Chemogenetic surface modified viral particles

Abstract

This disclosure relates to engineered viral capsid proteins comprising functional sequence motifs and the recombinant virions comprising the same. Furthermore, this disclosure concerns intermediates for the preparation of a surface modified rAAV virion. The surface modified rAAV virions are designed to selectively and / or more efficiently deliver gene therapy. The surface modified rAAV virions, when incorporated into a recombinant virion, can be used to treat an illness characterized by genetic abnormality.

Description

CHEMOGENETIC SURFACE MODIFIED VIRAL PARTICLES1. SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing and is hereby incorporated by reference in its entirety. Said XML copy, created on July 19, 2024, is named 59606WO_CRF_sequencelisting.xml, and is 69,704 bytes in size.2. BACKGROUND

[0002] Recombinant adeno-associated virus (AAV) vectors have been, or are currently in use, in numerous phase I / II / III clinical trials for a number of human diseases, and in some cases remarkable clinical efficacy has been achieved. AAV vector-mediated gene delivery was recently approved for the treatment of inherited blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including hemophilia and Duchenne muscular dystrophy.

[0003] Although AAV vectors are safer and less inflammatory than other viruses, toxi cities have occurred following administration of high doses of rAAVs for gene therapy. Thus, there is an urgent need for the development of the next generation of AAV vectors that are (1) safe, (2) effective, and (3) human tropic. Local administration of rAAVs to a target tissue or organ has been used to improve targeting and reduce systemic toxicity. Further, various natural and synthetic AAV variants have been tested to develop an AAV vector with desired tropism and specificity.

[0004] In general, the capsid is thought to be the primary determinant of infectivity and hostvector related properties such as adaptive immune responses, tropism, specificity, potency, and bio-distribution. Indeed, several of these properties are known to vary between natural serotypes and engineered AAV variants. Over the last decade, novel synthetic AAV variants have been developed by using a variety of capsid engineering techniques, one of which is the insertion of small peptide into an exposed loop of the capsid protein, called variable region VIII (VR8). In some circumstances, the insertion of a novel peptide into a wild type capsid changes the tropism of the variant.

[0005] To date, however, there is little understanding as to how changes on the AAV capsid alter their biological properties and AAV vectors with a desired tropism and specificity to therapeutic targets, such as the central nervous system (CNS); typically, such changes are determined empirically through screening of large libraries of variant capsids.

[0006] One limitation of AAV lies in their broad tropism, which results in transgene expression in other tissues beyond those where transgene expression is desired. It is also well recognized that host and vector-related immune challenges need to be overcome for longterm gene transfer.

[0007] It has also been noted that the introduction of a genetic modification in the capsid can change the tropism of the vector and can modify the production yield and other biological parameters in unpredictable ways.3. SUMMARY OF THE DISCLOSURE

[0008] The present disclosure provides new viral platforms that can provide capsids and recombinant virions possessing a higher transduction efficiency, and / or specificity for relevant target tissues that would improve transduction of specific cells of interest and / or that can be efficacious when delivered at a lower titer.

[0009] In some embodiments, the present disclosure is directed to capsids and recombinant virions possessing a higher transduction efficiency, and / or specificity for their targeted tissues that improve transduction of the targeted specific cells and / or that can be efficacious when delivered at a lower titer. In some embodiments, the disclosure provides an enzyme-mediated ligation to engineer an adeno-associated virus (AAV) vector in order to redirect viral tropism to target cells. In some embodiments, the sorting motif, recognized by the enzyme (e.g., sortase), is cloned into the desired VP sequence. Then, the “sortagged” AAVs (e.g., VR-IV AAV2 and VR-VIII AAV2) carrying the motif are conjugated to a molecule of interest that redirect viral vector to target cells.

[0010] In some embodiments, the enzyme-mediated ligation enables the conjugation between the AAV capsid proteins, carrying the sorting motif and a cyclooctyne containing molecule (e.g., dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof), which is then used to attach an azide-tagged ligand.

[0011] In some embodiments, the present disclosure provides an engineered AAV capsid protein obtained by combining the accuracy of an enzymatic approach with the modularity of a click-chemistry reaction to conjugate a ligand of interest to an AAV vector.

[0012] In certain aspects of the present disclosure, an engineered adeno-associated virus (AAV) capsid protein is provided wherein:the capsid protein comprises: a functional sequence motif, wherein the functional sequence motif has the sequence:X1X2X3X4X5X6; wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional; wherein the functional sequence motif is a substrate for a sortase transamidase enzyme.

[0013] In certain of these embodiments, the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU. In certain of these embodiments, the enzyme is a sortase A. In certain of these embodiments, the sortase is heptamutant (SrtA 7M) or a pentamutant (SrtA 5M). In certain of these embodiments, the enzyme is HeptaMutant Staphylococcus aureus Sortase A (SrtA7m). In some embodiments, the sortase is SrtA 5M.

[0014] In certain of these embodiments, the functional sequence motif is located within a variable region (VR) of the capsid primary sequence. In particular embodiments, the functional sequence motif is located in a surface accessible variable region that is able to be acted on by a transamidase enzyme.

[0015] In certain embodiments, the functional sequence motif begins after:S453 in VR-IV of an AAV1 capsid serotype;5452 in VR-IV or R585 VR-VIII of an AAV2 capsid serotype;S576 in VR-VIII of an AAV5 capsid serotype;5453 in VR-IV or S587 VR-VIII of an AAV6 capsid serotype;P454 in VR-IV or A587 VR-VIII of an AAV7 capsid serotype;T454 in VR-IV or Q589 VR-VIII of an AAV8 capsid serotype; and5454 in VR-VIII of an AAV9 capsid serotype.

[0016] In certain embodiments, the functional sequence motif begins after S452 in VR-IV or R585 VR-VIII of an AAV2 capsid serotype.

[0017] In certain embodiments, LPET (SEQ ID NO. 2) is inserted after S452 to form the functional sequence motif.

[0018] Another aspect of the present disclosure is the provision of a polynucleotide encoding the engineered rAAV capsid protein of any one of the previous embodiments.

[0019] Another aspect of the present disclosure is the provision of a vector comprising the provided polynucleotide. In certain embodiments, the vector comprises a promoter operably linked to the polynucleotide.

[0020] Another aspect of the present disclosure is the provision of a host cell comprising the engineered rAAV capsid protein provided herein, the polynucleotide provided herein, or the vector provided herein.

[0021] In certain aspects of the present disclosure is the provision of a recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein as provided herein. In certain of these embodiments, the rAAV virion further comprises an exogenous cargo polynucleotide. In certain of these embodiments, the exogenous cargo polynucleotide comprises a template for homology directed repair. In certain of these embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA. In alternative embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0022] In certain embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

[0023] In certain aspects of the present disclosure, a surface functionalized rAAV virion is provided according to any embodiment described herein, wherein the rAAV virion comprises an exogenous cargo polynucleotide and at least one cross linker reactive moiety covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

[0024] In certain aspects of the present disclosure, a surface functionalized rAAV virion, is provided wherein the virion comprises at least one engineered rAAV capsid protein according to any of the provided embodiments, and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.

[0025] In certain of these embodiments, the covalent linkage comprises an amide bond. In certain of these embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.

[0026] In certain aspects of the present disclosure, a surface functionalized rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of the provided embodiments, and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

[0027] In certain aspects of the present disclosure, a surface modified rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of the embodiments described herein, and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the virion comprises a crosslinked moiety that covalently connects a C-terminal amino acid of the N-terminal cleavage fragment and a ligand.

[0028] In certain embodiments, the covalent linkage comprises an amide bond.

[0029] In certain aspects of the present disclosure, a surface modified rAAV virion is provided, wherein the virion comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO 3), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable region IV, VIII, or IV and VIII, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the virion comprises a crosslinked moiety that covalently connects the T of the N-terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a ligand.

[0030] In certain embodiments, the crosslinked moiety comprises:

[0031] In certain embodiments, the ligand is a cell-type specific ligand. In further embodiments, the ligand is selected from enzymes (e.g., proteases, endohexosaminidases), peptide mimetic, ligands of endogenous receptors, lipid binding proteins, toxins, bacteriophage peptidases, nucleotides and RNA / DNA based ligands (e.g., aptamers), viral vectors, nanoparticles, lipids and combinations thereof.

[0032] In certain embodiments, the functional sequence motif attenuates or abrogates binding of the provided virion to mammalian cell polysaccharides or proteoglycans when compared to a reference virion having the same capsid protein sequence or sequences but without the mutation required for the formation of a functional sequence motif.

[0033] In certain aspects of the present disclosure, a pharmaceutical composition comprising a plurality of rAAV virions is provided according to any provided embodiment, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient or combination thereof.

[0034] In certain aspects of the present disclosure, a method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, the method comprising the steps of administering a therapeutically effective amount of the pharmaceutical composition according to the embodiments provided herein, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0035] In certain aspects of the present disclosure, a composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo is provided, the composition comprising: a therapeutically effective amount of the pharmaceutical composition according to any provided embodiment, wherein the recombinant polynucleotide cargo is capable of treating the disease.

[0036] In certain aspects of the present disclosure, use of the rAAV virion according to any of the described embodiments is provided, for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo.

[0037] In some embodiments, the present disclosure is directed to a method of preparing a surface functionalized rAAV virion, wherein the virion comprises at least one engineeredrAAV capsid protein according to any of the embodiments described herein, and an exogenous cargo polynucleotide, the method comprising: contacting the virion with (a) the enzyme and (b) the cross linker moiety, whereby the functional sequence motif is enzymatically cleaved to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; and at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, the attached drawings illustrate some, but not all, alternative embodiments. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities shown. These figures, which are incorporated into and constitute part of the specification, assist in explaining the principles of the disclosures.

[0039] FIG. 1 depicts the influence of three different nucleophiles, GGG-peg4-DBCO; Amine-DBCO, and Amine-peg4-DBCO, on reaction efficiency for AAV2-LPETG-VR-IV (top) or AAV2-LPETG-VR-VIII (bottom) vectors.

[0040] FIG. 2 depicts the influence of two sortases, SrtA7M and SrtA5M, and buffers on reaction efficiency for AAV2-LPETG- VR-IV / VR-VIII.

[0041] FIG. 3 depicts the results of the qPCR analysis. One-way Anova, p<0.01; Dunnett’s post-test; VR-IV / VR-VIII vs WT p=0.0012; n=4. The total amounts of viral genome (VG) obtained from the production of the three variants were compared to the wild-type one (WT). 4.17E8 HEK 293T / 17 cells were transfected and after three days the viral particles collected.

[0042] FIG. 4 depicts HEK 293T / 17 transduction. AAV vectors tested in HEK 293T / 17 to assess whether the insertion of the sortag motif altered the viral tropism. To verify that, transduction efficiency was compared to the WT AAV one. Whereas VR-IV AAV2 tropism transduction was similar to the WT AAV2 one, VR-VIII and VR-IV / VR-VIII AAV2 weren’t able to transduce HEK293T / 17 cells successfully. Scale bar 50 um.

[0043] FIG. 5 depicts the influence of sortase concentration on reaction efficiency for AAV2-LPETG-VR-IV / VR-VIII.

[0044] FIG. 6 depicts the influence of reaction time and temperature efficiency of modification of AAV2-LPETG-VR-IV / VR-VIII.

[0045] FIG. 7 depicts the influence of reaction clean-up upon efficiency of modification of AAV2-LPETG-VR-IV / VR-VIII.

[0046] FIG. 8 depicts the yield expressed as Viral Genomes per producing cell, for each AAA vector, namely AAV2-Delta-HSPG, AAV2-LPETG- VR-VIII, and AAV2-LPETG-VR- IV / VR-VIII viruses compared to wildtype AAV2. Data are expressed as viral genomes per producing cell to normalize for different scales of production across preparations.

[0047] FIG. 9 depicts the comparison of transduction efficiency in HEK 293 cells by wildtype AAV2, AAV2-Delta-HSPG, AAV2-LPETG-VR-IV, AAV2-LPETG- VR-VIII and AAV2-LPETG-VR-IV / VR-VIII vectors. The AAV2-LPETG- VR-VIII and AAV2-LPETG- VR-IV / VR-VIII vectors were also tested at 30x higher titres.

[0048] FIG. 10 depicts the effect of different linkers on the efficiency of conjugation with Sortase, by comparing transduction on PC12 cells with different vectors, from left: AAV2- LPETG- VR-VIII (VR-VIII) unmodified; AAV2-LPETG- VR-VIII (VR-VIII treated with SortA and exposed to WGA without linker (VR-VIII + SortA +WGA); AAV2-LPETG- VR- VIII (VR-VIII) modified with SortA in the presence of the different linkers (GGG-PEG4- DBCO; Amine-DBCO; Amine-PEG4-DBCO) and then reacted with WGA.

[0049] FIG. 11 - A depicts the concentration of substrate (nucleophile) and enzyme (SrtA 7M) that were tested. “Low concentration” was composed by 100 uM nucleophile and 7 uM SrtA 7M; “Medium concentration” condition was 500 uM nucleophile and 16 uM SrtA 7M; “High concentration” formed by 1 mM nucleophile and 20 uM SrtA 7M.

[0050] FIG. 11 - B depicts three different nucleophiles, GGG-peg4-DBCO; Amine-peg4- DBCO; Amine-DBCO, which were tested to verify which one was the best candidate for the ligation with the viral particles.

[0051] FIG. 11 - C depicts three buffers which were tested to optimize the protocol. Buffer 1 was composed by 50 mM ammonium carbonate; Buffer 2 was formed by 50 mM Tris-HCl, 150 mM NaCl; Buffer 3 was composed of 50 mM Tris-HCl, 150 mM NaCl and 0.5 mM CaCh.

[0052] FIG. 11 - D depicts the testing of three different temperatures for the enzymatic reaction: 4°C; 21°C; 37°C. At higher temperature, the AAV vectors were able to transduce a greater number of PC-12 cells. At 4°C there were a minimal number of positive cells.

[0053] FIG. 11 - E depicts the comparison between the catalytic activity of SrtA 7M and SrtA 5M. Scale bar 50 um.

[0054] FIG. 12 - A depicts quantification of Td-Tomato positive cells for the different concentration conditions. One-way Anova, p<0.0001; Tukey’s post-test; VR-IV AAV2 vs Low concentration p=0.0027; VR-IV AAV2 vs Medium concentration p<0.001; VR-IV AAV2 vs High concentration p<0.001; Low concentration vs Medium concentration p=0.0003; Low concentration vs High concentration p<0.0001; Medium concentration vs High concentration p=0.0011; n=3.

[0055] FIG. 12 - B depicts quantification of Td-Tomato positive cells for the different nucleophile conditions. One-way Anova, p<0.0001; Tukey’s post-test; VR-IV AAV2 vs GGG-peg4-DBCO p=0.0012; VRIV AAV2 vs Amine-peg4-DBCO p<0.0001; VR-IV AAV2 vs Amine-DBCO p<0.0001; GGGpeg4- DBCO vs Amine-peg4-DBCO p=0.0046; GGG- peg4-DBCO vs Amine-DBCO p<0.0001; Amine-peg4-DBCO vs Amine-DBCO p<0.0001; n=3.

[0056] FIG. 12 - C depicts quantification of Td-Tomato positive cells for the different buffer conditions. One-way Anova, p=0.3910; Tukey’s post-test n=3.

[0057] FIG. 12 - D depicts quantification of Td-Tomato positive cells for the different temperature conditions. One-way Anova, p=0.0121; Tukey’s post-test; 4°C vs 21°C p=0.0167; 4°C vs 37°C p=0.0115; 21°C vs 37°C p=0.8087.

[0058] FIG. 12 - E depicts quantification of Td-Tomato positive cells for the different sortase conditions.

[0059] FIG. 13 depicts viral purification after Amine-DBCO sortase-mediated ligation measured as efficiency of transduction. Scale bar 50 pm.

[0060] FIG. 14 - A depicts the purification of viral prep that was modified with the SortA immediately after the iodixanol gradient step and was then cleaned up (buffer exchanged) through Amicon Ultra Centrifugal Filter or using HPLC with a Capto Core 400 resin in terms of total Vg recovered.

[0061] FIGs. 14 - B depicts the purification of viral prep that was modified with the SortA immediately after the iodixanol gradient step and was then cleaned up (buffer exchanged) through Amicon Ultra Centrifugal Filter or using HPLC with a Capto Core 400 resin in terms of transduction efficiency.

[0062] FIG. 15 depicts comparison between transduction efficiency of the AAV2-LPETG- VR-IV and AAV2-LPETG-VR-IV / VR-VIII unmodified (left), modified with chemical reaction (middle) or with chemo-enzymatic reaction with SortA (right) and conjugated with WGA.

[0063] FIGs. 16A-C depict the comparison between transduction efficiency of the AAV2- LPETG-VR-IV (top) and AAV2-LPETG-VR-VIII (middle) and AAV2-LPETG-VR-IV / VR- VIII (bottom) either unconjugated (left), or conjugated to WGA using the chemo-enzymatic reaction with SortA (middle) or the chemical reaction (right); results are summarized in graphs on the right.

[0064] FIG. 17 depicts ex vivo comparison between unmodified VR-IV AAV2 vector (VR- IV) and modified with SortA-WGA in primary DRG neurons. Scale bar 50 um.

[0065] FIG. 18 - A depicts whole-mount DRG deriving from animal injected with different doses of VR-IV-AAV2 vectors modified with NGF at. lx=5E10VG; 2x=lEl lVG;3x=l .5E11 VG dose per mouse.

[0066] FIG. 18 - B depicts whole-mount DRG deriving from animal injected with different doses of unmodified VR-IV-AAV2 vectors at lx=5E10VG; 2x=lEl lVG; 3x=1.5VG per mouse, subcutaneously in the paw of mice.

[0067] FIG. 19 - A-C depict imaging by confocal microscopy of whole-mount DRGs from mice injected with unmodified VR-IV-AAV2 (“control”) or VR-IV-AAV2 conjugated to NGF (“NGF”) at 5xl0E5 Vg (A panels), IxlOEl l Vg (B panels), 1.5xl0El l Vg (C panels) per mouse and quantification of the co-localization between the AAV reporter TdTomato and TrkA is shown in the graphs

[0068] FIG. 20 - A depicts the imaging by confocal microscopy of spinal cord sections from mice injected in the periphery as in FIG 20 with VR-IV-AAV2 either non conjugated (Ctrl) or conjugated with NGF (NGF) at 4x and stained with IB4 or TrkA.

[0069] FIG. 20 - B depicts the confocal acquisition of spinal cord section from mice injected in the periphery as in FIG 20 with VR-IV-AAV2 either non conjugated (Ctrl) or conjugated with NGF (NGF) at lOx and stained with IB4 or TrkA.

[0070] FIG. 21 depicts VR-IV AAV2 transduction in spinal cord. Spinal cord slices (30 um) derived from injected adult mice with VR-IV conjugate with NGF were stained for TrkA receptor (green) to assess whether NGF exposed on the viral capsid was able to re-direct AAV tropism (reporter in red) to peptidergic neuros expressing TrkA. Scale bar 100 um.

[0071] FIG. 22 - A depicts the comparison between WT AVV2 conjugated to WGA with the chemical reaction and VR4 AAV2 conjugated to WGA using the chemoenzymatic reaction, at different doses of virus applied to the cells. Oneway Anova, p<0.0001; Tukey’s post-test; WT lx vs VR-IV lx p=0.0002; WT lx vs VR-IV 3x p=0.3320; WT lx vs VR-IV lOx p=0.6401; WT lx vs VR-IV 30x p=0.8472.

[0072] FIG. 22 - B depicts the comparison between WT AVV2 conjugated to WGA with the chemical reaction and VR-VIII AAV2 conjugated to WGA using the chemoenzymatic reaction, at different doses of virus applied to the cells One-way Anova, p=0.0004; Tukey’s post-test; WT lx vs VR-VIII lx p=0.0020; WT lx vs VR-VIII 3x p=0.0037; WT lx vs VR- VIII lOx p=0.9081; WT lx vs VR-IV 30x p=0.9860.

