue at the located position. Generally, a G amino acid residue at the located position indicates an AAV whose liver transfection is enriched, while an A amino acid residue at the located position indicates an AAV whose liver transfection is reduced. Petition 870260051179, dated 05 / 28 / 2026, page 15 / 239 5 / 39

[0014] In another aspect, the description provides evaluation methods for an AAV whose liver transfection is enriched or reduced. Such methods typically include sequencing a nucleic acid encoding an AAV capsid protein; locating an amino acid position within the AAV capsid protein corresponding to position 168 in an Anc80 capsid protein (SEQ ID No.: 1); and identifying an AAV capsid protein possessing an R amino acid residue at the located position or a K amino acid residue at the located position. Generally, an R amino acid residue at the located position indicates an AAV whose liver transfection is enriched, while a K amino acid residue at the located position indicates an AAV whose liver transfection is reduced.

[0015] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 1 [Anc80], where X3 at position 266 is selected from a G or A.

[0016] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 1 [Anc80], where X1 at position 168 is selected from an R or K.

[0017] In another aspect, this description provides an AAV having the sequence shown in SEQ ID N°: 2 [Anc80L65].

[0018] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 3 [Anc80L65 G266A].

[0019] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 4 [AAV9 G267A].

[0020] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 5 [AAV9 G267A S269T].

[0021] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 6 [AAV9 Anc80L65-VRI].

[0022] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 7 [AAV9 Anc80L65 G266A-VRI]. Petition 870260051179, dated 05 / 28 / 2026, p. 16 / 239 6 / 39

[0023] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 8 [AAV3B A266G].

[0024] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 9 [AAV3B A266G S267 N268T].

[0025] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 10 [AAV3B G265 A266A].

[0026] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No.: 11 [AAV3B G265 A266G].

[0027] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No.: 12 [AAV3B G265 A266A S268T].

[0028] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 13 [AAV3B G265 A266G S268T].

[0029] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 14 [AAV3B AAV9-VRI].

[0030] In another aspect, this description provides an AAV having the sequence shown in SEQ ID N°: 15 [AAV3B Anc80L65-VRI].

[0031] In yet another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 16 [AAV3B Anc80L65 G266A-VRI].

[0032] In another aspect, this description provides an AAV having the sequence shown in SEQ ID No: 17 [Anc80L65 R168K].

[0033] When used herein, tissue tropism refers to a natural tissue specificity for infection and / or transfection of a cell by a particular AAV. For example, many AAVs exhibit hepatic tropism, meaning that these AAVs preferentially infect and / or transfect liver cells rather than cells of other tissue types. Tissue tropisms are often based on specific surface proteins, for example, receptor proteins, found on the surface of cells in a specific tissue and / or in Petition 870260051179, dated 05 / 28 / 2026, page 17 / 239 7 / 39 specific AAV surface.

[0034] As used herein, alternation refers to a specific site or region within an AAV capsid protein associated with a tissue tropism of that AAV. Thus, when the naturally occurring amino acid at the alternation site or region, as described herein, is replaced by a different amino acid, the tissue tropism of that AAV is altered. Therefore, a hepatic alternation, for example, hepatic alternation 1, refers to a residue that can be switched so that transfection occurs predominantly or essentially entirely in liver cells or predominantly or essentially not in liver cells.

[0035] Similarly, when used herein, hepatic alternation region refers to the 20 amino acid residues located between two beta strands within which hepatic alternation resides, as defined in Figure 14. Therefore, a hepatic alternation region refers to a sequential series of residues that can be exchanged via import of heterologous AAVs or via de novo derivation, such that transfection occurs predominantly or essentially entirely in liver cells or predominantly or essentially not in liver cells. The hepatic alternation region overlaps with variable region I (VRI), therefore all alternation region exchanges use this nomenclature as an abbreviation.

[0036] When used here, alternation 2 refers to an exchange independent of alternation 1. Therefore, hepatic alternation 2 refers to another residue that is different from the residue in question in hepatic alternation 1 and that can be exchanged so that transfection occurs predominantly or essentially entirely in liver cells or predominantly or essentially Petition 870260051179, dated 05 / 28 / 2026, page 18 / 239 8 / 39 entirely not in liver cells.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the methods and compositions of matter pertain. Although methods and materials similar or equivalent to those described in this document may be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. DESCRIPTION OF THE DRAWINGS

[0038] The patent file or application contains at least one drawing executed in color. Copies of this patent publication or patent application with color drawings will be provided by the Institute upon request and payment of the necessary fee.

[0039] Figure 1 is a sustaining sequence Anc80 (SEQ ID Figure 1) shows eleven positions that were varied to generate an Anc80 library of variant 211(2048) used for in vivo evaluation. Positions X1 and X3, highlighted, were determined as hepatic alternation positions and correspond to residues 168 and 266 in the Anc80 capsid sequence. The residue at X3 is the dominant position that defines whether the vector effectively releases genes to hepatocytes and, as such, will be referred to as hepatic alternation or alternation. Position X1 is referred to here as alternation 2.

[0040] Figures 2A and 2B are 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 hepatic alternation. The hepatic alternation of glycine (G) / alanine (A) Petition 870260051179, dated 05 / 28 / 2026, p. 19 / 239 9 / 39 in nucleic acid and protein sequences are in bold and underlined.

[0041] Figures 3A and 3B are representations of 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 alteration of the hepatic alternation in AAV9, and a further alteration in the latter to match the corresponding location in Anc80L65. The altered residues of AAV9 are in bold and underlined.

[0042] Figures 4A and 4B are representations of 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 importation of the hepatic alternation region, both hepatic enrichment and target withdrawal, from Anc80L65 to AAV9. The imported sequences are in bold and underlined.

[0043] Figures 5A and 5B are the sequences of AAV3B A266G (SEQ ID No.: 8) and AAV3B A266G S267_N268T (SEQ ID No.: 9), respectively. Although reduced compared to the Anc80 liver alternation region, making identification uncertain, A266 of AAV3B may represent liver alternation in this serotype. AAV3B transduces murine livers poorly, suggesting that altering A266G may improve this function. Inserting a T creates a liver alternation region with greater identity to Anc80. Altered or inserted residues of AAV3B are in bold and underlined.

[0044] Figures 6A and 6B are the sequences of AAV3B G265_A266A (SEQ ID No: 10) and AAV3B G265_A266G (SEQ ID No: 11), respectively. The liver alternation region of AAV3B is reduced compared to the liver alternation region of Anc80. Insertion of liver enrichment G or liver bleaching A from the corresponding position in Anc80 may import this function. The Petition 870260051179, dated 05 / 28 / 2026, p. 20 / 239 10 / 39 altered or inserted AAV3B residues are in bold and underlined.

[0045] Figures 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 alternation region of AAV3B is reduced compared to the liver alternation region of Anc80. Inserting liver enrichment G or liver bleaching A from the corresponding position in Anc80 can import this function. Changing from S to T at position 268 creates a region with greater identity to Anc80. Altered or inserted residues of AAV3B are in bold and underlined.

[0046] Figures 8A, 8B, and 8C are the sequences of AAV3B AAV9VRI (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 importation of the hepatic alternans region, both hepatic enrichment and hepatic debleaching, from AAV9, Anc80L65, and Anc80L65 G266A to AAV3B. The imported sequences are in bold and underlined.

[0047] Figure 9 is an MA plot showing the abundance of 2048 Anc80 library members in the liver versus viral entry in C57 / BL6J mice. On the y-axis, zero indicates no change in entry, with positive and negative values ​​indicating relative enrichment or disentanglement, respectively. Of eleven alternating positions, the X3 position correlates with the observed bimodality.

[0048] Figure 10 is an MA plot showing the abundance of 2048 Anc80 library members in the liver versus viral input in the non-human primate rhesus monkey. On the y-axis, zero indicates no change in input, with positive and negative values ​​indicating relative enrichment or unencumbering, respectively. As well as Petition 870260051179, dated 05 / 28 / 2026, p. 21 / 239 11 / 39 occurs with the murine liver, of eleven alternating positions, the X3 position correlates with the observed bimodality.

[0049] Figure 11 is a bar graph illustrating the clonal validation of the Anc80 liver alternation vector in murine livers. Both Anc80L65 (SEQ ID No.: 2) and Anc80L65 G266A (SEQ ID No.: 3) were produced by triple transfection with the eGFP-expressing genome CB7.CI.eGFP.FF2A.hA1AT.RGB. These vectors were injected into 5 mice each, at doses of 1.25 and 12 gc / kg, and the mice were sacrificed three days later. Biodistribution of the recovered livers revealed a 100X enrichment in eGFP-encoding genomes per cell in the Anc80L65 alternation vector versus the Anc80L65 G266A alternation vector.

[0050] Figures 12A-12K are a series of 11 microscope images illustrating the eGFP staining results in the livers of the 10 mice plus one uninjected control mouse from Figure 11. There is markedly more eGFP staining in the livers of mice injected with the Anc80L65 on toggle vector compared to the Anc80L65 G266A off toggle vector.

[0051] Figure 13A is a schematic representation of the crystal structure of the VP3 capsid monomer of Adenoassociated virus 2 (AAV2). The location of the hepatic alternans (LT) is indicated by the arrow and the structure of the region encompassing the LT is in a box.

[0052] Figure 13B is a schematic representation of a crystal structure showing an overlay of the LT regions of AAVs 2, 3, 6, 8, and 9, highlighting the local secondary structure that defines the alternation location. The alternation region is defined as the residues located between and including the ascending (ea) and descending (ed) secondary structures, with primary functionality encoded from two to three N-terminal residues for α-alternation (α^. O Petition 870260051179, dated 05 / 28 / 2026, page 22 / 239 12 / 39 residue of LT is located within VR1, a loop connected by a beta-sheet, N-terminal to an alpha-helix of three residues.

[0053] Figure 14 is a sequence alignment of archetypes of AAV clades and clones (e.g., lines 1, 4, 8-10, and 13), clinically relevant AAV serotypes (both naturally occurring and engineered; 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 location of the ascending β (ba), descending β (bd), alternation (at), the alternation region, and the alternation position itself are indicated. The ascending β (ba) starts at a conserved tyrosine, and the descending β (bd) terminates at a conserved serine. The alternation represents the residue 266 in Anc80L65, 267 in AAV9, and 257 in AAV5, emphasizing that the position number is relative when defining the alternation.

[0054] Figure 15 is a table of sequences from the hepatic alternation region (SEQ ID Nos. 40-55, from top to bottom), oriented towards the α-alternation (NDN) at the C-terminus, of the Anc80L65, AAV3B, and AAV9 serotypes, as well as variants constructed to test the hepatic alternation hypothesis. The final two columns list the known on- or off-transduction efficiency in murine and primate liver of Anc80L65, AAV3B, and AAV9, and the in vivo results for the variants reported here, in bold. The hypothesis-predicted efficiency for untested variants is indicated in italics.

[0055] Figure 16 is a diagram showing a method for testing the portability of the hepatic alternation region for CAPS of heterologous AAVs and also for testing the observation of single residue alternation. Flanking the region are two conserved domains amenable to homology-directed region switching. SEQ ID N°: 86.

[0056] Figure 17 is a table showing the efficiency forecasts. Petition 870260051179, dated 05 / 28 / 2026, page 23 / 239 13 / 39 liver transduction (similar to Figure 15) of variants with swapped liver alternation regions (SEQ ID Nos. 56-63, top to bottom). The final two columns list in bold the on or off transduction efficiency determined in vivo in murine livers for these variants, as reported here. The efficiency predicted by the hypothesis for untested variants is indicated in italics.

[0057] Figures 18A and 18B are bar graphs reporting the in vitro transduction efficiencies of AAV9 and AAV9-based hepatic alternation variants. The variants were produced by triple transfection of 293 cells and packaged into a CMV-luciferase encoding genome. The variants were titrated and subsequently added to Huh7 cells at a multiplicity of infection (MOI) of 100,000. Huh7 cells are a hepatocellular carcinoma cell line, and the transduction efficiency as reported in normalized RLUs can be interpreted as a rough indicator of whether the variants were liver-switched or switched off. Altering the native AAV9 G267 to A (SEQ ID No.: 4) severely reduced the transduction efficiency of Huh7 cells, as did the G267A S269T double mutant (SEQ ID No.: 5).AAV9 does not appear to tolerate hepatic alternation region switching well, although alternation may be apparent in the relative efficiencies of the Anc80L65 (SEQ ID No: 6) and Anc80L65 G266A (SEQ ID No: 7) variants.

[0058] Figures 19A and 19B are bar graphs reporting the in vitro transduction efficiencies of AAV3B and AAV3B-based hepatic alternation variants. The variants were produced by triple transfection of 293 cells and packaged into a CMV-luciferase encoding genome. The variants were titrated and subsequently added to Huh7 cells at an MOI of 100,000. Huh7 cells are a hepatocellular carcinoma cell line, and the transduction efficiency as reported in normalized RLUs may Petition 870260051179, dated 05 / 28 / 2026, page 24 / 239 14 / 39 can be interpreted as a rough indicator of whether the variants were switched on or off in the liver. Altering the native AAV3B A266 to G (SEQ ID No.: 8) unexpectedly severely reduced the transduction efficiency of Huh7 cells, however, insertion of a T at the corresponding location in Anc80L65 (SEQ ID No.: 9) rescued this variant and outperformed AAV3B. Similarly, insertion of an A (SEQ ID No.: 10) or a G (SEQ ID No.: 11) before A266, with or without alteration of S268T (SEQ ID No.: 12 and SEQ ID No.: 13), results in a pattern of efficiency similar to hepatic switching with both +G variants outperforming AAV3B. AAV3B tolerates liver switching well, with the apparent switching in the AAV9 variant on the liver (SEQ ID No: 14) and the relative efficiencies of the Anc80L65 (SEQ ID No: 15) and Anc80L65 G266A (SEQ ID No: 16) variants.