[0073] FIG. 22 - C depicts WT AAV2 conjugated to WGA using the chemical reaction and the VR-IV / VR-VIII-AAV2 virus conjugated to WGA using the chemoenzymatic reaction at different doses of virus applied to the cells. One-way Anova, p=0.0006; Tukey’s post-test; WT lx vs VR-IV / VR-VIII lx p=0.0014; WT lx vs VR-IV / VR-VIII 3x p=0.0015; WT lx vs VR-IV / VR-VIII lOx p=0.0025; WT lx vs VR-IV / VR-VIII 30x p=0.3757. Scale bar 50 um.5. DETAILED DESCRIPTION5.1. Definitions

[0074] “AAV” is adeno-associated virus and may be used to refer to a virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.

[0075] The term “AAV capsid protein” or simply “capsid protein” refers to a VP1, VP2, or VP3 capsid protein of an AAV capsid.

[0076] The term “amino acid position” within an AAV capsid protein as here herein refers to a position of an amino acid residue, e.g., in an AAV VP1 protein sequence, counted from thefirst amino acid at the N terminus. As used herein, the term "amino acid" comprises naturally occurring L- and D- amino acids and artificial, i.e. non-naturally occurring, a-amino acids. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a naturally occurring L-a-amino acid.

[0077] For the avoidance of doubt, as used herein, the indication that an insertion site is at amino acid position X means that the targeting peptide is inserted between amino acids X and X+l, i.e., the targeting peptide is inserted after the amino acid at position X and before the amino acid at position X+l.

[0078] The term “parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intracistemal injection, or infusion techniques.

[0079] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.

[0080] The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, excipients, stabilizers and adjuvants. For examples of carriers, excipients, stabilizers and adjuvants, see Remington: The Science and Practice of Pharmacy, 23rd Ed., Pharmaceutical Press, 2020.

[0081] The abbreviation “rAAV” refers to a recombinant adeno-associated viral particle (capsid) composed of at least one AAV capsid protein and an encapsidated polynucleotide, sometimes referred to herein as a “genome”. rAAV can include a genome that comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome), such as a heterologous polynucleotide encoding a gene delivered to a mammalian cell such as sequence encoding a therapeutic protein.

[0082] The terms “percent sequence identity” (% sequence identity), “percent identical” (% identical) and the like refer to percent sequence identity between two nucleotide sequences or between two amino acid sequences calculated by aligning the two sequences, determining the number of matches of nucleotides or amino acid residues between the two sequences, dividing the number of matches by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues), and multiplying by 100 to arrive at a percent sequence identity value. For calculation of the percent sequence identity (% sequence identity), two or more sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (available atwww.ebi.ac.uk / Tools / psa / emboss_needle) with the following parameters: Matrix: BLOSUM62 (for protein sequences) or DNA full (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: false; End Gap Open: 10; and End Gap Extend: 0.5.

[0083] Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman (1981) Adv. Appl. Math 2:482, may permit optimal alignment of compared sequences; by the homology alignment algorithm (GAP) of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC / Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al. (1988) Nucleic Acids Res. 16: 10881-10890; Huang, et al. (1992) Computer Applications in the Biosciences 8: 155-165; and Pearson, et al. (1994) Meth. Mol. Biol. 24:307-331. An example of a good program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle (1987) J. Mol. Evol. 25:351- 260, which is similar to the method described by Higgins and Sharp (1989) CAB IOS 5: 151- 153 (and is hereby incorporated by reference). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., eds., Greene Publishing and Wiley-Interscience, New York (1995). An updated version of the BLAST family of programs includes the BLAST+ suite. (Camacho, C., et al. (2009 Dec 15) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0084] The terms “treatment”, “treating”, and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treatment” as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may bepredisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0085] The terms “variable region” or “VR” as used herein refer to one or more of nine sequence variable regions (e.g., VRI to VRIX) in an AAV capsid protein previously defined by comparison and alignment of various AAV capsid proteins. See e.g., Govindasamy et al., Structurally mapping the diverse phenotype of adeno-associated virus serotype 4, J. Virol. (2006); Meyer et al. Structure of the gene therapy vector, adeno-associated virus with its cell receptor, AAVR, eLife (2019). The VRs are known to contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic re-activity.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.5.2. Other interpretational conventions

[0087] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0088] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody or antigen binding fragment” includes a plurality of such antibodies and antigen binding fragments and reference to “the recombinant adeno-associated virus” includes reference to one or more recombinant adeno-associated viruses and equivalents thereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0089] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0090] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. The dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0091] Where a range of values is provided, it is understood that the recited endpoints of the range are included. In addition, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0092] Ranges recited herein are understood to be shorthand for all values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, for example, including subranges such as from 11 to 48 or 39 to 41.

[0093] The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10% of a stated value or of a stated limit of a range.5.3. AAV production platform

[0094] To standardize the modification process, the present disclosure focuses on a hybrid approach, where an insertion sequence is genetically incorporated into the virus (one or more capsid proteins) allowing for site specific functionalization of the capsid surface with DBCO. Earlier attempts focused on protein or peptide-based tags such as the Clip tag or Spytag but were hindered by substantial reductions in AAV yield upon incorporation of the tag into the capsid protein. Similar problems have also been reported for encoding of unnatural amino acids into the AAV capsid proteins (Kelemen RE, Mukherjee R, Cao X, Erickson SB, Zheng Y, Chatterjee A. A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus. Angew Chem Int Ed Engl 2016; 55: 10645-9).

[0095] The current application focuses on an alternative method of enzyme mediated functionalization / modification of the AAV capsid surface. Experiments with capsid sequences, engineered to include a sortase functional sequence motif (e.g., by insertion of an insertion sequence) that are reactive towards enzymatic bioconjugation, were conducted.Such experiments, termed “sortagging”, are based upon a transpeptidation reaction catalyzed by a bacterial sortase enzyme. In some embodiments, the present disclosure describes the formation of an LPXTG peptide sequence (SEQ ID NO: 3) into the protein of interest (the AAV capsid), and subsequent incubation with the Sortase A enzyme from Staphylococcus aureus and a labelled oligoglycine peptide. The LPXTG sequence (SEQ ID NO: 3) was then cleaved between the T and G generating a thioester-linked acyl enzyme intermediate, which is then acted on by an appropriate bifunctional linker comprising a ligand and an appropriate nucleophile (referred to as an enzyme reactive moiety, herein) to form a covalent bond with the thioester-linked acyl enzyme intermediate, resulting in site-specific ligation of the ligand (e.g., labelled oligoglycine) at the site of the original LPETG sequence (SEQ ID NO: 7).

[0096] Scheme 1 presents an illustration of enzyme-catalyzed ligation (SEQ ID NOS 3 and 83, respectively, in order of appearance).Embodiment of Enzyme-catalyzed LigationScheme 1. Illustration of enzyme-catalyzed ligation.

[0097] Based on what was known at the time, it was unknown whether bifunctional linkers comprising an enzyme reactive moiety and a crosslinker reactive moiety (e.g. , clickchemistry) could be incorporated directly into exposed loops of the AAV2 capsid via sortase mediated ligation.

[0098] In some embodiments, the present disclosure is directed to AAV2 mutants comprising functional sequence motifs that can be produced in high yields and that are characterized by unexpectedly strong silencing of native cell tropism. The present disclosure also provides for the bioorthogonal crosslinking of functionalized ligands to thereby improve tropism and to enhance transduction efficiency.

[0099] In certain embodiments, a ligand of the surface modified viral capsid binds to its cognate receptors on the surface of mammalian cells to mediate gene delivery selectively into cell types which display the appropriate cognate receptor thus enabling targeted viral gene delivery.

[0100] The Examples section below indicates that the disclosed enzyme mediated “chemo-genetic” hybrid modification approach has a number of advantages over AAV capsids surface modified by chemical conjugation alone as well as prior genetic approaches.

[0101] Unlike sequence modified capsids, for which few human tissue-specific variants have yet been defined, despite intensive research efforts, this approach leverages onthe vast number of known human cell receptor-ligand interactions to drive tropism. Unlike sequence modified capsids, for which tissue tropism must be empirically determined, in embodiments when we interfere with the natural cell binding site of the AAV capsid, the current approach provides high specificity of cell targeting, driven by the receptor specificity of the conjugated ligand. In alternative embodiments, when ligands are conjugated that increase transduction without interfering with the native capsid cellular binding site, greater transduction efficiency is obtained without significantly altering known tropism.

[0102] In addition, the approach disclosed herein is compatible with any AAV production platform, with some embodiments showing an enhanced viral yield. The methods provided herein are also inexpensive to perform, and scalable from small-scale research applications to clinical scale production. Moreover, the provided platform for modifying the surfaces of AAV capsids is modular, allowing for essentially any virus / ligand combination, and this should facilitate its translation from experiments in rodent models to treatment of human patients.5.4. Engineered Capsid Proteins

[0103] In certain aspects of the present disclosure, an engineered adeno-associated virus (AAV) capsid protein is provided wherein the capsid protein comprises: a functional sequence motif, wherein the functional sequence motif has the sequence: X1X2X3X4X5X6; wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional; wherein the functional sequence motif is a substrate for a transamidase enzyme.

[0104] In certain embodiments, the engineered AAV capsid protein of the present disclosure comprises one or more sequence changes. In certain embodiments, the sequence changes are selected from insertions, deletions, or substitutions, as compared to a known wild type protein. A single insertion, deletion, or mutation comprises one or more sequential amino acid residue. These engineered sequence changes serve to introduce the functional sequence motif into the capsid protein. In certain of these embodiments, the sequence change comprises at least one insertion of an insertion sequence that forms the functional sequence motif. In some embodiments, the engineered rAAV capsid protein comprises one or more sequence changes that serve to introduce the functional sequence motif. In some embodiments, the sequence changes are selected from insertions, deletions, or substitutions, compared to a wild type protein.5.4.1. Transamidation Enzymes

[0105] In certain embodiments, the functional sequence motif of the present disclosure comprises any known transamidase enzyme recognition sequence. Recognition sequences are specific amino acid sequences found in proteins that are recognized and targeted by transamidase enzymes, such as transpeptidases. Transpeptidases, such as sortase enzymes, are a group of enzymes that play a crucial role in the process of protein crosslinking, particularly in the formation of the bacterial cell wall. The transamidase recognition sequences typically consist of a specific arrangement of amino acids, such as a sequence of three to five amino acids. The exact composition and arrangement of the amino acids vary depending on the bacterial species.

[0106] The transamidase for use with the present disclosure can also be selected from those mutated from the natural sequence to include mutations. In some embodiments the enzyme is a transamidase harboring a mutation that increases the efficiency of the cleavage and hence the overall reaction. Exemplary mutated sortases for use with the present disclosure are those identified in J. Biol. Chem. (2020) 295:9, 2664-2675 and Scientific reports 2016 (6:31899) are incorporated herein by refence. In certain embodiments, the sortase for use with the present disclosure harbors one or more of the following mutations: P94R, E105K, E108A, D124G, D160N, D165A, D186G, Y187L, E189R, K190E, and K196T. In a particular embodiment the transamidase is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

[0107] In certain embodiments, the functional sequence motif, corresponding enzyme and enzyme substrate moiety for functionalizing a ligand are selected from those described in the Table 1 below.Table 1. Peptide Ligation5.4.1.1 Sortase Transamidase Enzyme

[0108] The term “sortase,” as used herein, refers to an enzyme able to carry out a transpeptidation reaction, e.g., conjugating the C-terminus of a protein to the N-terminus of a protein, via transamidation. Sortases are also referred to as transamidases, and typically exhibit both a protease and a transpeptidation activity. Various sortases from prokaryotic organisms have been identified. For example, some sortases from Gram-positive bacteria cleave and translocate proteins to proteoglycan moieties in intact cell walls. Among the sortases that have been isolated from Staphylococcus aureus, are sortase A (Srt A) and sortase B (Srt B). Thus, in certain embodiments, a transamidase used in accordance with the present disclosure is sortase A, e.g., from S. aureus, also referred to herein as SrtAaureus. In certain embodiments, a transamidase is a sortase B, e.g., from S. aureus, also referred to herein as SrtBaureus.

[0109] Sortases have been classified into 4 classes, designated A, B, C, and D, designated sortase A, sortase B, sortase C, and sortase D, respectively, based on sequence alignment and phylogenetic analysis of 61 sortases from Gram-positive bacterial genomes (Dramsi S, Trieu-Cuot P, Bieme H, Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol. 156(3):289-97, 2005; the entire contents of which are incorporated herein by reference). These classes correspond to the following subfamilies, into which sortases have also been classified by Comfort and Clubb (Comfort D, Clubb R T. A comparative genome analysis identifies distinct sorting pathways in grampositive bacteria. Infect Immun., 72(5):2710-22, 2004; the entire contents of which are incorporated herein by reference): Class A (Subfamily 1), Class B (Subfamily 2), Class C (Subfamily 3), Class D (Subfamilies 4 and 5). The aforementioned references disclose numerous sortases and recognition motifs. See also Pallen, M. J.; Lam, A. C.; Antonio, M.; Dunbar, K. TRENDS in Microbiology, 2001, 9(3), 97-101; the entire contents of which areincorporated herein by reference. Those skilled in the art will readily be able to assign a sortase to the correct class based on its sequence and / or other characteristics such as those described in Drami, et al., supra. The term “sortase A” is used herein to refer to a class A sortase, usually named SrtA in any particular bacterial species, e.g., SrtA from S. aureus. Likewise “sortase B” is used herein to refer to a class B sortase, usually named SrtB in any particular bacterial species, e.g., SrtB from S. aureus. The disclosure encompasses embodiments relating to a sortase A from any bacterial species or strain. The disclosure encompasses embodiments relating to a sortase B from any bacterial species or strain. The disclosure encompasses embodiments relating to a class C sortase from any bacterial species or strain. The disclosure encompasses embodiments relating to a class D sortase from any bacterial species or strain.

[0110] Amino acid sequences of Srt A and Srt B and the nucleotide sequences that encode them are known to those of skill in the art and are disclosed in a number of references cited herein, the entire contents of all of which are incorporated herein by reference. The amino acid sequences of S. aureus SrtA and SrtB are homologous, sharing, for example, 22% sequence identity and 37% sequence similarity. The amino acid sequence of a sortase- transamidase from Staphylococcus aureus also has substantial homology with sequences of enzymes from other Gram-positive bacteria, and such transamidases can be utilized in the ligation processes described herein. For example, for SrtA there is about a 31% sequence identity (and about 44% sequence similarity) with best alignment over the entire sequenced region of the S. pyogenes open reading frame. There is about a 28% sequence identity with best alignment over the entire sequenced region of the A. naeslundii open reading frame. It will be appreciated that different bacterial strains may exhibit differences in sequence of a particular polypeptide, and the sequences herein are exemplary.5.4.2. Functional Sequence motifs

[0111] In embodiments of the present disclosure, an engineered adeno-associated virus (AAV) capsid protein comprising one or more functional sequence motif(s), wherein the functional sequence motif comprises a sortase recognition motif.

[0112] In some embodiments of the present disclosure, the sortase is a sortase A (SrtA). SrtA recognizes the motif LPXTX (wherein each occurrence of X represents independently any amino acid residue), with common recognition motifs being, e.g., LPKTG (SEQ ID NO: 4), LPATG (SEQ ID NO: 5), LPNTG (SEQ ID NO: 6). In some embodimentsLPETG (SEQ ID NO: 7) is used as the sortase recognition motif. However, motifs falling outside this consensus may also be recognized. For example, in some embodiments the motif comprises an ‘A’ rather than a ‘T’ at position 4, e.g., LPXAG (SEQ ID NO: 8), e.g., LPNAG (SEQ ID NO: 9). In some embodiments the motif comprises an ‘A’ rather than a ‘G’ at position 5, e g., LPXTA (SEQ ID NO: 10), e g., LPNTA (SEQ ID NO: 11). In some embodiments the motif comprises a ‘G’ rather than ‘P’ at position 2, e.g., LGXTG (SEQ ID NO: 12), e.g., LGATG (SEQ ID NO: 13). In some embodiments the motif comprises an ‘I’ rather than ‘L’ at position 1, e.g., IPXTG (SEQ ID NO: 14), e.g., IPNTG (SEQ ID NO: 15) or IPETG (SEQ ID NO: 16). Additional suitable sortase recognition motifs will be apparent to those of skill in the art, and the disclosure is not limited in this respect. It will be appreciated that the terms “recognition motif’ and “recognition sequence”, with respect to sequences recognized by a transamidase, transpeptidase or sortase, are used interchangeably.

[0113] In some embodiments of the disclosure the sortase is a sortase B (SrtB), e.g., a sortase B of S. aureus, B. anthracis, or L. monocytogenes. Motifs recognized by sortases of the B class (SrtB) often fall within the consensus sequences NPXTX, e.g., NP[Q / K]- [T / sHN / G / s], such as NPQTN (SEQ ID NO: 17) or NPKTG (SEQ ID NO: 18). For example, sortase B of S. aureus or B. anthracis cleaves the NPQTN (SEQ ID NO: 17) or NPKTG (SEQ ID NO: 18) motif of IsdC in the respective bacteria (see, e.g., Marraffini, L. and Schneewind, O., Journal of Bacteriology, 189 (17), p. 6425-6436, 2007). Other recognition motifs found in putative substrates of class B sortases are NSKTA (SEQ ID NO: 19), NPQTG (SEQ ID NO: 20), NAKTN (SEQ ID NO: 21), and NPQSS (SEQ ID NO: 22). For example, SrtB from L. monocytogenes recognizes certain motifs lacking P at position 2 and / or lacking Q or K at position 3, such as NAKTN (SEQ ID NO: 21) and NPQSS (SEQ ID NO: 22) (Mariscotti J F, Garcia-Del Portillo F, Pucciarelli M G. The listeria monocytogenes sortase-B recognizes varied amino acids at position two of the sorting motif. J Biol Chem. 2009.)

[0114] In certain embodiments, the sortase enzyme, the functional sequence motif (or “motif’) and the enzyme substrate moiety are as described in Bradshaw, W.J., et al. (2015), Molecular Features of the Sortase Enzyme Family. FEBS J, 282: 2097-2114, which is incorporated herein in its entirety.

[0115] In some embodiments, the sortase is a sortase C (Srt C). Sortase C may utilize LPXTX as a recognition motif, with each occurrence of X independently representing any amino acid residue.

[0116] In some embodiments, the sortase is a sortase D (Srt D). Sortases in this class are predicted to recognize motifs with a consensus sequence NA-[E / A / S / H]-TG (SEQ ID NO: 71) (Comfort D, supra). Sortase D has been found, e.g., in Streptomyces spp., Corynebacterium spp., Tropheryma whipplei, Thermobifida fusca, and Bifidobacterium longhum.

[0117] LPXTA (SEQ ID NO: 10) or LAXTG (SEQ ID NO: 23) may serve as a recognition sequence for sortase D, e.g., of subfamilies 4 and 5, respectively subfamily-4 and subfamily-5 enzymes process the motifs LPXTA (SEQ ID NO: 10) and LAXTG (SEQ ID NO: 23), respectively). For example, B. anthracis Sortase C has been shown to specifically cleave the LPNTA (SEQ ID NO: 11) motif in B. anthracis BasI and BasH (see Marrafini, supra).

[0118] See Barnett and Scott for description of a sortase that recognizes QVPTGV (SEQ ID NO: 24) motif (Barnett, T C and Scott, J R, Differential Recognition of Surface Proteins in Streptococcus pyogenes by Two Sortase Gene Homologs. Journal of Bacteriology, Vol. 184, No. 8, p. 2181-2191, 2002; the entire contents of which are incorporated herein by reference). Additional sortases, including, but not limited to, sortases recognizing additional sortase recognition motifs are also suitable for use in some embodiments of this disclosure. For example, sortases described in Chen I, Dorr B M, and Liu D R., A general strategy for the evolution of bond-forming enzymes using yeast display. Proc Natl Acad Sci USA. 2011; 108(28): 11399, the entire contents of which are incorporated herein.

[0119] The use of sortases found in any gram-positive organism, such as those mentioned herein and / or in the references (including databases) cited herein is contemplated in the context of some embodiments of this disclosure. Also contemplated is the use of sortases found in gram negative bacteria, e.g., Colwellia psychrerythraea, Microbulbifer degradans, Bradyrhizobium japonicum, Shewanella oneidensis, and Shewanella putrefaciens. Such sortases recognize sequence motifs outside the LPXTX consensus, for example, LP[Q / K]T[A / S]T (SEQ ID NO: 25). In keeping with the variation tolerated at position 3 in sortases from gram-positive organisms, a sequence motif LPXT[A / S], e.g., LPXTA (SEQ ID NO: 10) or LPSTS (SEQ ID NO: 26) may be used.

[0120] Those of skill in the art will appreciate that any sortase recognition motif known in the art can be used in some embodiments of this disclosure, and that the disclosureis not limited in this respect. For example, in some embodiments the sortase recognition motif is selected from: LPKTG (SEQ ID NO: 4), LPITG (SEQ ID NO: 27), LPDTA (SEQ ID NO: 28), SPKTG (SEQ ID NO: 29), LAETG (SEQ ID NO: 30), LAATG (SEQ ID NO: 31), LAHTG (SEQ ID NO: 32), LASTG (SEQ ID NO: 33), LAETG (SEQ ID NO: 30), LPLTG (SEQ ID NO: 34), LSRTG (SEQ ID NO: 35), LPETG (SEQ ID NO: 7), VPDTG (SEQ ID NO: 36), IPQTG (SEQ ID NO: 37), YPRRG (SEQ ID NO: 38), LPMTG (SEQ ID NO: 39), LPLTG (SEQ ID NO: 34), LAFTG (SEQ ID NO: 40), LPQTS (SEQ ID NO: 41), it being understood that in various embodiments of the disclosure the 5th residue may be replaced with any other amino acid residue. For example, the sequence used may be LPXT, LAXT, LPXA, LGXT, IPXT, NPXT, NPQS (SEQ ID NO: 46), LPST (SEQ ID NO: 47), NSKT (SEQ ID NO: 48), NPQT (SEQ ID NO: 49), NAKT (SEQ ID NO: 50), LPIT (SEQ ID NO: 51), LAET (SEQ ID NO: 52), or NPQS (SEQ ID NO: 46). The disclosure encompasses embodiments in which ‘X’ in any sortase recognition motif disclosed herein or known in the art is amino acid, for example, any naturally occurring or any non-naturally occurring amino acid. In some embodiments, X is selected from the 20 standard amino acids found most commonly in proteins found in living organisms. In some embodiments, e.g., where the recognition motif is LPXTG (SEQ ID NO: 53) or LPXT, X is D, E, A, N, Q, K, or R. In some embodiments, X in a particular recognition motif is selected from those amino acids that occur naturally at position 3 in a naturally occurring sortase substrate. For example, in some embodiments X is selected from K, E, N, Q, A in an LPXTG (SEQ ID NO: 53) or LPXT motif where the sortase is a sortase A. In some embodiments X is selected from K, S, E, L, A, N in an LPXTG (SEQ ID NO: 53) or LPXT motif and a class C sortase is used.

[0121] In certain embodiments, the functional sequence motif is selected from one of: SEQ ID NOs 1-65. In certain of these embodiments, the functional sequence motif is LPETG (SEQ ID NO 7).

[0122] The term “sortase substrate,” as used herein refers to any molecule that is recognized by a sortase, for example, any molecule that can partake in a sortase-mediated transpeptidation reaction. A typical sortase-mediated transpeptidation reaction involves a substrate comprising a C-terminal sortase recognition motif, e.g., an LPXTX motif, and a second substrate comprising an N-terminal sortase recognition motif, e.g., an N-terminal polyglycine or polyalanine. A sortase substrate may be a peptide or a protein, for example, a target protein on the surface of a virus, or a peptide comprising a sortase recognition motif such as an LPXTX motif or a polyglycine or polyalanine, wherein the peptide is conjugatedto an agent, e.g., a small molecule, a binding agent, or a fluorophore. Accordingly, both proteins and non-protein molecules can be sortase substrates as long as they comprise a sortase recognition motif. Some examples of sortase substrates are described in more detail elsewhere herein and additional suitable sortase substrates will be apparent to the skilled artisan. The disclosure is not limited in this respect.

[0123] The term “sortagging,” as used herein, refers to the process of functionalizing the surface of the AAV virion with a reactive moiety, tag or ligand, for example, a protein, polypeptide, detectable label, binding agent, or click chemistry handle, via a sortase-mediated transpeptidation reaction. Examples of additional suitable tags or ligands include, but are not limited to, amino acids, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags or ligands will be apparent to those of skill in the art and the disclosure is not limited in this aspect. In some embodiments, a tag comprises a sequence useful for purifying, expressing, solubilizing, and / or detecting a polypeptide. In some embodiments, a tag can serve multiple functions. In some embodiments, the tag is relatively small, e.g., ranging from a few amino acids up to about 100 amino acids long. In some embodiments, a tag is more than 100 amino acids long, e.g., up to about 500 amino acids long, or more.

[0124] The term “target protein,” as used herein in the context of sortase-mediated modification of viral particles, refers to a capsid protein that comprises the surface of a virus that is the target of a sortase-mediated conjugation. For example, in an embodiment where AAV2 is modified by sortagging, e.g., by adding a targeting ligand to VR4 on the surface of AAV2 particle, VP1 of AAV2 is the target protein. The term “target protein” may refer to a wild type or naturally occurring form of the respective protein, or to an engineered form, for example, to a recombinant protein variant comprising a sortase recognition motif not contained in a wild-type form of the protein. The term “modifying a target protein,” as used herein in the context of sortase-mediated protein modification, refers to a process of altering a target protein comprising a sortase recognition motif via a sortase-mediated transpeptidation reaction. Typically, the modifying results in the target protein being conjugated to ligand, for example, a peptide, protein, binding agent, detectable label, or small molecule.5.4.3. AAV Sequence Changes

[0125] In certain embodiments, the engineered rAAV capsid protein comprises one or more sequence changes as described above. In certain embodiments, the sequence changecomprises the insertion of an exogenous peptide (insertion peptide) selected from LPET (SEQ ID NO. 2), LPETX (SEQ ID NO: 54) and LPETG (SEQ ID NO: 7).

[0126] The sequence change is present in one or more of VP1, VP2, and VP3 that comprise the virion capsid of the present disclosure.

[0127] In embodiments of the present disclosure, the functional sequence motif is located within a surface loop region selected from: the AB, BC, CD, DE, EF, FG, GH, and HI loops. For example, in certain embodiments, the functional sequence motif is located within a GH surface loop region.

[0128] In embodiments of the present disclosure, the functional sequence motif is located within one or more variable regions (VR) selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8 and VR9. In certain of these embodiments, the functional sequence motif is located within a surface accessible variable region the capsid primary sequence. In certain of these embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII. For example, in certain embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII. In certain embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV. In other embodiments, the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV and VIII. In certain of these embodiments having more than one functional sequence, each functional sequence motif is independently selected.

[0129] In certain embodiments, the engineered rAAV capsid protein comprises more than one functional sequence motif. In certain embodiments, the rAAV virion of the present disclosure comprises more than one engineered rAAV capsid protein. In certain embodiments, each functional sequence motif (in the same or in different capsid proteins) is the same. In alternative embodiments, each functional sequence motif is different.5.4.4. AAV Serotypes and Insertion sites

[0130] In the framework of the present disclosure, an AAV capsid includes any combination of capsid proteins from natural, genetically modified and artificially created (random mutations, sequence shuffling, in silico design, etc.) serotypes that are able to assemble and produce a new AAV virus capsid that is not known to exist in nature.

[0131] Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transducedifferent cell types. AAV2 was the first serotype cloned into a bacterial plasmid and has since been used as a comparison to identify other serotypes. Twelve serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) have been tested thoroughly for their ability to transduce specific cell types and differentiated between capsid protein motifs that bind specific cell surface receptors for cell attachment. In the context of this disclosure, an rAAV capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 is preferred. However, any other AAV capsid proteins can be used in the context of the present disclosure.

[0132] In one embodiment, the AAV capsid protein of the present disclosure comprises capsid proteins selected from those of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In particular, the capsid of the present disclosure comprises capsid proteins comprising those of AAV2, AAV9, and AAV8.

[0133] In certain embodiments, the engineered AAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 serotype. In some embodiments, the AAV capsid protein of the present disclosure is AAV1. In some embodiments, the AAV capsid protein of the present disclosure is AAV2. In some embodiments, the AAV capsid protein of the present disclosure is AAV4. In some embodiments, the AAV capsid protein of the present disclosure is AAV5. In some embodiments, the AAV capsid protein of the present disclosure is AAV8. In some embodiments, the AAV capsid protein of the present disclosure is AAV9.

[0134] In particular embodiments of the present disclosure, the AAV serotype, variable region, insertion location and insertion sequence that forms the functional sequence motif are according to Table 2.Table 2. AAV serotype, variable region, insertion location, and insertion sequence that forms the functional sequence motif.

[0135] In certain embodiments, the capsid is an AAV2 subtype, and the functional sequence motif begins after S452 or R585 or both S452 and R585. In certain of these embodiments, LPET (SEQ ID NO. 2) is inserted to form the functional sequence motif. In certain of these embodiments, LPETG (SEQ ID NO. 7) is inserted to form the functional sequence motif.

[0136] In certain embodiments, a “VR4 mutant” comprises a functional sequence motif formed by the insertion of LPET (SEQ ID NO.: 2), i.e., CTGCCCGAGACC (SEQ ID NO.: 55) between S452 and G 453 (in variable region IV) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes S(452)-L(453)-P(454)- E(455)-T(456)-G(457) and so on with the remaining AA residues incremented by the number of AAs inserted, in this case, four.

[0137] In certain embodiments, a “VR8 mutant” comprises a functional sequence motif formed by the insertion of LPET (SEQ ID NO.: 2), i.e., CTGCCCGAGACC (SEQ ID NO.: 55) between R 585 and G 586 (in variable region VIII) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R(585)-L(586)-P(587)- E(589)-T(590)-G(591) etc.

[0138] In certain embodiments, a “VR4 / VR8 mutant” comprises a functional sequence motif formed by the insertion of LPET (SEQ ID NO.: 2), i.e., CTGCCCGAGACC (SEQ ID NO.: 55) between S452 and G 453 (in variable region IV) of VP1 of AAV2 and a functional sequence motif formed by the insertion of LPET (SEQ ID NO.: 2), i.e., CTGCCCGAGACC (SEQ ID NO.: 55) between R585 and G586 (in variable region 8) of VP1 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R589-L590-P591-E592-T593-G594.

[0139] In certain embodiments, the functional sequence motif has a sequence of X1X2X3; wherein Xi, X2, and X3, are each independently selected from any amino acid residue.

[0140] In certain embodiments, the functional sequence motif has a sequence of X1X2X3, wherein two or more of Xi, X2, and X3, are selected from:Xi is valine (V);X2 is proline (P); andX3 is proline (P).

[0141] In certain embodiments, Xi, X2, and X3, are selected from:Xi is independently proline (P);X2 is independently selected from glycine (G) and glutamic acid (E); andX3 is independently selected from proline (P), and phenylalanine (F).

[0142] In certain embodiments, the functional sequence motif has a sequence selected from: PEF, PGF, or PEP.

[0143] In certain embodiments, the functional sequence motif has a sequence of X1X2X3X4; wherein Xi, X2, X3, and X4, are each independently selected from any amino acid residue.

[0144] In certain embodiments, the functional sequence motif has a sequence of X1X2X3X4X5; wherein Xi, X2, X3, X4, X5, are each independently selected from any amino acid residue. In certain embodiments, the functional sequence motif has a sequence of X1X2X3X4X5, and wherein:Xi is independently selected from lysine (L), valine (V), isoleucine (I) and asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), glycine (G), and asparagine (N).

[0145] In certain embodiments, two or more of Xi, X2, X3, X4, and X5, are selected from:Xi is independently selected from lysine (L);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), a glycine (G), and asparagine (N).

[0146] In certain embodiments, three or more of Xi, X2, X3, X4, and X5 are selected from:Xi is independently selected from lysine (L), valine (V), isoleucine (I) and an asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A) and glycine (G).

[0147] In certain embodiments, Xi is lysine (L); X2 is proline (P); X3 is glutamic acid (E), serine (S), or alanine (A); X4 is threonine (T); and X5 is alanine (A) or glycine (G).

[0148] In certain embodiments, the functional sequence motif comprises a sequence selected from: LPX3TG (SEQ ID NO: 3), LPX3TA (SEQ ID NO: 62), LPETG (SEQ ID NO: 7), and LPETA (SEQ ID NO: 63).

[0149] In certain embodiments, the functional sequence motif comprises a sequence selected from: NX2X3TNX6, and VPX3X4X5P.

[0150] In certain embodiments, the engineered rAAV capsid protein does not comprise additional mutations beyond the sequence changes that form the functional sequence motifs when compared to the wildtype capsid protein.

[0151] In some embodiments, the engineered rAAV capsid protein comprises a first functional sequence motif within variable region IV and a second functional sequence motif within variable region VIII. In some embodiments, the first and second functional sequence motifs are independently selected.

[0152] In some embodiments, the first and second functional sequence motifs are the same.5.5. Polynucleotide Encoding the Engineered rAAV Capsid Protein

[0153] Another aspect of the present disclosure is the provision of a polynucleotide encoding the engineered rAAV capsid protein of any one of the previous embodiments.5.6. Vector Comprising the Provided Polynucleotide

[0154] Another aspect of the present disclosure is the provision of a vector comprising the provided polynucleotide. In certain embodiments, the vector comprises a promoter operably linked to the polynucleotide.5.7. Host Cell Comprising the Engineered rAAV Capsid Protein

[0155] Another aspect of the present disclosure is the provision of a host cell comprising the engineered rAAV capsid protein provided herein, the polynucleotide provided herein, or the vector provided herein.5.8. rAAV Comprising the Engineered Capsid Protein

[0156] In certain aspects of the present disclosure is the provision of a recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein as provided herein. In certain of these embodiments, the rAAV virion further comprises an exogenous cargo polynucleotide. In certain of these embodiments, the exogenous cargo polynucleotide comprises a template for homology directed repair. In certain of these embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA. In alternative embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0157] In certain embodiments, the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.5.9. Surface Functionalized rAAV Virion5.9.1. Bifunctional Linker

[0158] The bifunctional linker in accordance with the present disclosure comprises two components, i) an enzyme substrate moiety, and ii) a crosslinker moiety or a ligand. Optionally, the bifunctional linker further comprises a spacer. In certain embodiments, the bifunctional linker comprises an enzyme substrate moiety, optionally a spacer, and acrosslinker moiety. In certain embodiments, the bifunctional linker comprises an enzyme substrate moiety, optionally a spacer, and a crosslinker moiety.

[0100] The crosslinker reactive moiety of the bifunctional ligand is preferably selected to be mutually and selectively reactive with the crosslinker reactive moiety of a functionalized ligand of the present disclosure.5.9.1.1 Enzyme Substrate Moiety (Nucleophile)

[0159] The enzyme substrate moiety as referred to herein is a nucleophilic chemical or biological functional group that is recognized as a substrate by the relevant enzyme and participates in bioconjugation (i.e., enzymatic ligation) for formation of a covalent attachment between a functional sequence motif of the present disclosure and a component of the enzyme substrate moiety.

[0160] In some embodiments, the enzyme substrate moiety comprises an amine. In certain embodiments, the enzyme substrate moiety comprises a primary amine. In further embodiments, the enzyme moiety comprises an unbranched primary amine, e.g. -CH2NH2.

[0161] In some embodiments, the enzyme substrate moiety comprises an amine as described in Glasgow JE, Salit ML, Cochran JR. In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant. Journal of the American Chemical Society 2016; 138:7496-9, the entire contents of which are incorporated herein.

[0162] In additional embodiments, the enzyme substrate moiety comprises one or more glycine residues. The glycine residues that comprise the enzyme substrate moiety may follow one or more other non-glycine residues. In certain embodiments, the enzyme substrate moiety comprises an oligopeptide comprising at least two glycine residues but up to ten. In an exemplified embodiment, the enzyme substrate moiety comprises a cysteine followed by four glycine resides, e.g., -CGGGG (SEQ ID NO: 72).5.9.1.2 Optional Spacers

[0163] The linker optionally further comprises one or more spacer moiety. The spacer moiety is not particularly limited and may be any spacer known in the art including but not limited to one or more divalent groups such as -CH2- (methylene), -O- (ether), - C(=O)- (carbonyl), and -N(R)- (amine, where R is H or C1-3 alkyl).5.9.2. Crosslinked Moiety - Q

[0164] In embodiments of the present disclosure, “crosslinked moiety” and “Q” are interchangeable and refer to the reaction product of a crosslinker moiety as described above.

[0165] In certain embodiments, the crosslinked moiety comprises a product of a reaction selected from: a CuAAC reaction, a SPAAC reaction, a SPANC reaction, an IEEDD reaction, a Staudinger ligation, a [4+1] cycloaddition reaction. In certain of these embodiments, the reaction is selected from: a SPAAC, a SPANC, and a IEEDD reaction.

[0166] In certain embodiments, the crosslinked moiety comprises a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N. In certain of these embodiments, the crosslinked moiety comprises

[0167] In an aspect of the present disclosure, the surface modified viral capsid comprises a moiety, Q, that is a moiety formed by the reaction between a crosslinker reactive moiety as described herein.

[0168] In certain embodiments, Q comprises the product of a CuAAC reaction. In certain embodiments, Q comprises the product of a SPAAC reaction. In certain embodiments, Q is the product of a SPANC reaction. In certain embodiments, Q comprises the product of an IEEDD reaction. In certain embodiments, Q comprises the product of a Staudinger ligation. In certain embodiments, Q comprises the product of a [4+1] cycloaddition reaction. In some embodiments, Q comprises the product of a strain promoted reaction, e g., SPAAC, SPANC, and IEEDD.

[0169] In certain embodiments, Q comprises a cyclic group. In certain embodiments, Q comprises a bicyclic group. In certain embodiments, Q comprises a tricyclic group. In certain embodiments, Q comprises a 5-8 membered carbocyclic ring comprising from 0 to 3 heteroatoms selected from O, S or N. In certain embodiments, Q comprises an eight membered ring comprising 0 to 1 heteroatom selected from O and N. In certain embodiments, Q comprises a five membered ring comprising 0 to 3 heteroatoms selected from O and N. In certain embodiments, Q is a triazole ring. In certain embodiments, Qcomprises a six membered ring comprising 0-3 heteroatoms selected from O and N. In certain embodiments, Q comprises a six membered ring comprising 2 N heteroatoms.

[0170] In some embodiments, where Q comprises a cyclic group, Q is according to a structure below, where Z is a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N:

[0171] In some embodiments, Q comprises a structure shown below:5.9.3. Enzyme Functionalized Virion

[0172] In certain aspects of the present disclosure, a surface functionalized rAAV virion is provided wherein the virion comprises at least one engineered rAAV capsid protein according to any of the provided embodiments, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.

[0100] In certain of these embodiments, the covalent linkage comprises an amide bond. In certain of these embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine. In certain of these embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a serine.

[0173] In certain of these embodiments, the surface functionalized rAAV virion of the present disclosure further comprises a surface modification as described in WO 2022 / 101363, the entire contents of which are incorporated herein.

[0174] In certain aspects of the present disclosure, a surface functionalized rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of the provided embodiments, and an exogenous cargo polynucleotide,and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.5.9.4. Chemically Functionalized Virion

[0175] In aspects of the present disclosure, a surface functionalized rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to any of the provided embodiments, the rAAV virion further comprises at least one cross linker reactive moiety covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif and an exogenous cargo polynucleotide.

[0176] In certain embodiments, the surface functionalized rAAV virion of the present disclosure comprises a surface modification as described in WO 2022 / 101363, the entire contents of which are incorporated herein.

[0177] In certain embodiments, the above-described amino acid residue that is attached to the crosslinker and that is not within a functional sequence motif is selected from a lysine, an N terminal amine and / or any non-natural amino acid residue known in the art. In certain embodiments, the amino acid residue in the viral capsid protein is an N terminal amine. In certain embodiments, the amino acid residue in the viral capsid protein is a nonnatural amino acid residue known in the art such as those disclosed in WO 2022 / 101363, the entire contents of which are incorporated herein.