[0059] Figures 20A-20C are graphs showing the results of in vivo kinetic expression of luciferase from mice injected with AAV9 and the AAV9-based variants G267A and G267A S269T. Mice were followed for 57 days, with in vivo imaging occurring throughout. Regions of interest were defined as total, liver, and hips (the major muscle groups of the thighs and buttocks). Both off-liver AAV9 variants have very low brightness observed in the liver region, although AAV9 G267A S269T has an overall total brightness comparable to AAV9. This double mutant may produce much of this total brightness in the hip region.

[0060] Figures 21A-21F are bar graphs supporting observed data from in vivo luciferase experiments. Mice injected with vectors expressing GFP AAV9, AAV9 G267A, or AAV9 G267A S269T were sacrificed 28 days after injection, and a biodistribution to both eGFP-containing (DNA) and eGFP-expressing (RNA) genomes was performed. While both mutants Petition 870260051179, dated 05 / 28 / 2026, page 25 / 239 15 / 39 of liver cells had DNA and RNA levels three orders of magnitude lower in the liver (Figures 21A-21B), while in cardiac cells AAV9 G267A and AAV9 G267A S269T were comparable to AAV9 (Figures 21C-21D). Significantly, in quadriceps muscle cells, AAV9 G267A S269T exceeded the gene release and expression levels of AAV9 (Figures 21E-21F).

[0061] Figures 22A and 22B are graphs showing the in vivo kinetic expression results of luciferase from mice injected with AAV3B or the AAV3B-based variants G265_A266A, G265_A266G, Anc80L65-VRI, Anc80L65 G266A-VRI, or AAV9. Mice were followed for 29 days, with in vivo imaging occurring throughout. Regions of interest were defined as total and liver. The on-liver variants of AAV3B emitted higher brightness than their off-liver counterparts, supporting the alternation hypothesis. In fact, both off-liver variants of AAV3B have a very low brightness observed in the liver region. Interestingly, the Anc80L65-VRI mutant performed as well as wild-type AAV3B; however, significantly, the simple insertion of a glycine created a vector that matched AAV9 for the liver region signal. The total signal, where the variant again matches AAV9, suggests that much of the AAV3B G265 A266G signal comes from the liver.

[0062] Figure 23 is the sequence of Anc80L65 R266K (SEQ ID N°: 17). The sequence is identical to Anc80L65, except for hepatic alternation 2. The arginine (R) / lysine (K) hepatic alternation is highlighted.

[0063] Figure 24 is a sequence alignment of archetypes of AAV clades and clones (e.g., lines 1, 4, 8-10, and 13), clinically relevant AAV serotypes (naturally occurring and modified; 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. A Petition 870260051179, dated 05 / 28 / 2026, page 26 / 239 16 / 39 location of Hepatic Alternation 2, the conserved orientation of proline and lysine and the non-canonical start codon of VP2 are indicated. Hepatic Alternation 2 represents residue 168 in Anc80L65 and AAV9, and 151 in AAV5, emphasizing that the position number is relative when defining the alternation.

[0064] Figures 25A and 25B represent heat maps and graphs. Figure 25A on the left represents an MA plot showing the abundance of fr 2048 Anc80 library members in murine hepatocytes, both C57BL / 6 and Figure 25B on the left represents an MA plot of a xenograft FRG mouse model, versus viral entry on day 28. The variants that are enriched in these cells have been boxed and the identity of each of the 11 alternation positions is indicated in the heat map on the right in both Figures. Of eleven alternation positions, the hepatic alternation position X3 correlates with enrichment. The alternation position X1 2, state 1, similarly correlates with enrichment, notably with the most enriched variants.

[0065] Figures 26A-26C are three left-hand MA plots showing the abundance of 2048 Anc80 library members in NHP (Figure 26A) on day 28, xenograft FRG mouse model (Figure 26B) on day 28, and human hepatocytes (Figure 26C) on day 3, in animal and in vitro models, versus viral entry. The variants that are enriched in these cells have been boxed, and the identity of each of the 11 alternation positions is indicated in the heat map to the right of each Figure. Of eleven alternation positions, the liver alternation position X3 correlates with enrichment. The alternation position X1, state 1, similarly correlates with enrichment, notably with the most enriched variants. Petition 870260051179, dated 05 / 28 / 2026, p. 27 / 239 17 / 39 DETAILED DESCRIPTION

[0066] Adeno-associated virus (AAV) is primarily hepatic. Considering that this tropism is beneficial for gene therapy treatments for diseases with a hepatic etiology, the number of viral particles containing the genome needed to effectively transduce this organ may represent a burden for both the patient and the provider. However, the related, contrasting treatment of diseases with non-hepatic etiology may be less effective or require a higher dosage because the liver acts as a sink for most of the therapeutic material released by AAVs. Furthermore, promising AAV serotypes do not transduce murine livers effectively, severely limiting the use of mouse models for clinically relevant studies and dosage assessments.

[0067] Previous approaches to identifying AAV sequences correlated with tropism have relied, for example, on comparing highly related existing serotypes with distinct characteristics, random domain exchanges between unrelated serotypes, or considering higher-order structure to identify motifs that define liver tropism. For example, mapping the determinants of AAV tropism has been performed by comparing highly related serotypes. One example is the single amino acid (E531K) change between AAV1 and AAV6 that enhances murine liver transduction into AAV1 (Wu et al., 2006, J. Virol., 80 (22): 11393-7). Another example is a reciprocal domain exchange between AAV2 and AAV8 that altered tropism but failed to define any robust tissue-specific targeting motifs (Raupp et al., 2012, J. Virol., 86 (17): 9396-408).Furthermore, the overall consideration of the structure highlighted only the gross differences between better or worse liver transducers that are more Petition 870260051179, dated 05 / 28 / 2026, page 28 / 239 18 / 39 observational rather than useful in practice (Nam et al., 2007, J. Virol., 81 (22): 12260-71). Identification of Hepatic Alternations in AAV Capsid Protein

[0068] This description describes the evaluation of an AAV capsid library rationally designed to identify a single-site amino acid change that results in bimodal murine liver tropism (e.g., a hepatic shunt). This description similarly describes a specific residue in an AAV capsid that a) enhances liver transduction in humans, or shunts the liver if desired, thereby allowing the effective dose to be reduced, and b) enhances murine liver transduction while minimally altering other favorable characteristics, allowing these serotypes to be used in murine disease models.

[0069] As described herein, the liver-specific tropism of an AAV capsid protein can be altered by changing a residue at position 266 in Anc80 (SEQ ID No.: 1). For example, changing a non-glycine (G) amino acid residue to a glycine (G) amino acid residue at this position results in enhanced or increased tropism or targeting of AAV to the liver; alternatively, changing a non-alanine (A) amino acid residue to an alanine (A) amino acid residue at this position results in reduced tropism or targeting of AAV to the liver (debleaching). Thus, the propensity of an AAV to infect and / or transfect the liver can be increased by altering a non-G residue at position 266 within the Anc80 AAV capsid protein (SEQ ID No.: 1) to a G residue, while the propensity of an AAV to infect and / or transfect the liver can be reduced by altering a non-A residue at position 266 within the Anc80 AAV capsid protein (SEQ Petition 870260051179, dated 05 / 28 / 2026, p. 29 / 239 19 / 39 ID No.: 1) for a residue of A.