[0178] In certain embodiments, the surface functionalized rAAV virion of the present disclosure wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and further comprises at least one cross linker reactive moiety covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif and an exogenous cargo polynucleotide.5.9.5. Crosslinker reactive moiety

[0179] In further embodiments of the present disclosure, the crosslinker reactive moiety participates in a bioorthogonal crosslinker or bioconjugation reactions. As used herein, the term bioorthogonal refers to any chemical process that can occur inside of living systems without interfering with native biochemical processes or can occur in vitro without interfering with biochemical / biological activity of the reaction products. A number of chemical bioconjugation strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry), between nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, the tetrazine ligation, e.g., the cycloaddition of s-tetrazine and trans-cyclooctene derivatives or isocyanide-based click reaction, and most recently, the quadricyclane ligation.

[0180] In certain of the embodiments of the present disclosure, each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne- nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels- Alder (IEEDD) reaction, and a Staudinger ligation and a [4+1] cycloaddition reaction.

[0181] In certain embodiments, the crosslinker reactive moiety comprises at least one of an eight membered ring and a triazole ring. In certain of these embodiments, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. In certain of these embodiments, the crosslinker reactive moiety is selected from a cyclooctyne and an azide. In certain of these embodiments, the cyclooctyne is selected from dibenzyl cyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof. In certain of these embodiments, the cyclooctyne is a DBCO. In certain of these embodiments, the reaction is an inverse electron demand Diels-Alder (IEEDD) reaction. In certain of these embodiments, the crosslinker reactive moiety is selected from a transcyclooctene and a tetrazine.5.9.5.1.1 CuAAC

[0182] In certain embodiments, the crosslinker reactive moiety is selected from chemical moieties that participate in a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). In certain embodiments, the crosslinker reactive moiety comprises an azide and an alkyne.Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinked moiety Q comprises a 5-membered heteroatom ring. In certain embodiments, the crosslinked moiety Q comprises a 1,4 triazole.5.9.5.1.2 SPAAC and SPANC

[0183] Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. Instead of copper, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC). Copper-free click chemistry has been adapted to use nitrones as the 1,3-dipole rather than azides and has been used in the modification of peptides.

[0184] In certain embodiments, the crosslinker reactive moiety is selected from chemical moieties that participate in a strain-promoted alkyne-nitrone cycloaddition (SPANC). In certain embodiments, the crosslinker reactive moiety comprises an azide and a nitrone. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinked moiety Q comprises an isoxazoline.

[0185] In some embodiments, the crosslinker reactive moiety is an azide and a nitrone, as illustrated below, where the R group represents the point of attachment to the capsid-reactive linker or functionalized ligand.azidenitrone

[0186] Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. For example, substitution on both the carbon and nitrogen atoms of the nitrone dipole, and acyclic and endocyclic nitrones are all tolerated.

[0187] In some embodiments, the crosslinker reactive moiety comprises a cyclooctyne analogue. In certain embodiments, the crosslinker reactive moiety comprises a cyclooctyne analogue, e.g., those illustrated below where the R group represents the point of attachment to the capsid-reactive linker or functionalized ligand. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.

[0188] In certain embodiments, the crosslinker reactive moiety comprises a dibenzylcyclooctyne analog selected from the group dibenzylcyclooctyne (DIBO), Dibenzoazacyclooctyne (DIBAC or DBCO), and biarylazacyclooctynone (BARAC). Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.

[0189] In certain embodiments, the crosslinker reactive moiety comprises a nitrone according to the structure below, where the R1 group represents the point of attachment to the capsid-reactive linker or functionalized ligand.

[0190] R2and R3are not particularly limited. In some embodiments, R2and R3are independently selected from hydrogen and C1-C4 alkyl groups such as methyl, ethyl, propyl and butyl groups. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein.

[0191] In certain embodiments, the crosslinker reactive moiety comprises a dibenzylcyclooctyne analog as identified above and either a 1,3 -nitrone or an azide. In certain embodiments, the crosslinker reactive moiety comprises a dibenzylcyclooctyne (or analog thereof) and either a 1,3 -nitrone or an azide, as shown below, where the Ri group represents the point of attachment to a viral capsid or a capsid-reactive linker, and wherein R2 group on either the azide or the nitrone represents the point of attachment to a functionalized ligand. Inalternative embodiments, the crosslinker reactive moiety comprises a dibenzylcyclooctyne (or analog thereof) and either a 1,3 -nitrone or an azide, as shown below, where the Ri group represents the point of attachment to a ligand and wherein R2 group on either the azide or the nitrone represents the point of attachment to a surface functionalized viral capsid or a capsid- reactive linker. Scheme 2 illustrates exemplary SPAAC and SPANC reactions of DIBO.Scheme 2. Exemplary SPAAC and SPANC reactions of DIBO.

[0192] In certain embodiments, the crosslinked moiety Q comprises a cyclic moiety according to any one of those illustrated below, where Ri and R2 represent the point of attachment to the viral capsid. R3 and R4 may be H or any substituent described herein, provided the substituted derivatives retain the desired chemical reactivity are also contemplated herein.5.9.5.1.3 IEDDA

[0193] In certain embodiments, the crosslinker reactive moiety comprises chemical moieties that participate in an inverse electron demand Diels-Alder (IEDDA) reaction. In certain embodiments, the crosslinker reactive moiety comprises an electron poor diene and an electron rich dienophile. Examples of such groups are known in the art and described elsewhere, for example, F. Thalhammer, et al., Tetrahedron Lett., 1990, 31, 6851-6854; and B. L. Oliveira, Chem. Soc. Rev., 2017, 46, 4895-4950. In some embodiments, the electron poor diene has an electron withdrawing group substituted on the diene as exemplified below. In some embodiments, the electron rich dienophile has an electron donating group substituted on the dienophile, as exemplified below. inverse electron demandElectron poor dieneelectron ncn oiienopnue

[0194] In certain embodiments, the crosslinker reactive moiety comprises chemical moieties that participate in a Diels-Alder [4+2]-cycloaddition, the reaction between a diene and a dienophile to form a six-membered ring in a 7t4s + 7t2s fashion via suprafacial / suprafacial interaction of 47t-electrons of the diene with the 27t-electrons of the dienophile. In contrast to a normal electron demand Diels- Alder reaction, where an electronrich diene reacts with an electron-poor dienophile, in an inverse-electron-demand Diels- Alder reaction (IEDDA), an electron-rich dienophile reacts with an electron-poor diene. Alkyne dienophiles directly yield the respective pyridazine upon reaction.

[0195] In certain embodiments, the crosslinker reactive moiety comprises a triazine (e.g., 1, 2, 4 triazine), a tetrazine (Tz) (e.g., 1,2,4,5-tetrazines, also referred to as an s- tetrazine) or a strained dienophile such as noroborene, transcyclooctene (TCO), cyclopropene, or N-acylazetine. In certain embodiments, the crosslinker reactive moiety comprises a crosslinker reactive moiety exemplified below, where the R group represents the point of attachment to a capsid-reactive linker, surface functionalized viral capsid, or functionalized ligand of the present disclosure. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein. In certain embodiments, the crosslinker reactive moiety comprises TCO and tetrazine.transcyclooctene cyclopropene N-acylazetine5.9.5.1.4 Staudinger ligation

[0196] In certain embodiments, the crosslinker reactive moiety is selected from a crosslinker reactive moiety that participates in a Staudinger reaction such as an azide, a phosphine (PPI12) or phosphite that are able to react to produce an iminophosphorane.

[0197] In certain embodiments, the crosslinker reactive moiety is a triphenylphosphine, such as the triphenylphosphine shown below where the R group represents the point of attachment to the capsid-reactive linker of the present disclosure. Derivatives of this crosslinker reactive moiety that retains the desired chemical reactivity is also contemplated herein.5.9.5.1.5 [4+1] Cycloaddition

[0198] In certain embodiments, the crosslinker reactive moiety is selected from a crosslinker reactive moiety that participates in an a [4+1] cycloaddition followed by a retro- Diels Alder elimination of N2, e.g., an isocyanide or a 1,2, 4, 5, tetrazine.

[0199] In some embodiments, the crosslinker reactive moiety is an isocyanide as shown below, where the R group represents the point of attachment, e.g., to the capsid- reactive linker. In some embodiments, the crosslinker reactive moiety is a 1,2, 4, 5, tetrazine as shown below, where the Ri or R2 group represents the point of attachment, e.g., to the ligand. Derivatives of these moieties that retain the desired chemical reactivity are also contemplated herein (Scheme 3).Scheme 3. Schematic representation of [4+1] cycloaddition.5.9.6. Tag reactions

[0200] In certain embodiments, the crosslinker reactive moiety is a bioorthogonal tag known in the art, such as a SNAP -tag, a CLIP tag, a Halo-tag, or LUMIO-tag or a chemical group that reacts with these tags, e.g., benzylguanine group, a benzylcytosine group, or a chloroalkane group. In certain embodiments, one member of the crosslinker reactive moiety comprises a SNAP -tag and the other member of the crosslinker reactive moiety comprises a benzylguanine group.5.10. Ligand Modified Virion

[0201] In certain aspects of the present disclosure, a surface modified rAAV virion is provided, wherein the virion comprises at least one engineered rAAV capsid protein according to any of the provided embodiments, and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the virion comprises a crosslinked moiety that covalently connects a C-terminal amino acid of the N-terminal cleavage fragment and a ligand.

[0202] In certain embodiments, the covalent linkage comprises an amide bond.

[0203] In certain embodiments, the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.5.10.1. Combination of Chemical and Enzyme Functionalized Virion

[0204] In certain aspects of the present disclosure, a surface modified rAAV virion is provided, wherein the virion comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO 3), wherein X3 is selected from any amino acid residue; wherein the functionalsequence motif is located within variable region IV, VIII, or IV and VIII, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the virion comprises a crosslinked moiety that covalently connects the T of a N-terminal cleavage fragment and a ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a ligand.

[0100] In certain of these embodiments, the functional sequence motif is individually selected. In certain of these embodiments, the ligand is individually selected.5.10.2. Ligands

[0205] The ligand for use with the present disclosure is not particularly limited, provided that the ligand is amenable to functionalization with a reactive moiety and subsequent conjugation to the viral capsid surface as described herein.

[0206] In some embodiments, the ligand is selected from a protein or oligonucleotide ligand having a cognate that is located on the surface of mammalian cells, such as a receptor. In some of these embodiments, the cognate receptor / ligand pair is involved in transduction of the surface modified viral capsid. In some embodiments, the ligand is a protein or aptamer known to promote permeabilization of cells.

[0207] In some embodiments, the ligand is a cell-type specific ligand. In certain embodiments, the ligand is selected from polypeptides, proteins, monosaccharides or polysaccharides, from steroid hormones, from RGD motif peptide, from vitamins, from small molecules or from targeting peptides. Also contemplated are antibodies (e.g., single chain) or fragments thereof, and nanobodies or DARPins (designed ankyrin repeat proteins) genetically engineered antibody mimetic proteins typically exhibiting highly specific and high- affinity target protein binding); enzymes such as proteases, glycosidases, lipases, peptidases; immunoglobulins such as CD47 (don't eat me signal); IgG proteases such as IdeZ and IdeS; protein based and small molecule adjuvants for vaccination.

[0208] According to one embodiment, a cell-type specific ligand is derived from proteins such as transferrin, Epidermal Growth Factor EGF, basic Fibroblast Growth Factor bFGF.

[0209] According to one embodiment, a cell-type specific ligand is derived from mono- or polysaccharides such as galactose, N-acetylgalactosamine and mannose.

[0210] According to one embodiment, a cell-type specific ligand is derived from vitamins such as folates.

[0211] According to one embodiment, a cell-type specific ligand is derived from small molecules including naproxen, ibuprofen or other known protein-binding molecules.

[0212] In certain embodiments, the ligand is selected from a protein ligand, such as a growth factor or a cytokine; a toxin subunit, such as a cholera toxin B subunit; a lectin, such as isolectin B4 or wheat germ agglutinin; an adhesion factor, such as lactadherin; an antibody or a single chain variable fragment thereof, such as an anti -CD-34 antibody; more specifically, an E. coli recombinant scFv CD-34 antibody fragment, a peptide, such as deltorphin opioid receptor ligand; and a gene editing nuclease, such as Cas9, a DARPin such as MP0112. In other embodiments, the ligand is an oligonucleotide, for example, an aptamer such as those described in Kelly, L., Maier, K.E., Yan, A. et al. A comparative analysis of cell surface targeting aptamers. Nat Commun 12, 6275 (2021), incorporated by reference herein.5.10.3. Functionalized Ligand

[0213] In an aspect of the present disclosure, a functionalized ligand is provided wherein the ligand as described above is functionalized to comprise a crosslinker reactive moiety, a bioorthogonal tag or an enzyme substrate as described herein according to peptide and oligonucleotide labeling techniques known in the art.

[0214] In some embodiments, the ligand is functionalized by reaction with a ligand- reactive linker.

[0215] In alternative embodiments, the functionalized ligand is a biological polypeptide mutated to include a non-natural amino acid comprising a crosslinker reactive moiety, enzyme substrate moiety or a bioorthogonal tag.

[0216] In some of these embodiments, the enzyme substrate moiety comprises an oligoglycine enzyme substrate moiety or amine moiety.5.10.3.1 Fusion Proteins with Tag-Reactive Molecules

[0217] In embodiments of the present disclosure, the ligand is a fusion protein comprising a tag that is able to bind to their corresponding counterpart with high affinity, such as SNAP -tag, CLIP -tag, Halo Tag, Lumio Tag, and others known to those in the art.5.10.4. Cargo

[0218] In certain embodiments, the exogenous cargo polynucleotide comprises a template for homology directed repair. In certain of these embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA. In additional embodiments, the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0219] The nucleic acid cargo packaged inside the surface modified rAAV capsid of the present disclosure can be any kind of nucleic acid molecule usefully transduced into cells by rAAV.

[0220] In some embodiments, the payload or cargo of the rAAV capsid is an expressible polynucleotide. In certain embodiments, the expressible polynucleotide encodes a protein (e.g., encoding a therapeutic protein). In certain embodiments, the expressible polynucleotide encodes a transgene. In certain embodiments, the expressible polynucleotide can be transcribed to provide a guide RNA, a trans-activating CRISPR RNA (tracrRNA), a messenger RNA (mRNA), a microRNA (miRNA), or a shRNA.

[0221] In some embodiments, the payload provides a DNA homology construct for homology directed repair.

[0222] In some embodiments, the exogenous cargo polynucleotide encodes intracellular antibodies, peptide toxins, optogenetic actuators, pharmacogenetic tools, CRISPR based-editors for precision gene editing, CRISPR-epigenetic tools to regulate gene expression, or suicide genes to induce cell death.

[0223] In some embodiments, said nucleic acid molecule is encoding intracellular antibodies (for example to neutralize certain proteins inside cells), nucleic acid molecules encoding peptide toxins (for example to block ion channels in the pain pathway), nucleic acid molecules encoding optogenetic actuators (for example to turn on or turn off neuronal activity using light), nucleic acid molecules encoding pharmacogenetic tools (for example to turn on or off neuronal signaling using chemical ligands that have no interfering pharmacological effect), nucleic acid molecules encoding CRISPR based-editors for precision gene editing, nucleic acid molecules encoding CRISPR-epigenetic tools to regulate gene expression, and / or nucleic acid molecules encoding suicide genes to induce cell death.

[0224] In some embodiments, when the cargo comprises a gene editing nuclease, such as Cas9, the cargo further comprises a nucleic acid molecule, such as a gRNA and / or a specific DNA to be inserted into a host genome. In certain of these embodiments, the cargo comprises a transgene known to be associated with a genetic disorder. In some embodiments, the gene editing nuclease system comprises a Cas9 orthologue, a gRNA, and a specific DNA to be inserted into a host genome.

[0225] The person of skill is aware of other gene editing nucleases, apart from Cas9, such as Cpfl, TALEN, ZFN, or a homing endonuclease. Further, it may be convenient to engineer using DNA-guided Argonaute interference systems (DAIS). Basically, said Argonaute (Ago) protein is heterologously expressed from a polynucleotide introduced into said cell in the presence of at least one exogenous oligonucleotide (DNA guide) providing specificity of cleavage to said Ago protein to a preselected locus. The TALEN and Cas9 systems are respectively described in WO 2013 / 176915 and WO 2014 / 191128. The Zinc- finger nucleases (ZFNs) are initially described in Kim, YG; Cha, J.; Chandrasegaran, S. ("Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain" (1996). Proc Natl Acad Sci USA 93 (3): 1156-60). Cpfl is a class 2 CRISPR Cas System described by Zhang et al. (Cpfl is a single RNA-guided Endonuclease of a Class 2 CRIPR-Cas System. (2015). Cell; 163:759-771). The argonaute (AGO) gene family was initially described in Guo S, Kemphues KJ. (Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser / Thr kinase that is asymmetrically distributed. (1995).Cell;81(4):611-20).5.11. Methods of Making Surface Modified rAAV Virions

[0226] Another aspect of this disclosure relates to a method of producing a surface- modified recombinant AAV virion.

[0227] The method comprises the step of crosslinking, i.e., covalently conjugating, a ligand to an engineered viral capsid protein via a linker comprising a crosslinked moiety, Q. In some embodiments, the ligand introduces at least one mammalian cell surface target binding site into said capsid, optionally wherein a natural cell surface target binding site in said capsid is removed, such as is previously removed.

[0228] In one embodiment, a method of a making a surface modified viral capsid described herein comprises the steps:1) obtaining a surface functionalized viral capsid by conjugating an engineered viral capsid protein as described herein with a bifunctional linker as described herein; ii) conjugating the surface functionalized viral capsid with a functionalized ligand to form a crosslinked moiety, Q; and iii) obtaining the surface modified viral capsid.

[0229] In another embodiment, a method of a making a surface modified viral capsid described herein comprises the steps: obtaining a surface functionalized viral capsid by enzymatic conjugation between:(i) a functional motif in a viral capsid protein; and(ii) an enzyme-reactive linker; wherein the linker comprises an enzyme substrate moiety and a crosslinker reactive moiety; and2) reacting the obtained surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive moiety.

[0230] In certain embodiments, the method further comprises the step of producing an AAV capsid comprising at least one viral capsid protein comprising at least one recognition sequence. Optionally, the recognition sequence is located in a surface loop region, such as variable region 4 or 8 or both 4 and 8. In certain embodiments the process of preparing the surface functionalized virion is according to Process A or Process B as illustrated in Scheme 4.Scheme 4. Schematic representation of preparing a chemically modified surface functionalized virion using Process A and Process B.

[0231] In certain embodiments the process of preparing the surface functionalized virion is according to Process A’ or Process B’ as illustrated in Scheme 5.Scheme 5. Schematic representation of preparing an enzymatically surface functionalized virion using Process A’ and Process B’.

[0232] Step A comprises conjugating a ligand functionalized to the surface of the modified viral capsid. In some embodiments, the conjugation is a chemical conjugation to a surface exposed amino acid in the primary sequence of a capsid protein. In some embodiments, the conjugation is an enzyme catalyzed conjugation (i.e., Scheme 5, Step A’).

[0233] Step B 1 comprises conjugating a bifunctional linker to the surface of the modified viral capsid. In some embodiments, the conjugation is a chemical conjugation to a surface exposed amino acid in the primary sequence of a capsid protein. In some embodiments, the conjugation is an enzyme catalyzed conjugation (i.e., Scheme 5, Step B’).

[0234] Step B2 comprises conjugating a functionalized ligand to the bifunctional linker using bio-orthogonal chemistry. In certain embodiments the process of preparing the surface functionalized virion is according to a process in Scheme 6 as illustrated below.Scheme 6. Schematic representation of preparing surface functionalized virion using chemical and enzymatic approaches.

[0235] In certain embodiments the process of preparing the surface functionalized virion is according to a process in Scheme 7 as illustrated below.Step A’Scheme 7. Schematic representation of preparing surface functionalized virion using chemical and enzymatic approaches.

[0236] In certain embodiments the process of preparing the surface functionalized virion is according to a process in Scheme 8 as illustrated below.Scheme 8. Schematic representation of preparing a surface functionalized virion using chemical and enzymatic approaches.5.12. Pharmaceutical Composition

[0237] In certain aspects of the present disclosure, a pharmaceutical composition comprising a plurality of rAAV virions is provided according to any provided embodiment, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient, or combination thereof.5.13. Formulations

[0238] Yet another embodiment of the disclosure pertains to the afore-described surface modified viral capsid for use in the treatment of a disease, wherein said AAV is administered to a subject in a liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.

[0239] In certain embodiments, a pharmaceutical composition is provided comprising a recombinant virion, the recombinant virion comprising a surface modified viral capsid as provided herein with a recombinant nucleic acid cargo contained therein, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and / or excipient. Such pharmaceutically acceptable carrier, diluents, solubilizer, filler, preservative and / or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

[0240] A further aspect of the present disclosure then relates to a pharmaceutical composition, comprising the surface modified viral capsid according to the present disclosure, together with at least one pharmaceutically acceptable carrier and / or diluent, i.e. in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. In some embodiments, the composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / medically accepted method.

[0241] A further aspect of the present disclosure then relates to a kit comprising: a) the surface modified viral capsid as disclosed and / or for use according to the present disclosure, or a pharmaceutical composition comprising the surface modified viral capsid as disclosed according to the present disclosure, b) written instructions to apply said surface modified viral capsid or said pharmaceutical composition to a target said; and optionally, a container holding the surface modified virion for use or the composition and the written instructions.

[0242] Another aspect of the present disclosure relates to the use of the abovedescribed kit for preventing, treating, and / or inhibiting a viral infection in a subject in need of said treatment.5.14. Methods of Treating Disease

[0243] The present disclosure also includes a method for treating a subject at risk for development and / or progression of a disease, including a monogenic or polygenic genetic disease, wherein a therapeutically effective amount of the AAV particle as provided by the present disclosure is administered to the patient. In this context, therapeutically effective describes an amount of AAV particles sufficient to treat the disease, such as a genetic disease, by resolution of symptoms. Therapeutically effective can also be an amount sufficient to prevent symptoms of a disease, such as a genetic disease, from occurring. Being at risk for the disease can result from, e.g., genetic and / or phenotypic symptoms, which predispose to the disease. In some embodiments, a patient at risk for a genetic disease has been determined to carry or be deficient in a gene associated with a genetic disease.

[0244] A further aspect of this disclosure then relates to a method for treating a disease that can be treated by gene therapy, the method comprising administering the surface modified rAAV virion according to the present disclosure to a subject in need thereof.

[0245] Cells and / or subjects to be treated with the surface modified rAAV virions of this disclosure are, in some embodiments, of mammalian origin, such as of human origin. Nevertheless, the present disclosure can advantageously be used also in veterinary medicine, cell culture procedures, or even in plant cell diseases, depending on the similarities of the mechanisms of entry into the cells. In some embodiments, said cell to be treated is a mammalian cell, a prokaryotic cell, or a plant cell. In particular embodiments, said cell to be treated is a human cell.

[0246] Yet another embodiment of the disclosure pertains to the afore-described method for treating a disease, comprising administering the surface modified rAAV virion according to the present disclosure to a subject in need thereof, wherein said surface modified rAAV virion is administered to a subject in a liquid, dry or semi-solid form, such as, for example, in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granulate, sugar-coated tablet, lozenge, pill, ampoule, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, moist compress, gargling solution, plant juice, nasal agent, inhalation mixture, aerosol, mouthwash, mouth spray, nose spray, or room spray.

[0247] The disease to be treated by the above method for treating a disease that comprises administering the surface modified rAAV virion to a subject. In certain embodiments, the disease selected from cancer, an inherited monogenic disease, such as inherited retinal disease, a genetic skin disease, such as Olmsted Syndrome or Familiar Primary Localized Cutaneous Amyloidosis, an infectious disease, adrenoleukodystrophy, alpha-1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, beta thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolemia, familial lecithin-cholesterol acyltransferase deficiency, Fanconi anemia, galactosialidosis, Gaucher's disease, gyrate atrophy, hemophilia A, hemophilia B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late infantile neuronal ceroid lipofuscinosis, leukocyte adherence deficiency, limb girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), sanfilippo B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogen storage disease type la), X-linked myotubular myopathy, anemia of end stage renal disease, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcers, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria,pediatric respiratory disease, respiratory syncytial virus, tetanus, tuberculosis, gynecological cancer, breast cancer, ovary cancer, cervix cancer, vulva cancer, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon, colorectal, liver metastases, post-hepatitis liver cancer, pancreas, gall bladder, hepatocellular carcinoma, genitourinary cancer, prostate, renal, bladder, ano-genital neoplasia, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell, mesothelioma, hematological cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, late infantile neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson disease, peripheral neuropathy, spinal muscular atrophy type 2, achromatopsia, age-related macular degeneration, choroideraemia, diabetic macular edema, glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative joint disease, severe inflammatory disease of the rectum, ulcerative colitis, chronic renal disease, diabetic ulcer, foot ulcer, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft versus host disease / transplant patients, oral mucositis, parotid salivary hypofunction, systemic scleoderma, type I diabetes, and wound healing, or combinations thereof.

[0248] Also provided is a method for treating a disease, comprising administering the surface modified rAAV virion according to the present disclosure to a subject in need thereof, wherein said surface modified rAAV virion is administered to said subject or to a cell, in the form of a pharmaceutical composition, e.g., in combination with pharmaceutically acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. In certain embodiments, said composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / medically accepted method.

[0249] Yet another aspect of this disclosure relates to the surface modified rAAV virion according to the present disclosure for use in the transfection of a cell, for example as a gene delivery tool in research. Said use can also be for cosmetic purposes, and the presentdisclosure includes a method for cosmetic treatment in analogy to the medical treatment as disclosed herein. For this, administering the surface modified rAAV virion according to the present disclosure to a subject or to a cell can be also achieved in form of a cosmetic composition, e.g. in combination with cosmetically safe and acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders or stabilizers. In certain embodiments, said composition is administered to said subject in form of sprays, coatings, foams, lotions, gels, mouthwash, oral formulations or injections. Said composition can be administered to said subject systemically, orally or by any other clinically / cosmetically accepted method.

[0250] The person of skill in the art is aware of methods of using vectors derived from AAV for transferring genes in vitro and in vivo, such as those that have been described in WO 93 / 09239, US4797368, US 5139941 and EP 488 528.

[0251] An additional aspect of the present disclosure relates to a kit comprising: a) the surface modified rAAV virion for the transfection of cells, b) written instructions to use the surface modified rAAV virion for the transfection of cells; and optionally, a container holding the surface modified rAAV virion and the written instructions.5.14.1. Indications

[0252] Another aspect of this disclosure relates to recombinant virions comprising the surface modified rAAV virion according to the present disclosure for use in the treatment of a disease, and methods of treating disease by administering an effective amount of recombinant virions comprising surface modified capsid as described herein. In certain embodiments, the compositions provided herein are for use in a treatment of a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo. In certain embodiments, the compositions provided herein are for use in a treatment comprising gene therapy.Furthermore, the disclosure provides for the use of the surface modified rAAV virion composition for the preparation of a medicament for gene therapy. Also, the disclosure provides for a method of treatment comprising gene therapy, wherein the method comprises the administration of a rAAV composition comprising the surface modified capsid.

[0253] The kind of disease that can be treated or prevented by administration of the rAAV according to the present disclosure is not particularly limited. Diseases include those diseases that can be treated by gene therapy, such as cancer, an inherited monogenic disease, such as inherited retinal disease, a genetic skin disease, such as Olmsted Syndrome orFamiliar Primary Localized Cutaneous Amyloidosis, an infectious disease, ataxia, adrenoleukodystrophy, alpha- 1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, beta thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolaemia, familial lecithin-cholesterol acyltransferase deficiency, Fanconi anaemia, galactosialidosis, Gaucher's disease, gyrate atrophy, hemophilia A and B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late infantile neuronal ceroid lipofuscinosis, leukocyte adherence deficiency, limb girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), Sanfilippo B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogen storage disease type la), X-linked myotubular myopathy, anemia of end stage renal disease, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcers, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, pediatric respiratory disease, respiratory syncytial virus, tetanus, tuberculosis, gynecological cancer, breast, ovary, cervix, vulva, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma, gastrointestinal cancer, colon, colorectal, liver metastases, post-hepatitis liver cancer, pancreas, gall bladder, hepatocellular carcinoma, genitourinary cancer, prostate, renal, bladder, ano-genital neoplasia, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal carcinoma, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell / non-small cell, mesothelioma, hematological cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell cancer, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, late infantile neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson disease, peripheral neuropathy, spinal muscular atrophy type 2,achromatopsia, age-related macular degeneration, choroideraemia, diabetic macular oedema, glaucoma, Leber congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, superficial corneal opacity, X-linked retinoschisis, arthritis (rheumatoid, inflammatory, degenerative), degenerative joint disease, severe inflammatory disease of the rectum, ulcerative colitis, chronic renal disease, diabetic ulcer / foot ulcer, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft versus host disease / transplant patients, oral mucositis, parotid salivary hypofunction, systemic scleroderma, type I diabetes, and / or wound healing.

[0254] In certain embodiments, the ataxia to be treated in accordance with the present disclosure is ataxia associated with a hereditary disorder consisting of degeneration of the cerebellum or of the spine and may present with overlapping cerebellar and sensory ataxia, even. Hereditary disorders causing ataxia include autosomal dominant ones such as spinocerebellar ataxia, episodic ataxia, and dentatorubropallidoluysian atrophy, as well as autosomal recessive disorders such as Friedreich's ataxia (sensory and cerebellar, with the former predominating) and Niemann Pick disease, ataxia-telangiectasia (sensory and cerebellar, with the latter predominating), and abetalipoproteinaemia. An example of X- linked ataxic condition is the rare fragile X-associated tremor / ataxia syndrome or FXTAS.

[0255] In certain embodiments, the indication to be treated is lipoprotein lipase deficiency, large B-cell lymphoma, beta thalassemia, mantle cell lymphoma, vascular endothelial growth factor peripheral artery disease, head and neck squamous cell carcinoma, spinal muscular atrophy, adenosine deaminase deficiency (ADA-SCID), melanoma in patients who have recurring skin lesions, B cell lymphoblastic leukemia, or Leber congenital amaurosis.

[0256] In certain embodiments, the indication to be treated include Charcot-Mari e- Tooth (all types), Gangliosidosis (all types), Genetic epilepsy (i.e. Dravet), tuberous sclerosis complex, Spinal cord injury, all demyelinating hereditary motor and sensory neuropathies (HMSN), Krabbe disease, fibrodysplasia ossificans progressive, Neurofibromatosis 1 and 2, essential tremor, fragile X syndrome, Lesch-Nyhan syndrome, myotonic dystrophy, multiple system atrophy (MSA), Zellweger syndrome, neuromyelitis optica, or Devic's disease, central pontine myelinolysis, myelopathies such as tabes dorsalis (syphilitic myelopathy), leukoencephalopathies such as progressive multifocal leukoencephalopathy, leukodystrophies, and Guillain-Barre syndrome and its chronic counterpart, chronic inflammatory demyelinating polyneuropathy.

[0257] In certain embodiments, the indication to be treated is anti-MAG peripheral neuropathy, or copper deficiency-associated conditions (peripheral neuropathy, myelopathy, and rarely optic neuropathy), or progressive inflammatory neuropathy.5.14.2. Modes of Administration

[0258] Another aspect of this disclosure relates to modes of administration of the surface modified rAAV virion according to the present disclosure for use in the treatment of a disease.

[0259] In some embodiments, the surface modified rAAV virion according to the present disclosure may be directly or indirectly administered using suitable means known in the art. Methods and uses of the disclosure include delivery and administration of the surface modified rAAV virion according to the present disclosure composition systemically, regionally or locally, or by any route, for example, by injection, infusion, orally (e.g., ingestion or inhalation), or topically (e.g., transdermally). Exemplary administration and delivery routes include intravenous (i.v.), intra-articular, intraperitoneal (i.p.), intra-arterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, parenterally, e.g., transmucosal, intra-cranial, intra-spinal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, intralymphatic, intrathecal, intra cisterna magna.

[0260] In certain embodiments, the mode of administration is systemic. Systemic administration includes systemic routes of injection, such as intramyocardially, intramuscularly, and intravascularly. In certain embodiments, the surface modified rAAV virion comprises an enzyme ligand (e.g., Tissue Plasminogen Activator) that permeabilizes the blood brain barrier, e.g., the pia matter, allow transduction of spinal cord and brain parenchyma following, e.g., intrathecal injection. In certain embodiments, the surface modified rAAV virion further comprises a cell-targeting ligand.

[0261] Improvements in means for providing an individual or a cell, tissue, organ of said individual with the surface modified rAAV virion according to the present disclosure composition are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect of the disclosure.

[0262] In certain embodiments, the step of administering the surface modified rAAV virion according to the present disclosure, the capsid composition is dissolved in a solution that is compatible with the delivery method. In certain embodiments formulation for intravenous, subcutaneous, intramuscular, intrathecal, intraarticular and / or intraventricular administration, is the capsid composition is formulated as a physiological salt solution.5.15. Examples5.15.1. Example 1. Optimizing Production of AAV2 Mutants Having Altered Tropism5.15.1.1 Methods5.15.1.1.1 Recombinant AAV2 virion production

[0263] Independently, an LPET sequence (SEQ ID NO: 2) was inserted via site directed mutagenesis into the coding sequence of an AAV2 capsid protein at the following locations:-variable region IV (i.e., “VR4” or “VR-IV”) between S452 and G453 of the wild type VR4, to produce the sequence segment: S452-L453-P454-E455-T456-G457 (the engineered capsid comprising this protein is referred to herein as AAV2-LPETG- VR-IV), see FIG. 1;-variable region VIII (i.e., “VR8” or “VR-VIII”) between R585 and G586 (in variable region 8) of VP1, VP2 and VP3 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R(585)-L(586)-P(587)-E(589)-T(590)-G(591) (the engineered capsid comprising this protein is referred to herein as AAV2-LPETG- VR-VIII), see FIG. 2; and-both sites (i.e. “VR-IV / VR-VIII”) between S452 and G453 of the wild type VR-IV, to produce the sequence segment: S452-L453-P454-E455-T456-G453), and between R585 and G586 (in variable region VIII) of VP1, VP2 and VP3 of AAV2. In this case, this section of the sequence of the engineered capsid protein becomes R589-L590-P591-E592-T593- G594 (the engineered capsid comprising this protein is referred to herein as AAV2-LPETG- VR-IV / VRVIII), thereby providing at least one LPETG recognition sequence (SEQ ID NO: 7) within an engineered capsid peptide.

[0264] The recombinant AAV2 virions AAV2-LPETG- VR-IV, AAV2-LPETG- VR- VIII and AAV2-LPETG- VR-IV / VR-VIII carrying tdTomato under a CAG promoter as acargo were produced in HEK293 cells as according to known protocols (Grieger JC, Choi VW, Samulski RJ. Production and characterization of adeno-associated viral vectors. Nat Protoc 2006; 1 : 1412-28, incorporated herein by reference in its entirety). Cells were harvested around 72 hours post transfection, lysed with Triton X-100 at 0.5%, nuclease treated, concentrated by tangential flow filtration, and purified using isopycnic ultracentrifugation (Dias Florencio G, Precigout G, Beley C, Buclez PO, Garcia L, Benchaouir R. Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors. Mol Ther Methods Clin Dev 2015; 2: 15024, incorporated herein by reference in its entirety). Vector genome titration was performed using Q-PCR with primers targeting the promoter region of the viral cargo (Grieger JC 2006).

[0265] For rAAV production, 40x106 of HEK 293T / 17 cells were plated in a two- layer cell stack. Reached -80% confluency, cells were transfected with 600 pg of DNA of capsid plasmid (WT; Addgene, #104963 or VR variants), helper plasmid pFdelta6 (Addgene, #112867) and cargo vector either pAAV-CAG-GFP (Addgene, #37825) or pAAV-CAG- TdTomato (Addgene, #59462) at a ratio of 1 :2: 1 (capsid:helper:cargo), using linear polyethylenimine (PEI). Transfections were performed at a ratio of 1 pg of DNA to 11 pL of linear PEI (0.33mg / mL). Linear PEI solution was added to the DNA one, vortexed and incubated for 15 minutes at room temperature. Fresh media, together with 20 mL of the DNA-linear PEI mixture was added to a final volume of 200 mL to the cell stack and fresh media was replaced within 24 hours after transfection. 72 hours after transfection, Triton X- 100 and RNase were added to the culture medium at a final concentration of 0.05% and 25 mg / mL, respectively and cells were kept at 37°C for 1 hour, shaking. Residual cells and media were centrifuged at 3700 x g, at 18°C for 30 minutes. The supernatant was then filtered through 500 mL Stericup quick release filter 0.22pm (Millipore, #S2GPU02RE) and concentrated using a peristaltic pump equipped with a filtering polyether sulfone membrane cartridge with a cutoff of 100 kDa (Sigma Aldrich, #Z615390) to obtain 30 mL. Viral particles were then purified through an assembled gradient of OptiPrep Density Gradient Medium (Optiprep, #1114542) by ultracentrifugation (Beckmann, optima XE-90 ultracentrifuge) at 44.400 x g for 2 hours at 18°C. Viral particles concentrated in the 40% iodixanol phase was collected, diluted to a final volume of 15 mL with 200 mM NaCl / 0.001% PLURONIC F-68 in PBS IX and filtered using a 0.22 pm Millex-GP Syringe Filter Unit (Millipore, #SLGP033R). Once filtered, the solution was loaded in a 15 mL 100kDa Amicon Ultra Centrifugal Filter (Millipore, UFC9100) and five centrifugation rounds were run at 3700 x g, for 20 min at 4 °C until a final volume of 200-250 pL was reached. Finally, 10 pL aliquots were prepared, snap-frozen in liquid nitrogen and stored at -80°C. All plastic materials and filters were previously equilibrated using decreasing concentrations of PLURONIC F-68 in PBS (0.1%; 0.01%; 0.001% supplemented with 200 mM NaCl).

[0266] Full AAV particles were quantified through quantitative PCR (q-PCR) following “AAV titration by qPCR using SYBR green technology” by Addgene (https: / / www.addgene.org / protocols / aav-titration-qpcrusing-sybr-green-technology / ). Standard curve was obtained using pAAV-CAG-TdTomato plasmid and amplification of the cargo plasmid were obtained by using primers targeting the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).5.15.1.1.2 DBCO functionalized AAV2 mutants

[0267] 8E+10 VG of purified vector mutant was incubated with the identified SortaseA enzyme and ImM of an enzyme reactive bifunctional linker (i.e., nucleophile linker) comprising a crosslinker moiety described below in the identified buffer. In this example, DBCO is used as the crosslinker moiety and various enzyme reactive nucleophiles are explored to produce a DBCO modified AAV2 mutant. Excess enzyme and nucleophile were removed using a lOOKda MWCO centrifugal filter. In some embodiments, capsid protein further comprises a nucleophile linker.

[0268] In some embodiments, the nucleophile linker is selected from GGG-PEG4- DBCO, Amine-PEG4-DBCO, and Amine-DBCO. In some embodiments, the nucleophile is GGG-PEG4-DBCO. In some embodiments, the nucleophile is Amine-PEG4-DBCO. In some embodiments, the nucleophile is Amine-DBCO.5.15.1.1.3 Functionalized WGA

[0269] WGA (0. InMol) was functionalized with a crosslinker reactive moiety, an azide moiety in this example, by reaction with 20-fold molar equivalent of the bifunctionallinker Azide-PEG4-NHS in PBS at pH7.2 for 3 hours at room temperature. Unreacted linker was removed using a lOKDa MWCO centrifugal filter.