[0070] In some embodiments, an amino acid position corresponding to position 266 in Anc80 in other known capsid proteins of, for example, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, may be altered to an amino acid residue of G or an amino acid residue of A depending on the desired specific liver tropism. In some embodiments, the insertion of an amino acid residue of G or an amino acid residue of A may alter the specific liver tropism as desired. In some embodiments, additional alterations, insertions, or deletions in the hepatic alternation region may increase the specific liver tropism as desired. In some embodiments, the entire native hepatic alternation region, containing the alternation residue, may be replaced by a heterologous capsid or de novo synthesized sequence to achieve the desired, enhanced, or reduced hepatic tropism.

[0071] In some embodiments, capsid proteins may be genetically modified or designed to have the desired amino acid residue corresponding to position 266 in Anc80 (SEQ ID No: 1) so that, when combined with other viral components and assembled into AAV viral particles, the AAV viral particles are liver-enriched or liver-debleached (compared to, for example, the capsid sequence before being altered, or a corresponding capsid sequence not having a G or not having an A at the relevant position, or a corresponding capsid sequence having an A instead of a G or a G instead of an A at the relevant position).

[0072] As described herein, the hepatic tropism of an AAV capsid protein can similarly be altered by changing a residue at position 168 in Anc80 (SEQ ID No.: 1). For example, changing a non-arginine amino acid residue to an arginine amino acid residue (R) at this position results in hepatic tropism. Petition 870260051179, dated 05 / 28 / 2026, p. 30 / 239 20 / 39 enhanced or increased AAV; alternatively, changing a non-lysine amino acid residue to a lysine amino acid (K) residue at this position results in reduced hepatic tropism of AAV (de-targeting). Thus, the propensity of an AAV to infect and / or transfect the liver can be increased or potentiated by changing a non-R residue at position 168 within the Anc80 AAV capsid protein (SEQ ID No.: 1) to an R residue, while the propensity of an AAV to infect and / or transfect the liver can be reduced by changing a non-K residue at position 168 within the Anc80 AAV capsid protein (SEQ ID No.: 1) to a K residue.

[0073] In some embodiments, an amino acid position corresponding to position 168 in Anc80 in other known capsid proteins of, for example, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, may be changed to an R amino acid residue or a K amino acid residue depending on the specific liver tropism desired.

[0074] It should be understood that amino acids with similar properties (e.g., polarity, acidity / basicity, hydrophobicity, charge and / or size) to the amino acids that confer tropism described herein could be used (e.g., instead of G or A at position 266 relative to Anc80 or instead of R or K at position 168 relative to Anc80).

[0075] As described herein, the specific liver tropism of an AAV capsid protein can be altered by changing additional residues within the hepatic alternans region (as defined in Figure 14). In some embodiments, a naturally occurring hepatic alternans region can be replaced with a hepatic alternans region of a heterologous serotype to have the desired hepatic tropism (e.g., to increase hepatic tropism or Petition 870260051179, dated 05 / 28 / 2026, page 31 / 239 21 / 39 reduce the hepatic tropism of the resulting AAV vector). In some embodiments, a naturally occurring hepatic alternans region can be synthesized de novo to have the desired hepatic tropism (e.g., to increase hepatic tropism or reduce hepatic tropism of the resulting AAV vector). As described herein, when the heterologous or de novo derived hepatic alternation region contains an amino acid residue of G at a position within an AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No.: 1), the hepatic tropism of the resulting AAV vector is enhanced, and when the heterologous or de novo derived hepatic alternation region contains an amino acid residue of A at a position within an AAV capsid protein corresponding to position 266 in an Anc80 capsid protein (SEQ ID No.: 1), the hepatic tropism of the resulting AAV vector is reduced.

[0076] Based on the findings reported here, it is clear that the AAVs can be evaluated for those enriched in liver cells or debled from liver cells based on the capsid protein sequence and, specifically, the nucleic acid sequence encoding the amino acid residue corresponding to positions 266 and / or 168 in Anc80 (SEQ ID No.: 1). Nucleic acid sequencing methods are well known in the art and include, without limitation, chain termination methods (e.g., Sanger sequencing method) or chemical degradation methods (e.g., MaxamGilbert sequencing method). Many variations and improvements have been developed and are used in the art for sequencing, including automated sequencing methods and high-throughput sequencing methods.

[0077] When used here, enriched or enrichment Petition 870260051179, dated 05 / 28 / 2026, page 32 / 239 22 / 39 refers to an increase in the number of AAV genomes in liver cells compared to the number of AAV genomes in liver cells when the capsid protein lacks a G amino acid residue at an amino acid position corresponding to position 266 and / or an R amino acid position corresponding to position 168, in Anc80 (SEQ ID No: 1) (e.g., the original or wild-type sequence or the sequence before being altered). When used herein, debleached or debleaching refers to a decrease in the number of AAV genomes in liver cells compared to the number of AAV genomes in liver cells when the capsid protein lacks an amino acid residue of A at an amino acid position corresponding to position 266, and / or an amino acid position of K corresponding to position 168, in Anc80 (SEQ ID No: 1) (e.g., the original or wild-type sequence, or the sequence before being altered).

[0078] Assessment methods for the number of AAV genomes in the liver are known in the art and typically include in vitro transduction of immortalized and / or primary hepatocytes, as well as in vivo systemic injections of wild-type and humanized mice (see, for example, Grimm et al., 2008, J. Virol., 82: 5887-911; Lisowski et al., 2014, Nature, 382: doi: 10.1038 / nature12875).

[0079] Representative capsid proteins that confer efficient (or enhanced) or inefficient (or reduced) hepatic tropism are provided here. The sequences of representative capsid proteins that confer efficient hepatic tropism (e.g., resulting in an enrichment of AAV genomes in liver cells) are shown in SEQ ID No.: 2 [AAV9 Anc80 + 266G], SEQ ID No.: 6 [AAV9 Anc80 + 266G VRI], SEQ ID No.: 9 [AAV3B A266G S267 N268T], SEQ ID No.: 11 [AAV3B G265 A266G], SEQ ID No.: 13 [AAV3B G265 A266G S268T], SEQ ID No.: 14 [AAV3B Petition 870260051179, dated 05 / 28 / 2026, p. 33 / 239 23 / 39 AAV9 + 267G -VRI], SEQ ID No: 15 [AAV3B Anc80 + 266G-VRI], while representative capsid protein sequences conferring reduced hepatic tropism (e.g., resulting in debleaching of AAV particles to the liver) are shown in SEQ ID No: 3 [Anc80 + 266A], SEQ ID No: 4 [AAV9 G267A], SEQ ID No: 5 [AAV9 G267A S269T], SEQ ID No: 7 [AAV9 Anc80 + 266A-VRI], SEQ ID No: 10 [AAV3B G265 A266A], SEQ ID No: 12 [AAV3B G265 A266A S268T], SEQ ID No: 16 [AAV3B Anc80 + 266A-VRI], SEQ ID NO: 17 [Anc80L65 R168K].