[0270] Azide-PEG4-NHS was obtained from Thermo Fisher (Catalogue Number26130):5.15.1.1.4 Crosslinking to produce WGASurface modified AAV2 mutant

[0271] DBCO functionalized AAV2 mutant was then incubated with WGA-PEG4- Azide for one hour at room temperature and overnight at 4 °C. Excess unreacted functionalized ligand was removed by passing through a lOOKDa MWCO centrifugal unit, and the WGA surface modified AAV product was resuspended in PBS.5.15.1.1.5 In Vitro Transduction assay: PC12 cells

[0272] PC12 cells were maintained at 37C in DMEM / F12 medium containing 10% horse serum 5% fetal bovine serum, and 100 U of penicillin / streptomycin. PC12 cells were incubated with WGA modified AAV2 vectors in PBS overnight. Media was then replaced, and cells were maintained at 37C for 5 days labelling with Hoechst and imaging with a Zeiss AxioObserver Al microscope.5.15.1.1.6 Cell maintenance

[0273] PC-12 cells (ATCC®, #CRL-1721TM) were used to verify the transduction efficiency of VR-IV, VR-VIII and VR-IV / VR-VIII AAV2 after capsid modification with WGA through either chemical or chemoenzymatic engineering. PC-12 cells were cultured in PC-12 cell media composed by 50% Dulbecco’s Modified Eagle Medium with GlutaMAX™ supplement (DMEM-GlutaMAX™, Gibco, #31966-047) and 50% DMEM / F12 (Gibco, #11320033) supplemented with 5% fetal bovine serum (FBS, Euroclone, #ECS0180L), 10% horse serum (HS, Gibco, #16050-122), 1% penicillin-streptomycin (pen / strep, Euroclone, #ECB3001D), HEPES IM. Cells were maintained at 37 °C and 5% CO2 in a humidifiedincubator. Each plastic support was coated with poly-L-lysine (lOOpg / mL) (Sigma- Aldrich, #P2636).

[0274] HEK 293T / 17 cells (ATCC®, #CRL-11268) were used to produce rAAVs and verify the transduction efficiency of VR-IV, VR-VIII and VR-IV / VR-VIII AAV2 before capsid modification with WGA through either chemical or chemoenzymatic engineering. HEK 293T / 17 were cultured in DMEM-GlutaMAX™ (Gibco, #31966-047) supplemented with 10% FBS (Euroclone, #ECS0180L) and 1% pen / strep (Euroclone, #ECB3001D). Cells were maintained at 37 °C and 5% CO2 in a humidified incubator.5.15.1.2 Investigated Variables

[0275] Enzyme reactive moiety type (also referred to herein as nucleophile type): ImM of either GGG-Peg4-DBCO, Amine-DBCO, or Amine-Peg4-DBCO was added to the reaction with either AAV2-LPETG- VR-IV or AAV2-LPETG- VR-VIII using SrtA7M.

[0276] GGG-Peg4-DBCO was produced via reaction of a GGGC (SEQ ID NO: 75) peptide with Dibenzocyclooctyne-PEG4-maleimide obtained from Millipore Sigma (product number 760676) to form the succinimidyl thioether conjugate referred to GGG-PEG4-DBCO shown below:

[0277] Buffer composition and sortase type. Experiments were performed with either SrtA7M or SrtA5M.

[0278] The reaction was performed in either 50mM Tris-HCl, 150mM NaCl in the presence or absence of 0.5mM CaCh, or lOOmM ammonium bicarbonate for SrtA7M. Because SrtA5 requires calcium, experiments were performed in either 50mM Tris-HCl, 150mM NaCl, lOmM CaCh or lOOmM ammonium bicarbonate, lOmM CaCh. The double mutant AAV2-LPETG-VR-IV / VR-VIII and ImM amine-DBCO was used for this optimization.

[0279] Sortase concentration'. The reaction was performed with either 5pM or 20pM SrtA7M using the double mutant AAV2-LPETG-VR-IV / VR-VIII and ImM amine-DBCO linker for 16 hours at 37C.

[0280] Temperature and duration of reaction'. The reaction was allowed to proceed for either 2 hours at 4°C, 2 hours at room temperature, 2 hours at 37°C, 16 hours at room temperature, or 16 hours at 37°C, using the double mutant AAV2-LPETG-VR-IV / VR-VIII, ImM amine-DBCO and SrtA7M.

[0281] Clean-up of the reaction. The reaction was either reacted directly with WGA- Azide or washed one or four times with an Amicon lOOKda centrifugal filter, using the double mutant AAV2-LPETG-VR4 / VR8, ImM amine-DBCO and SrtA7M.

[0282] Enzyme production. Sortase A 5M and 7M'. BL21 E. coli were transformed with either SrtA 5M plasmid (Addgene #75144) or SrtA 7M plasmid (Addgene #51141) onto LB-kanamycin plates and incubate at 37°C overnight. One colony from the plate were inoculated into 50 mL of Terrific Broth (TB)-kanamycin medium with IX phosphate buffer (lOx 0.17 M KH2PO4, 0.72 M K2HPO4) and shacked at 220 r.p.m. at 37°C overnight. 50 mL of the overnight culture were diluted into 1 L of TB-kanamycin medium with IX of phosphate buffer and shacked at 220 r.p.m. at 37°C measuring the absorbance at 600 nm every 30 minutes. Reached an absorbance at 600 nm between 0.6-0.8, isopropyl-P-d- thiogalactopyranoside was added at a final concentration of 0.5 mM and the culture incubated for 4h at 30°C. Culture medium was centrifuged at 6,000 g for 15 min at 4°C; the supernatant was discharged and the pellet frozen. The day after, pellet was resuspended with the lysis buffer, incubated for 30 min in ice and sonicated 60% amplitude five times for 30 sec with interval of 30 sec. The suspension was centrifuged for 45 min at 40,000 r.p.m. at 4°C. Two poly-prep column with 2 mL of Ni-NTA agarose resin was packed, washed with 20 mL ofwater to remove the trace of EtOH, and equilibrated with 7.5 mL of washing buffer. The supernatant was loaded into the column and incubated for Ih at RT while rotating. Then, flow-through was collected and four rounds of washing with 3 mL of washing buffer alternate by 3 min of incubation were performed. The same rounds were performed with the elution buffer and collecting the phases. The eluate was washed with PBS in 10 kDa Amicon Ultra Centrifugal Filter (Millipore, UFC9010) to eliminate the imidazole present on the elution buffer. Protein concentration was calculated at the UVVIS at 280 nm, diluted at the desired concentration in PBS and 10% (vol / vol) glycerol, aliquoted, snap frozen in liquid nitrogen and stored at -80°C. The composition of the buffers if presented in Table 3.Table 3. The composition of the lysis buffer, washing buffer, and elution buffer.

[0283] The first experiments were planned to assess whether the rAAVs, produced with the LPET (SEQ ID NO: 2) insertion in the VR-IV, VR-VIII or VR-IV / VR-VIII, were still able to assemble, pack and efficiently transduce. The three different AAVs were produced carrying as cargo a fluorescence reporter either Td-Tomato or Green Fluorescence Protein (GFP). Once produced, quantitative PCR (qPCR) was performed to assess the viral titer (FIG. 3).

[0284] FIG. 3 depicts the results of the qPCR analysis. The total amounts of viral genome (VG) obtained from the production of the three variants were compared to the wildtype one (WT). 4.17E8 HEK 293T / 17 cells were transfected and after three days the viral particles collected. It emerged no significant difference between the WT and VR-IV and VR- VIII, whereas the production of VR-IV / VR-VIII leads to a significant increase of total VG.

[0285] Primers, annealing in the WPRE region of the plasmid, were used in order to evaluate the amount of full particle present in the preparation. Surprisingly, VR-IV / VR-VIII AAV preparation showed a significant increase in the total yield. Indeed, WT and VR-IV AAV productions led to the collection of 1E11-1E12 total VG, whereas VR-IV / VR-VIII one allowed the collection of more than 20 times more, approximately 2E13 on average. Thisincrease in the total VG could be due to a modification of the capsid ability of VR-IV / VR- VIII AAV2. Indeed, it may alter either the ability to successfully pack the cargo plasmid or to an alteration during the virus production. It may also be due to a reduction of HEK293T / 17 infection by secreted AAV during production which leads to a higher accumulation of AAV particles in the cell media.

[0286] Empty AAVs have a mass approximately of 3.7 MDa, whereas full AAVs have one around 4.1 to 5.3 MDa depending on the cargo length. In all the preparations, there was a high level of proteins and DNA fragments corresponding to the peaks in the region below 2 MDa. Notably, VR-IV AAV2 showed the higher packaging capacity, with a percentage of full particle of 61.8%, partial particle 23.9% and empty particle of 14.3%, whereas VR-VIII AAV2 had 18.8% of full particle and VR4 / VR8 42%. It was found that WT AAV2 showed a very low percentage of full particle, around 4%. It is to be declared that VR- IV AAV2 was produced recently to the analysis date (two months before) whereas the other three vectors were produced at least one year before. Thereby, a small variance could be due to the length of storage, although they were all stored at -80°C. Further analysis will be performed with freshly produced affinity purified vectors. Nevertheless, the high percentage of full particle for VR-IV AAV suggested that modification of variable region 4 could lead to an improved efficiency of the packaging ability of AAV.

[0287] To further confirm the correct structure of the novel AAVs, transduction was evaluated via infection of HEK 293T / 17 cells. This cell line is a derivative human cell line which is highly permissive to AAV and should be successfully transduced by AAV2. To assess that, 4E9 VG of vectors were compared to the same amount of WT (FIG. 4). FIG. 4 depicts HEK 293T / 17 transduction. AAV vectors were first tested in HEK 293T / 17 to assess whether the insertion of the sortag motif altered the viral tropism. To verify that, transduction efficiency was compared to the WT AAV one. Whereas VR-IV AAV2 tropism transduction was similar to the WT AAV2 one, VR-VIII and VR-IV / VR-VIII AAV2 weren’t able to transduce HEK293T / 17 cells successfully.

[0288] As expected, one day post infection, WT AAV2 successfully transduced HEK 293T / 17, detectable thanks to the expression of the delivered reporter gene, GFP. At the same level, also VR-IV AAV2 was efficiently able to transduce HEK293T / 17 cells. On the other hand, VR-VIII and VR-IV / VR-VIII AAV2 were not able to transduce HEK 293T / 17 cells. It indicated that modification of variable region VIII, where the critical amino acid residues for the binding to heparan sulphate proteoglycan are, lead to an alteration of the viral tropismwhich, as consequence, did not enable the infection and the resulting expression of the reporter gene. Nevertheless, the inability of these vectors to transduce HEK293T / 17 cells could be considered as an advantage, indeed, the creation of silent vectors could lead to an optimal re-targeting after coupling of a ligand. For this reason, all the three viral vehicles were further testing to verify whether, after capsid engineering, they could acquire a novel tropism.5.15.1.3 Results5.15.1.3.1 Enzyme reactive moiety - Nucleophile type

[0289] While the GGG-Peg4-DBCO, and Amine-Peg4-DBCO nucleophiles offered some improvement over transduction efficiency, particularly with the AAV2-LPETG-VR-IV mutant, the Amine-DBCO nucleophile was clearly superior for both VR-IV and VR-VIII mutant vectors (FIG. 1).5.15.1.3.2 Buffer composition and sortase type

[0290] As shown in FIG. 2, SrtA7 outperformed the SrtA5 mutants in all tested buffers. lOOmM ammonium bicarbonate was found to be a superior buffer for the reaction.5.15.1.3.3 Sortase concentration

[0291] The reaction was more efficient at 20pM SrtA7 compared to 5pM (FIG. 5).5.15.1.3.4 Temperature and duration of reaction

[0292] Reaction efficiency was improved with increasing temperature and time (FIG. 6).5.15.1.3.1 Purification lOOKda centrifugal filters were used to remove excess sortase and amine-DBCO from the reaction. In the absence of clean-up, the AAV2-LPETG-VR-IV / VR-VIII vector was unable to transduce PC 12 cells, presumably because excess amine-DBCO reacted with all freeWGA-Azide. After a single wash, infectivity was restored, and this was improved further by 4 washes (FIG. 7). Increases in infectivity occurred despite a reduction in the physical titer of AAV2-LPETG-VR-IV / VR-VIII of 30% after 1 wash and 41% after 4 washes (as measure by ddPCR) we believe this is due to non-specific binding of the vector to column surfaces.

[0293] In summary, the optimal reaction conditions determined from these are experiments are 20uM of SrtA7M with ImM Amine-DBCO in lOOmM ammonium bicarbonate buffer, at 37°C for 16 hours, followed by 4 washes through a lOOKda centrifugal filter.5.15.2. Example 2. Production Yields of Mutant Capsids5.15.2.1 Background

[0294] Here we summarize production yields for the AAV2-Delta-HSPG, AAV2- LPETG-VR-VIII, and AAV2-LPETG-VR-IV / VR-VIII viruses compared to wildtype AAV2. Data are expressed as viral genomes per producing cell to normalize for different scales of production across preparations.5.15.2.2 Methods5.15.2.2.1 AAV production

[0295] The following recombinant AAV vectors were produced:1. Wildtype AAV2,2. AAV2-Delta-HSPG (harboring mutations at R585A+R588A),3. AAV2-LPETG-VR-VIII (LPET (SEQ ID NO: 2) insertion in the variable region VIII: R585-L586-P587 -E588-T589-G590), and4. AAV2-LPETG-VR-IV / VR-VIII (LPET (SEQ ID NO: 2) insertions in VR-IV and VR- VIII: S452-L453-P454-E455-T456-G453, R589-L590-P591-E592-T593-G594).Recombinant AAV vectors harboring these mutations or insertions and carrying either tdTomato or eGFP under a CAG promoter as a cargo were produced in HEK293 cells as described previously (Grieger JC 2006). Cells were harvested 72 hours post transfection, lysed with Triton X-100 at 0.5%, nuclease treated, concentrated by tangential flow filtration, and purified using isopycnic ultracentrifugation (Florencio DG 2015). Vector genome titration was performed using Q-PCR with primers targeting the promoter region of the viral cargo (Grieger JC 2006). Viral genomes per producing cell for each variant was calculated by normalizing to the number of cells present at transfection.5.15.2.3 Results

[0296] As shown in FIG. 8, all 3 mutant vectors had higher yields than wildtype AAV2 (in VG per cell: WT: 2056, Delta: 12048, VR-VIII: 19277, VR-IV / VR-VIII: 37681), and this correlated with increased silencing capacity of the mutant. Thus, the Delta-HSPG mutant, which still displays moderate transduction of HEK293 cells, had a 6-fold increase in yield, the AAV2-LPETG- VR-VIII mutant, which is more silent, had a 10-fold increase in yield, and the AAV2-LPETG- VR-IV / VR-VIII which is almost completely silent in HEK293 cells had a 20-fold increase in yield compared to the wildtype. These data would suggest that by abrogating natural tropism of the virus, yields can be increased, perhaps as a result of decreased binding to (and infection of) HEK293 cells during production.5.15.3. Example 3. Unexpected Increase in Transduction in VR- VIII Mutants with Short Amine-DBCO Bi-functional Linkers5.15.3.1 Background

[0297] We first optimized reaction conditions and established that enzymatic functionalization of both AAV2-LPETG-VR-IV and AAV2-LPETG- VR-VIII mutants occurred optimally at enzyme concentrations of 20pM and nucleophile concentrations of ImM in lOOmM ammonium bicarbonate (see Example 1). Here, conjugation using different enzyme reactive moieties / nucleophiles (e.g., GGG-PEG4-DBCO, Amine-DBCO and Amine- PEG4-DBCO) was then tested by bioconjugation to WGA-PEG4-Azide and then evaluated by measuring transduction efficiency in PC12 cells. (Glasgow JE, Salit ML, Cochran JR. In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant. Journal of the American Chemical Society 2016; 138:7496-9, incorporated herein by reference in its entirety)5.15.3.1.1 Recombinant AAV2 mutant production

[0298] AAV2-LPETG-VR-IV and AAV2-LPETG- VR-VIII mutants were prepared as described in Example 1.5.15.3.1.2 Enzyme functionalization of Mutant Virions

[0299] Purified AAV2-LPETG-VR-IV or AAV2-LPETG- VR-VIII mutants were incubated for 16 hours at room temperature with 5uM and 20uM concentrations of SrtA7M and ImM an enzyme reactive linker selected from: GGG-PEG4-DBCO, Amine-DBCO andAmine-PEG4-DBCO, combined in either lOOmM tris HCL or lOOmM ammonium bicarbonate buffers, to produce a DBCO functionalized AAV. Excess enzyme and nucleophile were removed using a lOOKda MWCO centrifugal filter.5.15.3.1.1 Crosslinking to Produce WGA Surface Modified AAV2 Mutant

[0300] As described in Example 1.5.15.3.1.2 In Vitro Transduction Assay: PC12 cells

[0301] As described in Example 1.5.15.3.2 Results

[0302] Weak transduction of cells (as evidenced by weak expression of the fluorescent cargo) by unmodified AAV2-LPETG-VR-IV vector (FIG. 1, top row, panel 1). However, transduction was significantly increased upon targeting via enzyme functionalization / conjugation to WGA (FIG. 1, top row, panels 2-4). Surprisingly, the amine DBCO nucleophiles performed far better than oligoglycine DBCO, with the short Amine- DBCO outperforming the longer Amine-PEG4-DBCO (FIG. 1, top row, panel 3).

[0303] In experiments with AAV2-LEPTG-VR-VIII, fewer cells were positive (FIG. 1, bottom row, panel 1) indicating that the LPET (SEQ ID NO: 2) insertion alone essentially completely abrogated HSPG binding. Intriguingly, only amine DBCO nucleophiles increased transduction efficiency of this mutant (FIG. 1, bottom row, panels 2-4), and at levels much lower compared to the mutant with VR-IV insertion alone (FIG. 1, top row). Without being constrained by theory, one possibility is that this may be due to reduced accessibility to functionalization and subsequent modification of the VR-VIII loop compared to VR-IV.5.15.4. Example 4. Reduced Transduction of unmodified VR-VIII Mutants5.15.4.1 Transduction assay

[0304] The aim of these experiments was to investigate whether sortag insertions themselves, without further functionalization, impact the virion functionality (such as transduction). Thus, we evaluated transduction efficiency (via expression of florescent cargo gene) of WGA-conjugated wildtype AAV2, AAV2-LPETG- VR-IV, AAV2-LPETG- VR-VIII and AAV2-LPETG-VR-IV / VR-VIII vectors and tested for transduction of PC12 cells over a range of titers. By using a conjugated ligand (WGA), we reasoned that we would bypass cellattachment through HPSG binding, enabling us to determine whether other functionalities such as internalization, endosomal escape, nuclear trafficking etc. were altered in these vectors.5.15.4.2 Methods

[0305] The following recombinant AAV vectors were obtained or produced as described in Example 1 : Wildtype AAV2, AAV2-LPETG-VR-IV, AAV2-LPETG- VR-VIII, AAV2-LPETG- VR-IV / VR-VIII.

[0306] For chemical surface functionalization, a range of titers of wildtype AAV2, AAV2-LPETG-VR-IV, AAV2-LPETG- VR-VIII or AAV2-LPETG- VR-IV / VR-VIII was combined with DBCO-PEG4-NHS in 20ul PBS pH 7.2 for 3 hours at room temperature. Excess DBCO-PEG4-NHS and byproducts were removed using a lOOKda MWCO centrifugal filter.

[0307] WGA (0. InMol) was reacted with 20-fold molar equivalent of Azide-PEG4- NHS in PBS at pH7.2 for 3 hours at room temperature. Unreacted linker was removed using a lOKDa MWCO centrifugal filter. DBCO chemically functionalized vectors were then incubated with WGA-PEG4-Azide for one hour at room temperature and overnight at 4C.Excess unreacted ligand was removed by passing through a lOOKDa MWCO centrifugal unit, and the conjugated product was resuspended in PBS.5.15.4.3 In Vitro Transduction assay: PC12 cells

[0308] PC12 cells were maintained at 37C in DMEM / F12 medium containing 10% horse serum 5% fetal bovine serum, and 100 U of penicillin / streptomycin. PC12 cells were incubated with WGA modified AAV2 vectors at a range of titers in PBS overnight. Media was then replaced, and cells were maintained at 37C for 5 days labelling with Hoechst and imaging with a Zeiss AxioObserver Al microscope.5.15.4.4 Results

[0309] VR-IV: As shown in FIG. 1, a titer of 1E+9 VG of both WGA conjugated AAV2-LPETG-VR-IV and wildtype AAV2 vector produced equivalent levels of transduction in PC12 cells, while lower titers of WGA-AAV2 were less effective.