[0080] As explained in more detail below, the sequences of the two AAV capsid proteins exhibiting the bimodal liver transduction pattern, SEQ ID No.: 2 and SEQ ID No.: 3, originate from an Anc80 support sequence with the sequence shown in SEQ ID No.: 1, where position 266, shown with an X3 in Figure 1, is a G or an A. Similarly, the sequences of the two AAV capsid proteins exhibiting the bimodal liver transduction pattern, SEQ ID No.: 16 and SEQ ID No.: 17, originate from an Anc80 support sequence having the sequence shown in SEQ ID No.: 1, where position 168, shown with an X1, is an R or a K. Nucleic Acids Encoding an AAV Capsid Protein Containing a Hepatic Alternation

[0081] As described herein, altering or inserting an amino acid residue in the AAV capsid protein between a G amino acid residue and an A amino acid residue at position 266 alternates the resulting AAV's hepatic tropism between enrichment and detangling. Similarly, altering the amino acid residue in the AAV capsid protein between an R amino acid residue and a K amino acid residue at position 168 alternates the resulting AAV's hepatic tropism between enrichment and detangling. Changes to a sequence are typically made Petition 870260051179, dated 05 / 28 / 2026, p. 34 / 239 24 / 39 at the nucleic acid level, and the changes are translated into the encoded amino acid sequence (e.g., the encoded protein). When used here, nucleic acids may include DNA and RNA, including those containing one or more nucleotide analogs or structural modifications. A nucleic acid may be single-stranded or double-stranded, which generally depends on its intended use.

[0082] Alterations can be introduced into nucleic acids using various methods, many of which are well known in the art. For example, alterations can be introduced into nucleic acids using mutagenesis (e.g., site-directed mutagenesis, PCR-mediated mutagenesis) or by chemically synthesizing the nucleic acid molecule including the desired alteration(s). See, for example, Sambrook, Fritsch & Maniatis (Molecular Cloning: A Laboratory Manual, 1989, 2nd ed.) and Dieffenbach & Dveksler (PCR Primer: A Laboratory Manual, 2003, 2nd ed.).

[0083] Nucleic acids can be obtained (e.g., isolated) using routine techniques in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology and / or the polymerase chain reaction (PCR). General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids can likewise be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0084] An isolated nucleic acid molecule is a nucleic acid molecule that is free of sequences that naturally flank one or both ends of the nucleic acid in Petition 870260051179, dated 05 / 28 / 2026, page 35 / 239 25 / 39 genome of the organism from which the nucleic acid molecule is derived (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease digestion). Such an isolated nucleic acid molecule is usually introduced into a vector (e.g., a cloning vector or an expression vector) for ease of handling or to generate a fusion nucleic acid molecule, discussed in more detail below. Additionally, an isolated nucleic acid molecule may include an engineered nucleic acid molecule, such as a recombinant or synthetic nucleic acid molecule.

[0085] Polypeptides can be obtained (e.g., purified) from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide can similarly be purified, for example, by expression of a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of the purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0086] As used herein, a purified polypeptide is a polypeptide that has been separated or purified from naturally occurring cellular components. Typically, a polypeptide is considered purified when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight free of naturally occurring proteins and molecules with which it is naturally associated. Since a chemically synthesized polypeptide is, by nature, separated from its naturally occurring components, a synthetic polypeptide is purified. Petition 870260051179, dated 05 / 28 / 2026, page 36 / 239 26 / 39

[0087] Nucleic acids can be propagated within a vector. Vectors can include viral vectors or non-viral vectors and likewise can include expression vectors. Many vectors are commercially available and vectors can be easily produced using routine recombinant DNA techniques in the art. A vector containing a nucleic acid may have expression elements that, in some cases, can be operatively linked to that nucleic acid. Vectors may also include sequences, such as those encoding a selectable marker (e.g., an antibiotic resistance gene). A vector containing a nucleic acid may encode a chimeric or fusion polypeptide (i.e., a polypeptide operatively linked to a heterologous polypeptide, which may be at the N-terminus or C-terminus of the polypeptide).Representative heterologous polypeptides are those that can be used in the purification of the encoded polypeptide (e.g., 6xHis tag, glutathione S-transferase (GST)).

[0088] Expression elements are known in the art and include nucleic acid sequences that direct and regulate the expression of coding sequences. An example of an expression element is a promoter sequence. Expression elements may likewise include introns, enhancer sequences, response elements, or inducible elements that modulate the expression of a nucleic acid. Expression elements may be of viral origin or, for non-viral molecular biology techniques (e.g., simple growth of a plasmid vector), expression elements may be, without limitation, of bacterial, yeast, insect, or mammalian origin, or expression elements may be a combination of elements from different origins. When used herein, operationally linked means that a promoter or other expression element(s) are positioned in Petition 870260051179, dated 05 / 28 / 2026, page 37 / 239 27 / 39 a vector in relation to a nucleic acid in order to direct or regulate the expression of the nucleic acid. In some cases, operationally linked means that two sequences are in the frame.

[0089] Methods of introducing a viral vector into a host cell are known in the art and typically take advantage of the virus's natural ability to infect. Methods of introducing a nonviral vector into a host cell are known in the art. When used herein, host cell refers to the particular cell into which the viral or nonviral vector is introduced and likewise includes the progeny or potential progeny of such cell. A host cell may be a prokaryotic or eukaryotic cell as appropriate. For example, nucleic acids may be expressed in bacterial cells, such as E. coli, or in insect cells, yeast cells, or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells). Other suitable host cells are known to those skilled in the art.Nonviral nucleic acids can be introduced into host cells, both in vivo and in vitro, using known methods such as, but not limited to, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer. Methods of Using an AAV Capsid Protein Containing a Liver Alternate

[0090] An AAV virus may include a transgene (in cis or trans with other viral sequences) to release to a cell. A transgene may be, for example, a reporter gene (e.g., beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent polypeptide (GFP), chloramphenicol acetyltransferase (CAT), or luciferase), or fusion polypeptides that include a domain. Petition 870260051179, dated 05 / 28 / 2026, page 38 / 239 28 / 39 antigen label, such as hemagglutinin or Myc) or a therapeutic gene (e.g., genes encoding 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, RNAs (e.g., inhibitory RNAs or catalytic RNAs), or target antigens (e.g., oncogenic antigens, autoimmune antigens)).

[0091] The particular therapeutic gene will depend, at least in part, on the particular disease or deficiency to be treated. Simply by way of example, gene transfer or gene therapy may be applied to the treatment of hemophilia, retinitis pigmentosa, cystic fibrosis, Leber congenital amaurosis, lysosomal storage disorders, inborn errors of metabolism (e.g., inborn errors of amino acid metabolism, including phenylketonuria, inborn errors of organic acid metabolism, including propionic acid, inborn errors of fatty acid metabolism, including medium-chain acylCoA dehydrogenase (MCAD) deficiency), cancer, achromatopsia, cone-rod dystrophies, macular degenerations (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,diabetes, congestive heart failure, hypercholesterolemia, hearing loss, coronary heart failure, familial renal amyloidosis, Marfan syndrome, fatal familial insomnia, Creutzfeldt-Jakob disease, sickle cell disease, Huntington's disease, frontotemporal lobe degeneration, Usher syndrome, lactose intolerance, lipid storage disorders (e.g., Niemann-Pick disease, type C), Batten disease, choroideremia, lipid storage disease, Petition 870260051179, dated 05 / 28 / 2026, page 39 / 239 29 / 39 glycogen type II (Pompe disease), ataxia telangiectasia (Louis-Bar syndrome), congenital hypothyroidism, severe combined immunodeficiency (SCID), and / or amyotrophic lateral sclerosis (ALS). Also by way of example, gene transfer or gene therapy can be applied to the treatment of retinal dystrophy (e.g., (LUXTURNA™) (voretigene neparvovec-rzyl), which is a single gene therapy product indicated for the treatment of patients with retinal dystrophy associated with confirmed RPE65 biallelic mutation (Spark Therapeutics Inc., Philadelphia, PA)).

[0092] A therapeutic gene may likewise be, for example, an immunogen that is useful for immunizing an individual (e.g., a human, an animal (e.g., a companion animal, a farm animal, an endangered animal)). For example, immunogens may be obtained from an organism (e.g., a pathogenic organism) or an immunogenic portion or component thereof (e.g., a toxin polypeptide or a byproduct thereof). By way of example, pathogenic organisms from which immunogenic polypeptides may be obtained include viruses (e.g., picornaviruses, enteroviruses, orthomyxoviruses, reoviruses, retroviruses), prokaryotes (e.g., Pneumococci, Staphylococci, Listeria, Pseudomonas) and eukaryotes (e.g., amebiasis, malaria, leishmaniasis, nematodes). It would be understood that the methods and compositions described herein by such methods should not be limited by any particular transgene.

[0093] An AAV virus, usually suspended in a physiologically compatible carrier, can be administered to an individual (e.g., a human or non-human mammal) using standard techniques. Suitable carriers include saline solution, which may be formulated with a variety of buffering solutions (e.g., phosphate-buffered saline), lactose, sucrose, Petition 870260051179, dated 05 / 28 / 2026, page 40 / 239 30 / 39 calcium phosphate, gelatin, dextran, agar, pectin, and water. The AAV virus is administered in sufficient quantities to transduce or infect the relevant cells and to provide sufficient levels of gene transfer and expression to deliver a therapeutic benefit without undue adverse effects. Conventional and pharmaceutically acceptable administration routines include, but are not limited to, direct organ delivery, such as to the liver or lung, oral, intranasal, intratracheal, inhalation, intravenous, intramuscular, intraocular, subcutaneous, intradermal, transmucosal, or other administration routines. Administration routines may be combined if desired.

[0094] The dose of AAV virus administered to an individual will depend primarily on factors such as the condition being treated and the individual's age, weight, and health. For example, a therapeutically effective dosage of an AAV virus to be administered to a human individual is generally in the range of about 0.1 ml to about 10 ml of a solution containing concentrations of about 1 x 10¹ to 1 x 10¹² viral genome copies (GCs) (e.g., about 1 x 10³ to 1 x 10⁹ GCs). Transduction and / or expression of a transgene can be monitored at various time points after administration by DNA, RNA, or protein assays. In some cases, transgene expression levels can be monitored to determine the frequency and / or amount of dosing. Dosing regimens similar to those described for therapeutic purposes can likewise be used for immunization.

[0095] According to the present invention, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques can be employed within the experiment in the technique. Such techniques are fully explained in the literature. The invention will also be described in the following examples, which do not limit the scope of the methods. Petition 870260051179, dated 05 / 28 / 2026, p. 41 / 239 31 / 39 and compositions of the subject matter described in the claims. EXAMPLES Example 1 - Materials and methods for identifying a hepatic alternation of AAV

[0096] An Anc80 library of variant 211(2048) of AAV sequences (see, for example, US Patent No. 9,695,220) was generated by varying the eleven positions (Xi to X11 in Figure 1) within the Anc80 support sequence (SEQ ID No: 1; Figure 1). Each variant was cloned into a mammalian expression plasmid and transfected into HEK293 cells with pRep and pAd Helper accessory plasmids to produce the library in viral vector form. This library was then used for in vivo liver localization assessment (e.g., enrichment vs. debleaching). Briefly, in one experiment, three mice were injected with 2.7311 gc total (~1e13 gc / kg) with the Anc80 vector library. On day 3 after the injection, the mice were sacrificed, and their livers were harvested and frozen.In another experiment, two rhesus monkeys were injected with 1.6e12 gc / kg of the Anc80 vector library, with the study ending and liver harvesting occurring on day 28 after injection. Total genomic DNA was extracted from the livers, and the population of viral variants present in the tissue was quantified by next-generation sequencing. As described below, position X3, highlighted in the Anc80 support sequence shown in Figure 1, was determined to be a hepatic alternation position. Position X3 corresponds to residue 266 in the Anc80 support sequence.

[0097] The results of the in vivo evaluation experiments are presented in Figure 9 and Figure 10. A graph showing the abundance of Anc80 library members of variant 2048 in the liver (y-axis) relative to viral entry (x-axis) is shown. On the axis Petition 870260051179, dated 05 / 28 / 2026, page 42 / 239 32 / 39 y, zero indicates no change in input, with positive and negative values ​​indicating relative enrichment or debleaching, respectively. Of the eleven alternating positions within the Anc80 sustaining sequence, position X3 correlated with the observed liver bimodality.

[0098] Subordinate, however, relevant for liver enrichment or clearance is the identity of the amino acid residue at position X1, corresponding to residue 168 in the Anc80 support sequence. A methodology called alternation heatmap allowed consideration of the identity of each alternation position in a visual format and, by selecting groups of variants by a desired characteristic, a heatmap can reveal multiple influential alternation positions, if present. Using the same data from Figure 9 and Figure 10, both wild-type C57BL / 6 mice (Figure 25) and rhesus monkeys (Figure 26) have X3 = 1 (G) and X1 = 1 (R) present in the most enriched Anc80 variants in the liver.Supporting the relevance of the X1 position, a similar pattern was observed for murine and human hepatocytes harvested from the FRG mouse model of human liver xenotransplantation, as well as in primary human hepatocytes cultured under conditions mimicking liver architecture in vivo (standardized micrococulture, MPCC). Example 2 - Generate and test a hepatic alternation of AAV

[0099] Specific sequences of the Anc80 variant library 2048 (see, for example, U.S. Patent No. 9,719,070, which is incorporated herein by reference in its entirety) containing liver alternation were generated (Figure 2). Anc80L65 (SEQ ID No.: 2) contains a G at position 266 and shows liver enrichment, and Anc80L65 G266A (SEQ ID No.: 3) contains an A at position 266 and shows liver disengagement. The G / A liver alternation is Petition 870260051179, dated 05 / 28 / 2026, p. 43 / 239 33 / 39 highlighted and color-coded in Figure 3 to be consistent with the color code shown in Figure 2.