[0310] VR-VIII and VR-IV / VR-VIII: In contrast, titers of 1E+10 VG of WGA conjugated AAV2-LPETG- VR-VIII (FIG. 2) and AAV2-LPETG- VR-IV / VR-VIII (FIG. 3)were ineffective at transducing PC 12 cells, and 30-fold higher titers of these mutants were required to reach similar levels to the wildtype vector (3E+11 VG compared to 1E+10VG).

[0311] These data indicate that insertion of the LPET sequence (SEQ ID NO: 2) at VR-IV does not alter the functionality (such as internalization, endosomal escape, nuclear trafficking as mentioned above) of the AAV2-LPETG- VR-IV vector. However, insertion at VR8, renders both the AAV2-LPETG- VR-IV and LPETG-VR-IV / VR-VIII vectors less effective at transduction.5.15.5. Example 5. Reduced Transduction Even at High Titer of VR-VIII Mutants in Permissive HEK2935.15.5.1 Background

[0312] Here we compared the efficiency of AAV2 and the mutant vectors AAV2- Delta-HSPG, AAV2-LPETG- VR-IV, AAV2-LPETG- VR-VIII or AAV2-LPETG-VR- IV / VR-VIII in transducing HEK293 cells, a cell line which is highly permissive to AAV2.5.15.5.2 Methods5.15.5.2.1 AAV production

[0313] The following recombinant AAV vectors were obtained or produced as described in Example 1 : Wildtype AAV2, AAV2-LPETG-VR-IV, AAV2-LPETG- VR-VIII, AAV2-LPETG-VR-IV / VR-VIII.5.15.5.2.2 In Vitro Transduction assay: HEK293 cells

[0314] HEK293 cells were maintained at 37°C in DMEM / F12 medium containing 10% fetal bovine serum, and 100 U of penicillin / streptomycin. Cells were incubated with wildtype and mutant AAV2 vectors at the indicated titer in medium overnight. Medium was then replaced and cells were maintained at 37C for 48 hours before imaging with a Zeiss AxioObserver Al microscope.5.15.5.2.3 Results

[0315] As shown in FIG. 9, HEK293 cells are robustly transduced at similar levels by wildtype AAV2 and AAV2-LPETG- VR-IV at a titer of 1E+10 VG. Transduction is reduced somewhat with the AAV2-Delta-HSPG mutant, and essentially abolished with the AAV2- LPETG- VR-VIII and AAV2-LPETG-VR-IV / VR-VIII mutants at 1E+10VG. However, at 30 times higher titer (3E+11 VG), the AAV2-LPETG- VR-VIII mutant retains some activitywhile the double AAV2-LPETG- VR-IV / VR-VIII mutant remains silent. Thus, insertion of an LPET sequence (SEQ ID NO: 2) into VR-VIII and VR-IV / VR-VIII regions of AAV2 improves upon previous methods to produce a silent AAV2 that relied on mutating R585 and R588 to alanine.5.15.6. Example 6. VR-VIII Mutants Not Efficiently Enzyme Modified5.15.6.1 Background

[0316] Here we demonstrate that insertion of the LPET (SEQ ID NO: 2) to form theLPETG functional sequence motif (SEQ ID NO: 7) (also referred to as a “sortase sequence” herein) into VR-VIII of AAV2 results in AAV vectors which are not efficiently surface functionalized by sortase mediated conjugation. To evaluate whether mutants harboring the VR-VIII mutant can be surface modified, we subjected the VR-VIII mutant to the process of sortagging and WGA conjugation to determine whether the modified mutants capable of transduction are obtained.5.15.6.2 Methods5.15.6.2.1 AAV production

[0317] AAV2-LPETG- VR-VIII recombinant AAV vector was produced as described in Example 1.5.15.6.2.2 Sortagging and coupling of WGA to AAV2-LEPTG- VR-VIII

[0318] 8E+10 VG of purified AAV2-LPETG- VR-VIII was incubated for 24 hours at37C with 20pM heptamutant Sortase A (https : / / www(dot)addgene(dot)org / 51141 / ) and either ImM GGG-PEG4-DBCO, Amine-DBCO or Amine-PEG4-DBCO, in lOOmM ammonium bicarbonate buffer. Excess enzyme and nucleophile were removed using a lOOKda MWCO centrifugal filter. WGA (0. InMol) was reacted with 20-fold molar equivalent of Azide- PEG4-NHS in PBS at pH7.2 for 3 hours at room temperature. Unreacted Azide groups were removed using a lOKDa MWCO centrifugal filter.

[0319] Each DBCO modified AAV2-LEPTG- VR-VIII was then incubated with WGA-PEG4-Azide for one hour at room temperature and overnight at 4C°. Excess unreacted ligand was removed by passing through a lOOKDa MWCO centrifugal unit, and the conjugated product was resuspended in PBS. As a negative control, AAV2-LPETG- VR-VIIIwas also incubated with 20pM Sortase and WGA-Azide, but without the amine containing nucleophile.5.15.6.2.3 In vitro transduction assay in PC12 cells

[0320] PC12 cells were maintained at 37C in DMEM / F12 medium containing 5% horse serum 5% fetal bovine serum, and 100 U of penicillin / streptomycin. PC12 cells were incubated with WGA modified AAV2 vectors in PBS overnight. Media was then replaced, and cells were maintained at 37 °C for 5 days labelling with Hoechst and imaging with a Zeiss AxioObserver Al microscope.5.15.6.3 Results

[0321] Very weak transduction of cells by unmodified AAV2-LPETG-VR-VIII vector was observed (FIG. 10) and control vector treated with sortase (SrtA) and WGA in the absence of nucleophile. Of the nucleophiles tested, only amine DBCO nucleophiles increased transduction efficiency of AAV2-LPETG-VR-VIII, and only at low levels (FIG. 10).Without being bound by theory, on possibility is that the reduced expression of the fluorescent cargo is due to reduced surface accessibility of the functional sequence motif in the VR-VIII loop compared to location in the VR-IV loop.5.15.7. Example 7. Sortase-mediated ligation: the first step to functionalize an AAV vector5.15.7.1 Sortase-mediated ligation of a viral particle to a desired moiety

[0322] Here we compared sortase-mediated modification of AAV2-LPETG- VR-VIII and AAV2-LPETG- VR-IV / VR-VIII with standard NHS-mediated chemical modification of the same vectors. As a readout of modification, we conjugated the ligand Wheat Germ Agglutinin (WGA) and tested for improved transduction of PC12 cells.

[0323] The technique relies on the capability of sortase A to ligate a nucleophile, carrying a dibenzocyclooctyne (DBCO) molecule, to the “sortag” site exposed on the viral structure. As result, DBCO is attached to the capsid structure and can be exploited to perform a click chemistry reaction with an azide linked to a specific ligand. The ligand would retarget the viral vectors in order to transduce specifically cell types expressing the corresponding receptor.

[0324] Several combinations of reaction buffer, time, temperature and concentration of substrates and enzyme were tested. In this series of experiment, VR-IV AAV2 was functionalized with WGA since this lectin is able to bind to N-Acetylglucosamine residues present on cell membranes. PC-12 cells were selected as in vitro system because they are not permissive to unmodified AAV2. In this way, only vectors successfully modified with the chemo-enzymatic method and conjugated to the WGA ligands would have been able to efficiently transduce cells. The success of the strategy was monitored by the expression of the reporter gene delivered by the VR-IV AAV2 particles.5.15.7.2 Methods

[0325] An LPET sequence (SEQ ID NO: 2) was inserted into the coding sequence of AAV2 at variable region 8 between R585 and G586 (VR-VIII, sequence: R585-L586-P587- E588-T589-G590) to generate AAV2-LPETG- VR-VIII, and at variable region IV between S452 and G453 (VR-IV, sequence: S452-L453-P454-E455-T456-G453) to generate the double mutant AAV2-LPETG-VR-IV / VR-VIII. Recombinant AAV2 vectors harboring these insertions and carrying either tdTomato or eGFP under a CAG promoter as a cargo were produced in HEK293 cells as described previously (Grieger JC 2006). Cells were harvested 5 days post transfection, lysed with Triton X-100 at 0.5%, nuclease treated, concentrated by tangential flow filtration, and purified using isopycnic ultracentrifugation (Florencio 2015). Vector genome titration was performed using Q-PCR with primers targeting the promoter region of the viral cargo (Grieger JC 2006).5.15.7.2.1 Sortagging, NHS-mediated chemical modification, and coupling of WGA to AAV2-LEPTG- VR-VIII

[0326] For sortag-mediated modification, 8E+10 VG of purified A A V2 -LPET G- VR-VIII or AAV2-LPETG-VR-IV / VR-VIII was incubated for 24 hours at 37C with 20pM heptamutant Sortase A (https: / / www(dot)addgene(dot)org / 51141 / ) and either ImM GGG- PEG4-DBCO, Amine-DBCO or Amine-PEG4-DBCO, in lOOmM ammonium bicarbonate buffer. Excess enzyme and nucleophile were removed using a lOOKda MWCO centrifugal filter.

[0327] For chemical modification, 8E+10 VG of purified AAV2-LPETG- VR-VIII or AAV2-LPETG-VR-IV / VR-VIII was reacted with 0.42pMol DBCO-PEG4-NHS in 20ul PBS pH7.2 for 3 hours at room temperature. Excess DBCO-PEG4-NHS and byproducts were removed using a lOOKda MWCO centrifugal filter.

[0328] WGA (0. InMol) was reacted with 20-fold molar equivalent of Azide-PEG4- NHS in PBS at pH7.2 for 3 hours at room temperature. Unreacted Azide groups were removed using a lOKDa MWCO centrifugal filter. Sortase or NHS-mediated DBCO modified AAV2-LEPTG-VR-VIII or AAV2-LPETG-VR-IV / VR-VIII was then incubated with WGA-PEG4- Azide for one hour at room temperature and overnight at 4C. Excess unreacted ligand was removed by passing through a lOOKDa MWCO centrifugal unit, and the conjugated product was resuspended in PBS.5.15.7.2.2 In vitro application to PC12 cells

[0329] PC12 cells were maintained at 37C in DMEM / F12 medium containing 5% horse serum 5% fetal bovine serum, and 100 U of penicillin / streptomycin. PC12 cells were incubated with WGA modified AAV2 vectors in PBS overnight. Media was then replaced, and cells were maintained at 37C for 5 days labelling with Hoechst and imaging with a Zeiss AxioObserver Al microscope.5.15.7.3 Results

[0330] The first tested parameter was the concentration of substrate (nucleophile) and enzyme (SrtA 7M) (FIG. 11-A). Because the sortase-mediated ligation is a reversible enzymatic reaction, a high concentration of nucleophile has to be used in order for the reaction to produce the final products.

[0331] Three different combinations were tested to engineer VR4 AAV2: “Low concentration” was composed by 100 uM nucleophile and 7 uM SrtA 7M; “Medium concentration” condition was 500 uM nucleophile and 15 uM SrtA 7M; “High concentration” formed by 1 mM nucleophile and 20 uM SrtA 7M. The same amount of unmodified VR-IV was used as control. Starting from the “Low concentration” condition, there was an increased number of positive cells compared to the control, but as expected, the best result was achieved using the “high concentration” condition, where the highest number of positive cells was reached (FIG. 12 - A). FIG. 12 - A depicts quantification of Td-Tomato positive cells for the different concentration conditions. The results highlighted the “High concentration” was the best condition. Therefore, these concentrations of sortase and nucleophile were kept while testing the other parameters.

[0332] The native nucleophile in the sortase-mediated ligation is a poly-glycine chain, composed by at least three glycines. To verify which one was the best nucleophile in thecoupling with the AAV particles, three different compounds were tested: GGG-peg4-DBCO; Amine-peg4-DBCO; Amine-DBCO (FIG. 11 - B). Surprisingly, Amine-DBCO led to a higher number of positive cells, showing that, in our in vitro assay, it could be conjugated in a more efficient way than the natural substrate. Furthermore, it suggested that the polyethylene glycol units negatively interfere with the enzymatic reaction since its absence in the Amine- DBCO condition compared to the Amine-peg4-DBC0 increase the percentage of positive cells (FIG. 12 - B). FIG. 12 - B depicts quantification of Td-Tomato positive cells for the different nucleophile conditions. The results highlighted the “Amine-DBCO” was the best condition. It is to keep in consideration that GGG-peg4-DBCO was produced in the laboratory by a click-chemistry reaction between the oligopeptide GGGC (SEQ ID NO: 75) and Malamide-peg4-DBC0, and no analyses were conducted to assess the purity of the reaction product.

[0333] Another important parameter to consider is the reaction buffer. Indeed, Sa- SrtA and SrtA 5M are calcium-dependent enzymes, whereas the more recent version, SrtA 7M, is calcium-independent. Different buffers are used in several protocols, therefore, to assess which was the most suitable one for AAV modification, the three most used buffers in literature were tested: Buffer 1 composed by 50 mM ammonium carbonate; Buffer 2 formed by 50 mM Tris-HCl, 150 mM NaCl; Buffer 3 composed of 50 mM Tris-HCl, 150 mM NaCl and 0.5 mM CaCh (FIG. 11 - C). Three buffers were tested to optimize the protocol. In particular, Buffer 1 composed by 50 mM ammonium carbonate; Buffer 2 formed by 50 mM Tris-HCl, 150 mM NaCl; Buffer 3 composed by 50 mM Tris-HCl, 150 mM NaCl and 0.5 mM CaCh. Notably, the transduction level was similar in each condition and buffer 1 was arbitrary chosen for future experiments. FIG. 12 - C depicts quantification of Td-Tomato positive cells for the different buffer conditions. No significant differences were observed among the three buffers (FIG. 12 - C); therefore, the choice of the buffer was done arbitrary and following experiments were all conducted in Buffer 1.

[0334] Another variable which could interfere with the enzymatic reaction is the temperature at which the reactions is performed. Also in this case, it is a parameter which has to be tested empirically and which can be critical for the enzyme performances. Three different temperatures were chosen: 4°C; 21°C (room temperature); 37°C (FIG. 11 - D). At higher temperature, the AAV vectors were able to transduce a greater number of PC-12 cells. In each condition, the enzymatic reaction was performed for a duration of 16 to 18 hours. At 4°C there were a minimal number of positive cells, so this condition was immediatelydiscarded. FIG. 12 - D depicts quantification of Td-Tomato positive cells for the different temperature conditions. The highest number of positive cells were observed when the sortasemediate ligation was performed at 37°C (FIG. 12 - D), thereby this temperature was chosen as the optimal one to conduct the sortase-mediated ligation.

[0335] Several other variants were produced from the wild-type SrtA in order to improve the catalytic efficiency of the enzyme or to be calcium-independent. To assess whether SrtA 7M was the most favourable sortase variant to use in AAV engineering, it was compared with the SrtA 5M (FIG. 11 - E). FIG. 11 - E depicts the comparison between the catalytic activity of SrtA 7M and SrtA 5M. The experimental result suggested the SrtA 7M were more prone to modifying the AAV particles. SrtA 7M carries the same five mutation of the 5M plus two more which avoid the needs of calcium to perform the sortagging.Therefore, the enzymatic activity of the SrtA 5M and SrtA 7M should be the same respectively in presence and absence of calcium. Both of them were produced in the laboratory following the same protocol and visualized through SDS-PAGE to confirm the dimension. Once empirically compared, SrtA 7M in 50 mM ammonium bicarbonate were more efficient in the sortase-mediated ligation than SrtA 5M in 50 mM ammonium bicarbonate, 10 mM CaCh (FIG. 12 - E). FIG. 12 - E depicts quantification of Td-Tomato positive cells for the different sortase conditions. The results highlighted there was a higher number of Td-Tomato positive cells when SrtA 7M was used. SrtA 7M was chosen for further investigation.

[0336] In conclusion, this series of experiments led to the design of an optimal protocol to ligate AAV particles to a moiety of interest. Summarizing, AAV vectors, carrying the LPETG motif (SEQ ID NO: 7), were reacted with 20 uM SrtA 7M and 1 mM Amine- DBCO in 50 mM ammonium bicarbonate for 18h at 37°C. Then, the excess of amine-DBCO was removed to avoid that the unbound DBCO competed with the azide-ligand.

[0337] The sortase-mediated ligation is the “first step” of the chemogenetic technology developed in this thesis and it delineates the success of the capsid engineering. Therefore, it was essential to verify all the possible variables in order to achieve the highest modification efficiency.

[0338] One of the main limitations of the sortase-mediated ligation is the fact that a high excess of nucleophile is needed in order to move the reaction towards the final products. This excess of Amine-DBCO has to be removed from the viral preparation, otherwise it willcompete for the binding with the Azide-ligand, as shown in FIG. 13. FIG. 13 depicts viral purification after Amine-DBCO sortase-mediated ligation. Once performed the sortase- mediated ligation, the unbound nucleophile Amine-DBCO, has to be removed in order for the click-chemistry reaction to occur. Indeed, when it is not removed (No Amicon Ultra Centrifuge filter sample), the unbound nucleophile competes with the bound Amine-DBCO to react with the Azide-ligand. As result, there was no Td-Tomato signal in PC-12 cells after five days of infection. In contrast, when the excess of unbound Amine- DBCO is washed away (Amicon Ultra Centrifugal filter sample), the hybrid approach allows a correct conjugation between viral preparation and azide-ligand, leading to a successful transduction. Scale bar 50 pm.

[0339] In this experiment, after having performed the sortase mediated ligation, viral samples were either run or not through a 0.5 mL 100 kDa Amicon Ultra Centrifugal Filter in order to remove the unbound Amine-DBCO. As shown, when sample did not go through the filter, the excess of Amine-DBCO competed for the binding with Azide-peg4-WGA and did not lead to an efficient coupling. As consequence, there were no positive cells for Td-Tomato signal.

[0340] On the other side, when viral preparation run through the 100 kDa Amicon Ultra Centrifugal Filter, the unbound Amine-DBCO, having a weight smaller than 100 kDa, was presumably removed and conjugation with Azide-peg4-WGA enabled to successfully transduce PC-12 cells.

[0341] In order to accelerate the procedure and having a viral vector “ready -to-use”, the entire viral preparation underwent bulk sortase-mediated ligation immediately after iodixanol centrifugation. At this point the sample was purified to remove the excess of unbound Amine-DBCO and subsequently ali quoted, snap-frozen and store at -80°C. To delete the unbound nucleophile, two different methods were performed to assess which one was the most suitable for our application. In the first case, the entire viral preparation coupled to the Amine-DBCO was run through a 15 mL 100 kDa Amicon Ultra Centrifugal Filter, the same one used to remove the excess of iodixanol. Thereby, while performing the buffer exchange of the sample, the excess nucleophile was also removed. In the second case,

[0342] HPLC was used with a size-exclusion resin. Indeed, the size-exclusion resin should enable one to remove all the contaminants which are not bound to the viral vectors. In particular, Capto Core 400 (Cytiva) is a resin designed for intermediate purification andpolishing of viruses. As with the Amicon Ultra Centrifugal Filter, the entire viral preparation was sortagged with Amine-DBCO, then it was purified via Capto Core 400 HPLC. At that point the flow-through was collected, concentrated and aliquoted.

[0343] The two methods were compared to identify which one allowed to collect a higher amount of purified AAV vectors. FIG. 14 - A depicts the purification of viral prep through Amicon Ultra Centrifugal Filter. FIGs. 14 - B depicts the purification of viral prep through Capto Core 400. The comparison was performed analysing the total VG collected (FIG. 14 - A) and evaluating the transduction level (FIG. 14 - B). Once modified with Azide-PEG4-WGA and added at PC-12 cells, the same concentration of AAV purified with the two different methods led to a slightly different transduction level. In particular, purifying the vector with the HPLC method led to more loss of AAV compared to the purification via Amicon Ultra Centrifugal Filter.

[0344] Indeed, Amicon enabled the collection of two times more of AAV, approximately 8E11 total VG, compared to 4.5 VG collected with the HPLC method. Furthermore, comparing the same amount of engineered VR4 AAV, it appeared that there was a higher transduction for the sample purified via Amicon filter than the one via HPLC, suggesting either a better removal of the excess of Amine-DBCO when using the Amicon filter or that the ligation of Amine-DBCO to the AAV vector could have interfered with the efficiency of the Capto Core 400 resin during the purification process. From these experiments, we decided to purify future viral preparation with the Amicon Ultra Centrifugal Filter, although further investigation will be taken to optimize the purification via HPLC.