[00100] Each individual variant is derived by site-directed mutagenesis or isothermal cloning (Gibson) by combining PCR-generated and gene-synthesized fragments. These variants are cloned into a standard rep / cap trans plasmid for vector production. Vector variants expressing GFP and alpha-1-antityrpin are then produced by the Gene Transfer Vector Core at the Grousbeck Gene Therapy Center.

[00101] Three 8-week-old male mice per variant and parental control are injected with 1 and 11 copies of the total genome (~ 5e12 gc / kg) and liver tissue is harvested on day 3. The number of genome copies in the liver is determined by qPCR and expressed as an absolute value and as a proportion to the parental control. Figure 11 shows that Anc80L65 (SEQ ID No: 2), containing a G at position 266, shows liver enrichment, and Anc80L65 G266A (SEQ ID No: 3), containing an A at position 266, shows liver debleaching. Furthermore, Figure 12 shows that genome expression, as observed by eGFP staining of liver tissue, is more evident when using Anc80L65 (SEQ ID No.: 2) containing a G at position 266 versus Anc80L65 G266A (SEQ ID No.: 3) containing an A at position 266. Example 3 - Identify comparable hepatic alternans residues in other serotypes.

[00102] Figure 13A shows the crystal structure of the AAV2 VP3 capsid monomer. The location of the hepatic alternanance (LT) is indicated by the arrow, and the structure of the region encompassing LT is in a box. The hepatic alternanance residue may be located 2-3 residues n-terminal to α-alternance at a position comparable to position 266 of Anc80. Figure 13B is the crystal structure of AAV2 VP3. Petition 870260051179, dated 05 / 28 / 2026, page 44 / 239 Figure 34 / 39 shows an overlay of the LT regions of AAVs 2, 3, 6, 8, and 9 VP3s, highlighting the local secondary structure that defines the alternation location. The alternation region is defined as the residues located between and including β-ascending (βα) and β-descending (βd) secondary structures, with the primary functionality encoded from two to three N-terminal residues for α-alternation (α^). The LT residue is located within VR1, a beta-sheet-linked, N-terminal loop to a three-residue alpha-helix.

[00103] Figure 14 is an alignment of primary VRI amino acid sequences of archetypal and clinically relevant serotypes encompassing hepatic alternation, with Anc80L65 located in row 15. The β-ascending ^a), β-descending ^d), α-alternation (α^) location in the alternation region and the alternation itself are highlighted. β-ascending ^a) starts at a conserved tyrosine, β-descending ^d) ends at a conserved serine. For many serotypes, the comparable position to Anc80 266 can be easily inferred by close identity. In others, notably the B clade viruses AAV2 and AAV3, and the related viruses AAV4 and Rh32.33, the comparable position is more ambiguous or less evident. However, the primary sequence can serve as a guide to identifying hepatic alternans in other serotypes.As shown in Figure 5, the alternation represents residue 266 in Anc80L65, residue 267 in AAV9, and residue 257 in AAV5, emphasizing that the position number is relative when defining the alternation. Example 4 - Generate and test the hepatic alternation of AAV in other serotypes.

[00104] Similarly, Figure 15 is a sequence alignment of the Liver Alternation Regions, oriented towards the α-alternation (NDN) at the C-terminus, of the Anc80L65, AAV3B, and AAV9 serotypes, as well as variants constructed to test the hepatic alternation hypothesis. Anc80L65, AAV3B, and AAV9, and other variants Petition 870260051179, dated 05 / 28 / 2026, page 45 / 239 35 / 39 were tested in murine (M) or primate (P) livers in vivo, and the murine liver transduction efficiencies for these serotypes are indicated by an on or off in the last two columns. The phenotype for completed experiments is indicated in bold; phenotypes for experiments still in progress are indicated in italics.

[00105] In addition to the single-residue hepatic alternanance identified here, the entire hepatic alternanance region may import alternanance function into a heterologous serotype, as well as retain other desirable characteristics of the hepatic alternanance serotype. Figure 16 describes a method for testing the portability of the hepatic alternanance region quickly and easily, taking advantage of the conserved domains flanking these residues. Amplification or de novo synthesis of this ~100-110 bp region allows homology-directed assembly into a heterologous serotype.

[00106] Figure 17 lists the hepatic alternans region changes assembled as part of this study and as illustrated in Figure 16. The final two columns report the in vivo hepatic alternans function in mice and primates, as determined by this study in bold, or the predicted hepatic alternans function in italics.

[00107] In vitro transduction of Huh7 hepatocellular carcinoma cells served to test the viability of all hepatic alternation variants, as well as to suggest hepatic alternation for the on or off phenotype of these variants in vivo. When normalized to titer, most of the manipulated variants surprisingly exhibited some, if not robust, viability as determined by vector-released luciferase expression, as reported in Figure 18 and Figure 19. Furthermore, the amount of luciferase activity observed corresponded to the predicted hepatic alternation phenotype, where those variants with a liver-on G position comparable to Anc80 had higher RLU values ​​than their liver-off A counterparts. Petition 870260051179, dated 05 / 28 / 2026, page 46 / 239 36 / 39

[00108] It is important to highlight that the alternation was demonstrated in vivo at the comparable position to Anc80 266, which is position 267 in AAV9. The alteration of G267 to A in AAV9 reduces the luciferase signal in vivo, the genome copies per cell, and the expression of the released marker by orders of magnitude in the liver, as reported in Figure 20 and Figure 21. Furthermore, the need to alter other residues in the alternation region is demonstrated by the superior performance of the AAV9 G267A S269T double mutant versus the AAV9 G267A alternation mutant alone. The double mutant not only exhibits the liver-off phenotype, but the results suggest a gene release benefit to the heart and skeletal muscle.

[00109] Another study with NHPs, a human liver xenotransplantation model in FRG mice and an in vitro human liver model, identified a second position that influences the release of the liver gene by AAV, here called hepatic alternation 2. This alternation is encoded by residue X1 in the Anc80 support, which corresponds to position 168 in Anc80L65.

[00110] Figure 22 demonstrates by luciferase expression in vivo that a putative liver-off alternation virus, AAV3B, can be transformed into liver-on by altering A266 to G and inserting a C-terminal of two T residues (SEQ ID No: 9), or by inserting a G before A266 with or without alteration of the residue at position 268 from S to T (SEQ ID No: 11). The expectation of liver-off is observed with the corresponding A-off insertions of these last two variants (SEQ ID No: 10). Furthermore, swapping the liver-off alternation region of native AAV3B to the liver-on alternation regions of AAV9 (SEQ ID No: 14) and Anc80L65 (SEQ ID No: 15) similarly improves murine liver transduction. Switching to Anc80L65 G266A in the liver reduces luciferase expression in vivo compared to its counterpart on Anc80L65 (SEQ ID N°: 16). Petition 870260051179, dated 05 / 28 / 2026, page 47 / 239 37 / 39

[00111] Figure 24 identifies the location and local context of X1 in clinically relevant serotypes and AncAAVs. It is located on a positively charged motif near the N-terminus of VP2, easily identifiable by sequence alignment in all AAVs except AAV5, whose N-terminus of VP2 is 17-20 residues shorter. However, all serotypes can define hepatic alternans 2 by their relationship to conserved flanking residues: an N-terminus of two proline residues and a C-terminus of one lysine residue.

[00112] Figure 25 and Figure 26 illustrate the relevance of hepatic alternation 2 for liver enrichment. In Figure 25, experiments questioned the enrichment of Anc80 variants in both wild-type C57BL / 6 mice and the murine hepatocyte component of the FRG human liver xenograft mouse model. In Figure 26, experiments questioned the enrichment of Anc80 variants in rhesus monkey livers, the reciprocal human hepatocytes from the FRG experiment, and in human hepatocytes cultured in vitro using a technique called standardized microco-culture (MPCC). In each case, when the enriched liver variants were selected from the accompanying MA plots, the relevance of hepatic alternation X3 was clear. Furthermore, the relevance of the X1 position was apparent, with the variants changing from key 0 = K to key 1 = R as the enrichment increases in order from bottom to top.This pattern suggests that hepatic alternation 2 at position X1 is independent of hepatic alternation at position X3, but alternation at X3 is the dominant position.

[00113] AAV3B has gained popularity as a clinically relevant serotype since the discovery that it depends on the human hepatocyte growth factor receptor (HuHGFR) for efficient hepatocyte entry (Ling et al., 2010, Hum. Gen. Ther, 21 (12): 1741-1747) Petition 870260051179, dated 05 / 28 / 2026, page 48 / 239 38 / 39 Prior to this detection, this serotype was rejected by gene therapists due to its poor liver clearance performance in mice. However, most preclinical disease models are in mice, and these mouse models play an essential role, for example, in determining the efficacy, safety, and dose determination of gene therapy. Engineering AAV3B to retain its desirable therapeutic characteristics while improving its liver clearance performance in mice to primate-equivalent levels would be extremely valuable to the industry. AAV3B has a shortened VRI and hepatic alternation region compared to Anc80, making it difficult to assign a residue as being a comparable Anc80 position 266 (see Figure 14 and Figure 15).

[00114] Altering A at position 266 to G does not improve gene release to Huh7 cells; in fact, this alteration may produce a loss-of-function phenotype for AAV3B. Reinforcing the need to consider the entire hepatic alternation region in desirable engineering features, either inserting a T at the position comparable to Anc80 in AAV3B in conjunction with the alteration of A266 to G, or inserting an A or G at what would be comparable in Anc80 creates variants that exhibit on- and off-liver phenotypes in Huh7 cells and in vivo (Figure 19 and Figure 22). Furthermore, AAV3B appears to tolerate hepatic alternation region changes well, and importing on-hepatic alternation regions from AAV9 and Anc80L65 improves murine Huh7 hepatocyte gene release and in vivo release of these variants versus AAV3B alone. Importing the liver's off-switch region Anc80L65 G266A reduces this capacity to previous levels (Figure 19 and Figure 22).It is important to emphasize that the single insertion of a G between G265 and A266 creates a serotype based on 3B. Petition 870260051179, dated 05 / 28 / 2026, p. 49 / 239 39 / 39 performs as well as AAV9 in mice, as determined by the luciferase signal in the liver (Figure 22). OTHER MODALITIES

[00115] It should be understood that, although the methods and compositions of the subject have been described herein in conjunction with a number of different aspects, the preceding description of the various aspects is intended to illustrate and not to limit the scope of the methods and compositions of the subject. Other aspects, advantages and modifications are within the scope of the following claims.

[00116] Methods and compositions are disclosed that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the methods and compositions described. These and other materials are described in this document, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are described. That is, although specific reference to each of the various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described in this document. For example, if a particular composition of material or a particular method is disclosed and discussed and a series of compositions or methods are discussed, each and every combination and permutation of the compositions and methods will be specifically contemplated, unless specifically indicated otherwise.Similarly, any subset or combination of these will also be specifically considered and described. Petition 870260051179, dated 05 / 28 / 2026, page 50 / 239

Claims

1 / 3 CLAIMS 1. Capsid protein of an adeno-associated virus (AAV), characterized in that it has a sequence selected from the group consisting of 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: 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 AAV9-VRI], SEQ ID NO: 15 [AAV3B Anc80L65-VRI], SEQ ID NO: 16 [AAV3B Anc80L65 G266AVRI] and SEQ ID NO: 17 [Anc80L65 R168K].

2. Capsid protein of an adeno-associated virus (AAV), characterized by having the sequence SEQ ID NO: 4 [AAV9 G267A] or SEQ ID NO: 5 [AAV9 G267A S269T].

3. AAV capsid protein, according to claim 2, characterized in that it has the sequence SEQ ID NO: 4 [AAV9 G267A].

4. AAV capsid protein, according to claim 2, characterized in that it has the sequence SEQ ID NO: 5 [AAV9 G267A S269T].

5. Nucleic acid molecule, characterized in that it encodes the AAV capsid protein, as defined in any one of claims 2 to 4.

6. Recombinant AAV, characterized in that it comprises the capsid protein of AAV, as defined in any one of claims 2 to 4.

7. Adeno-associated virus (AAV), characterized in that it comprises a capsid protein selected from the group consisting of: Petition 870260051179, dated 05 / 28 / 2026, page 51 / 239 2 / 3 (a) an AAV9 capsid protein comprising a modification in its hepatic alternation site to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 58); (b) an Anc80L65 capsid protein comprising a modification in its hepatic alternation site to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63); (c) an Anc80L65 capsid protein comprising a modification in its hepatic alternation site to comprise AAV9-VR1 (NSTSGGSSNDN; SEQ ID NO: 62); and (d) an AAV3B capsid protein comprising an S268T modification.

8. Adeno-associated virus (AAV), according to claim 7, characterized in that the capsid protein is an AAV9 capsid protein comprising a modification in its hepatic switch site to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 58).

9. Adeno-associated virus (AAV), according to claim 7, characterized in that the capsid protein is an Anc80L65 capsid protein comprising a modification in its hepatic switch site to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63).

10. Adeno-associated virus (AAV), according to claim 7, characterized in that the capsid protein is an Anc80L65 capsid protein comprising a modification in its hepatic switch site to comprise AAV9-VR1 (NSTSGGSSNDN; SEQ ID NO: 62).

11. Adeno-associated virus (AAV), according to claim 7, characterized in that the capsid protein is an AAV3B capsid protein comprising an S268T modification. Petition 870260051179, dated 05 / 28 / 2026, p. 52 / 239 3 / 3 12. Method for altering the tissue tropism of an adeno-associated virus (AAV), characterized in that it comprises: (a) modifying the hepatic alternation site in an AAV9 capsid protein to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 58); (b) modifying the hepatic alternation site in an Anc80L65 capsid protein to comprise AAV3B-VR1 (SQSGASNDN, SEQ ID NO: 63); (c) modifying the hepatic alternation site in an Anc80L65 capsid protein to comprise AAV9-VR1 (NSTSGGSSNDN; SEQ ID NO: 62); or (d) modifying the hepatic alternation site in an AAV3B capsid protein to comprise an S268T modification.

13. Adeno-associated virus (AAV), characterized in that it is generated by the method as defined in claim 12. Petition 870260051179, dated 05 / 28 / 2026, p. 53 / 239

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