[0345] For the AAV2-LPETG-VR-IV vector, we observed efficient transduction of PC12 cells upon chemical modification using NHS-PEG4-Azide, but only minimal transduction by the sortase modified vector (FIG. 15). In contrast, both chemical and sortase modified AAV2-LPETG-VR-IV / VR-VIII double mutant vector transduced PC12 cells efficiently (FIG. 15). This would indicate that the VR-VIII loop is not sufficiently exposed to allow for adequate modification by sortase, but that inclusion of the LPETG sequence (SEQ ID NO: 7) in the VR-IV loop increases access to allow for a similar efficiency of transduction to that observed with chemical modification.5.15.8. Example 8. Comparison between unconjugated (control) and conjugated (chemo-enzymatic approach) vectors (VR- IV or VR-VIII) to azide-tagged WGA ligand in PC12 cells5.15.8.1 Conjugation between AAV capsid proteins

[0346] Here we demonstrate that sortase-mediated ligation enables the conjugation between the AAV capsid proteins, carrying the sorting motif and a DBCO molecule, which is then exploited to attach an azide-tagged ligand.

[0347] The chemogenetic approach was compared to a chemical technology. The efficiency was valuated depending on the number of transduced cells. The sorting motif, recognized by SrtA, was cloned into the VP sequence. Then, the “sortagged” AAVs (VR-IV AAV2 and VR-VIII AAV2) carrying the motif are conjugated to a molecule of interest that redirect viral vector to target cells.5.15.8.2 Methods5.15.8.2.1 Molecular cloningCloning pAA V2 / 2 LPET (SEQ ID NO: 2) VR-IV, VR-VIII and VR-IV / VR-VIII

[0348] Plasmid pAAV2 / 2 (Addgene #104963) was used as parental plasmid for the creation of pAAV2 / 2 VR-IV, pAAV2 / 2 VR-VIII, pAAV2 / 2 VR-IV / VR-VIII. Circular PCR was performed using the following primers:

[0349] Primers were designed to have the LPET encoding sequence (SEQ ID NO: 2) (capital letter) and homology arms on the parental plasmid. PCR was performed incubating 150 ng of parental plasmid, 3 uM forward and reverse primer, 5% DMSO, Phusion High- Fidelity PCR Master Mix (ThermoFisher Scientific, #F530L). The PCR mix was incubated with Dpnl (NEB, #R0176S) that specifically recognize methylated site on parental vector.The mix was then purified with the kit NucleoSpin PCR and Cleanup (Macherey -Nagel, #FC140609N) according to manufacturer’s instruction, and plasmid concentration is measured at the Nanodrop Spectrophotometer. One Shot™ Stbl3™ chemically competent E. coli (ThermoFisher Scientific, #C737303) were transformed with the final plasmid onto LB- ampicillin plates at 37°C overnight. Five colonies were separately inoculated in 3 mL of LB- ampicillin medium at 37°C 150 r.p.m. overnight. Plasmid DNA was isolated, purified with the kit QIAprep Spin Miniprep Kit (Qiagen #27104) and sequenced via Sanger analysis by Eurofins Genomic, using the following primers, to verify the success of the cloning strategy.

[0350] Positive construct was amplified and isolated through MaxiPrep (Macherey- Nagel, FC140414M) according to manufacturer’s instruction.5.15.8.3 Results

[0351] Once having verified the chemogenetic technology was effectively able to engineer AAV by coupling viral particles to a ligand and boosting the viral transduction, the strategy was compared to another technique based on chemical engineering of viral capsid, and, from now on, referred as chemical modification. This technology is also based on coupling AAV particles to a ligand of interest to retarget the viral tropism, but it differs in the “first step”. Indeed, while the chemogenetic approach is based on the binding of Amine- DBCO to the capsid surface via sortase activity, the chemical modification relies on the exposure of DBCO via reaction between N-hydroxysuccinimide (NHS) ester (carrying the DBCO molecule) and the unprotonated amine group of exposed lysine residues on the capsid surface. As a result, in both technologies DBCO is exposed superficially and the azide-ligand can be used to re-target the AAV particles or boost the transduction.

[0352] The three variants (VR-IV; VR-VIII; VR-IV / VR-VIII) were engineered either with the hybrid or the chemical approach in order to compare the two technologies and verify whether the hybrid strategy was as efficient as the chemical one.

[0353] In the case of VR-IV AAV2 (FIG. 16 - A), 8E9 VG were modified via either the hybrid or the chemical approach whereas 8E9 VG of unmodified viral vectors were used as control. PC-12 cells were used as an in vitro system in order to verify the transductionefficiency of the engineered vehicles. Five days post infection, reporter fluorescence was recorded, and percentage of positive cells were calculated.

[0354] The graph in FIG. 16 - A shows the percentage of reporter fluorescence positive cells over the total number of cells stained with the Hoechst staining. As expected, in the control condition (unmodified VR-IV AAV2) a small number of cells became positive for the reporter transgene. Notably, VR-IV AAV2 modified with the hybrid approach showed a transduction similar to the one obtained with the chemical strategy, underlying that the chemogenetic technology is a valid method to engineer AAV vectors.

[0355] For what concern VR-VIII AAV2 (FIG. 16 - B), 2E10 VG were modified via either the hybrid or the chemical approach whereas 2E10 VG of unmodified viral vectors were used as control. In the control condition (unmodified VR-VIII AAV2), there were not any positive cells for the reporter gene.

[0356] Surprisingly, it was shown that VR-VIII modified via the hybrid approach transduced a significant higher percentage of positive cells than the one transduced via the chemical approach. This suggested that depending on the insertion site of the sortag motif, the hybrid approach could boost more efficiently the transduction of PC-12 cells compared to the chemical strategy.

[0357] Lastly, 8E10 VG of VR-IV / VR-VIII AAV2 (FIG. 16 - C) were modified via either the hybrid or the chemical approach, while 8E10 VG of unmodified viral vectors were used as control. As for VR-VIII, in the control condition (unmodified VR-IV / VR-VIII AAV2) there were not any positive cells for the reporter gene. Notably, also for this vector the hybrid approach showed a transduction similar to the one obtained with the chemical strategy.

[0358] Among the three “sortagged” AAV2 the most promising seemed to be the VR- IV variants. Indeed, although all of them reached a percentage of positive cells around 55- 60%, an increase titre of VR-VIII and VR-IV / VR-VIII was needed to obtain that.Nevertheless, the three variants were further analyzed before choosing which one to test in vivo.

[0359] VR-IV AAV2 particles were modified via either hybrid or chemical approach. In both cases, approximately 60% of cells resulted positive for the reporter gene. One-way Anova, p<0.0001; Tukey’s post-test; WT vs hybrid approach p<0.0001; WT vs chemicalapproach p<0.0001; n=3 B. VR-IV AAV2 particles were modified via either hybrid or chemical approach.

[0360] AAV engineered via hybrid approach transduce a significant higher number of cells compared to the AAV modified via the chemical approach. One-way Anova, p<0.0001; Tukey’s post-test; WT vs hybrid approach p<0.0001; WT vs chemical approach p<0.0001; hybrid vs chemical p=0.0032; n=3 C. VR-IV / VR-VIII AAV2 particles were modified via either hybrid or chemical approach. In this case, there is not a significance difference between the two strategies. One-way Anova, p<0.001; Tukey’s post-test; WT vs hybrid approach p=0.0002; WT vs chemical approach p=0.0006; n=3. Scale bar 50 um.5.15.9. Example 9. VR-IV AAV2 transduction in ex vivo system5.15.9.1 Increase in transduction in DRG primary culture as a result of chemo-enzymatic engineering of VR-IV AAV2

[0361] Here we demonstrate that chemo-enzymatic engineering of VR-IV AAV2 with azide-tagged WGA boosted viral transduction in DRG primary culture.

[0362] To assess whether the chemoenzymatic approach was successfully able to retarget VR-IV AAV2 in a more complex system than PC 12 cell line, VR-IV AAV2, coupled to WGA ligand, was tested in ex vivo primary dorsal root ganglion (DRG) neuronal culture. Notably, unmodified VR-IV AAV2 transduced a low number of fibroblast and neurons, whereas VR-IV AAV2 conjugated to WGA via chemoenzymatic strategy successfully transduced DRG neurons (FIG. 17).5.15.9.2 Methods

[0363] VR-IV AAV2 was modified via chemoenzymatic approach in order to couple a ligand of interest, WGA, to the viral capsid. The modification improves the transduction efficiency of the vector compared to the control, consisting of unmodified VR-IV AAV2.5.15.9.2.1 Primary cell culture of DRG neurons

[0364] DRG neurons extracted from adult C57 / B16 mice and collected in sucrose- based artificial cerebrospinal fluid (ACSF; 234 mM Sucrose, 2.5 mM KC1, 0.5 mM CaCh, 10 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCCh, 11 mM Glucose). DRG neurons were enzymatically digested in collagenase type IV (lOng / pL, Sigma-Aldrich #C5138) for 26minutes at 37°C, followed by centrifugation at 700g for 3 minutes and remotion of the supernatant. The pellet was then resuspended and incubated with 0.05% Trypsin EDTA (Gibco, #25300-054), for 26 minutes at 37°C and mechanically isolated by repeated pipeting. Cell debris was removed by passing cell suspension through a 100pm filter (BD™, #340632). The cell suspension was pelleted and resuspended in DMEM-GlutaMAX™ (Gibco, #31966- 047) supplemented with 10% FBS (Euroclone, #ECS0180L) and 1% pen / strep (Euroclone, ECB3001D) and plated in 35mm glass-bottom (MatTek) dish coated with poly-L-lysine (lOOpg / mL) and 2% Matrigel (SIAL, #356231). Cells were maintained at 37 °C and 5% CO2 in a humidified incubator.5.15.9.3 Results

[0365] VR-IV AAV2 was modified via chemoenzymatic approach in order to couple WGA to the viral capsid. The modification improves the transduction efficiency of the vector compared to the control, consisting of unmodified VR-IV AAV2. The chemoenzymatic approach can efficiently boost the viral transduction by attaching a ligand of interest.Furthermore, the ligand maintains its ability to bind specifically to the complementing receptor present on cell surface.5.15.10. Example 10. VR-IV AAV2 transduction in an in vivo system5.15.10.1 Targeting viral tropism in vivo in mice

[0366] Here, we demonstrate that AAV conjugation with azide-tagged NGF re-target the sortagged VR-IV AAV2 towards TrkA / p75+ neurons (68.4%) in vivo.

[0367] In vitro and ex vivo characterization of VR-IV proved the chemoenzymatic approach was successfully able to engineer AAV particles. Therefore, the next step was to understand whether the technology and the “sortagged” AAV2 could be used to re-target viral tropism in vivo in mice.5.15.10.2 Methods5.15.10.2.1 Subcutaneous administration of AAV2

[0368] 5E10 VG (1X), 1E11 VG (2X), 1.5E11 VG (3X) of VR-IV AAV2 were injected subcutaneously in the hind paw of adult mice. Engineered VR-IV particles with NGFR121W were successfully able to infect DRG neurons, especially those present in DRG L4. Among the three different concentrations, IX, 2X, 3X, there was no difference in thetransduction level. Unmodified VR-IV AAV have different behavior depending on the dose. Indeed, IX condition is not sufficient to transduce DRG neurons, whereas 2X and 3X led to the infection of several DRG neurons.5.15.10.3 Results

[0369] Adult mice were injected subcutaneously in the hind paw with VR-IV AAV2 coupled to the Nerve Growth Factor (NGFR121W) ligand via the hybrid approach. Once injected subcutaneously, NGFR121W ligand should re-direct AAV tropism to peptidergic neurons, which express its corresponding receptor, the tropomyosin receptor kinase A (TrkA) with p75. The binding between NGFR121W and TrkA / p75 allows the internalization of the viral particles which is transported to the soma and the transgene cassette, in our case composed of Td-Tomato fluorescence reporter, is transcribed. This process leads to transduction of lumbar DRG neurons (L3, L4, L5) ipsilateral to the injection site.

[0370] Three different concentrations of either unmodified or modified virus were tested to verify whether also in vivo a higher amount of VR-IV AAV2 compared to WT AAV2 was needed. In details, normally used concentration (5E10 VG; IX), two-fold (1E11 VG; 2X) and threefold (1.5E11 VG; 3X) more than the one used for subcutaneous injection was administered for VR-IV AAV2. When modified with NGFR121W, VR-IV AAV2 at IX, 2X and 3X gave similar results (FIG. 18-A): a high percentage of positive neurons in L3, L4 and L5 became positive for Td-Tomato reporter.

[0371] The transduction included both soma and projection. On the other side, in the control condition (FIG. 18-B), unmodified VR-IV AAV2 was not able to transduce DRG neurons when administered at lower dose (IX), but some positive neurons were present in 2X and 3X conditions.

[0372] This implied that unmodified AAV retains its ability to transduce DRG neurons, however, its infectivity is dose-dependent, and, reducing the viral concentration, it is possible to avoid non-target cells and have a specific delivery.

[0373] AAV conjugation with azide-tagged NGF re-target the "sortagged" VR-IV AAV2 towards TrkA / p75+ neurons (68.4%) in vivo.5.15.11. Example 11. Whole-mount DRG deriving from animal injected with AAV2 vectors either unmodified (control group) or modified with NGF (NGF group)5.15.11.1 Chemogenetic approach for redirecting the viral vectors to cells

[0374] Here we demonstrate that the chemogenetic approach allows to redirect the viral vector to cells expressing the complementary receptor of the ligand of interest, with around 68-76% of transduction in target cells.

[0375] To verify whether positive DRG neurons correspond to peptidergic one, and therefore assess whether the exposed NGFR121W on the capsid surface re-directs the viral tropism specifically to peptidergic neurons, an immunostaining assay was performed in whole-mount DRGs deriving from the injected adult mice.5.15.11.2 Methods5.15.11.2.1 General protocol

[0376] Whole-mount DRGs derived from injected adult mice were stained for TrkA receptor (green) to assess whether NGFR121W exposed on the viral capsid was able to redirect AAV tropism to peptidergic neuros expressing TrkA.

[0377] 5E10 VG of either modified (NGF) or unmodified (Control) VR4 AAV2 was injected intraplanatarly. Three-week post infection DRGs were collected, and immunofluorescence assay performed. Quantification of the staining was performed. In the NGF condition, 68.4% of Td-Tomato positive neurons resulted also TrkA positive, whereas in the CTR condition, since there was not Td-tomato positive cells, there was no colocalization with TrkA positive neurons, suggesting the efficiency of the modification in targeting neurons expressing TrkA receptor. 16.1% of the TrkA+ neurons were Td-tomato positive in the experimental condition, whereas none of the TrkA positive neurons express Td-tomato in the control one.

[0378] 1E11 VG of either modified (NGF) or unmodified (Control) VR4 AAV2 was injected intraplanatarly. Three-week post infection DRGs were collected and immunofluorescence assay performed. A difference between control and NGF condition was detectable. Indeed, in the NGF condition, 71.6% of Td-Tomato positive neurons resulted also TrkA positive, whereas in the control condition only 28.4% of Td-Tomato positive neurons colocalized with TrkA staining, suggesting that when not modified AAVs tropism is morevariable. Also, at this viral dose, 16.3% of the TrkA+ neurons were Td-tomato positive in the experimental condition, whereas 0.6% of the TrkA positive neurons express Td-tomato in the control one.

[0379] 1.5E11 VG of either modified (NGF) or unmodified (Control) VR-IV AAV was injected intrapl anatarly. Three-week post infection DRGs were collected, and immunofluorescence assay performed. As for the previous condition, a difference between control and NGF condition was detectable even though the in the control Td-Tomato positive neurons colocalizing with TrkA staining increased to 55.2% versus the 77.8% for the modified vector. On the other side, the percentage of TrkA+ neurons which were also expressing Td-Tomato remain similar to the previous conditions, with 10.6% and 1.1% respectively for experimental and control conditions.5.15.11.2.2 Immunofluorescence assay

[0380] Immunofluorescence was performed on tissues dissected from injected animals. Spinal cords, liver and skin were fixed with 4% paraformaldehyde (PF A) overnight at 4°C, while DRG were fixed in 2% PFA overnight at 4°C. After fixation and 4 rounds of PBS IX washes, spinal cords, liver and skin were incubated in 30% sucrose solution at 4°C overnight, whereas DRG in ScalA2 solution (4 M urea, 10% glycerol, 0.1% Triton X-100) at 4°C overnight.

[0381] Tissue sections were embedded in Killik O.C.T. Compound (Bio-optica, #059801) and cut into 30 pm slices through a cryostat. Slices of tissue were then blocked with 0.3% Triton X-100 and 2% serum (generally from the specie on which the secondary antibodies were harvested) in PBS IX for at least one hour for the tissue slice. After that, primaries antibodies were diluted in the blocking solution and kept overnight at 4°C on slices, placed in a moist chamber. The day after, extensive rinses with 0.3% Triton X-100 in PBS were performed and samples were then incubated with secondary antibodies for 2 hours at room temperature for slice. Again, samples were extensive rinses with 0.3% Triton X-100 in PBS, mounted on glass slides with ProLong Diamond Antifade Mountant (ThermoFisher, #P36970) and imaged using a Nikon AIR confocal microscope.Whole-DRG DRG were processed in a whole-mount approach. DRG were incubated for 24 hours in the blocking solution, composed by 0.3% Triton X-100 and 2% serum (generally from the specie on which the secondary antibodies were harvested) in PBS IX at 4°C. Theday after, DRG were incubated with primary antibodies diluted in blocking solution for 72 hours at 4°C. After extensive rinses with 0.3% Triton X-100 in PBS, DRG were incubated with secondary antibodies diluted in blocking solution for 72 hours at 4°C. DRG were extensively rinsed with 0.3% Triton X-100 in PBS, mounted on glass slides with ProLong Diamond Antifade Mountant (ThermoFisher, #P36970) and imaged using a Nikon AIR confocal microscope.5.15.11.2.3 Image analysis and statistics

[0382] Images were acquired at the Nikon AIR confocal microscope system with different objective depending on the purpose of the experiment (lOx, 20x or 60x). Quantification of positive cells for the reporter was performed via Volocity® software. Images underwent immunofluorescence assay were visualized via Image J.

[0383] Colocalization quantification was performed using Just Another Colocalization Plug in (JaoCP) in Image J. Values were representative of the Mender’s Coefficient which give the percentage of the fluorphore #1 overlapping with flurophore #2 and vice versa. Graphs and statistical analysis were obtained with GraphPad Prism (version 9.5.1). Samples having a normal distribution were analysed through one-way Anova. Values are presented as mean ± standard error of the mean (s.e.m). Error bars in graph represent the s.e.m. unless stated otherwise. * p<0.05; ** p<0.005; *** p<0.001; **** p<0.0001.5.15.11.3 Results

[0384] Colocalization of Td-Tomato signal and anti-TrkA staining was investigated.

[0385] FIG. 19 - A depicts the confocal acquisition of whole-mount DRGs from VR- IV AAV2 injected animal and colocalization quantification of Td-tomato and TrkA positive neurons at 5E10 VG of either modified (NGF) or unmodified (Control) VR-IV AAV2 injected intraplanatarly. In IX VR-IV AAV2 (FIG. 19 - A), Td-Tomato signal was present only in DRG neurons infected with modified AAV, whereas none of the neurons expressed Td-Tomato reporter in the control condition. Colocalization quantification of lumbar DRGs showed that, on average, 68.4% of neurons expressing Td-tomato were positive for anti-TrkA staining in the experimental condition. Since there were no Tdtomato positive neurons in the control, the quantification of this group resulted in 0% (FIG. 19 - A). To understand how much the capsid engineering boosts the viral transduction, it was also quanfied how many TrkA+ neurons were transduced (FIG. 19 - A). It emerged that of the total of TrkA+ neurons, 16.1% were infected by the AAV2.

[0386] FIG. 19 - B depicts the confocal acquisition of whole-mount DRGs from VR- IV AAV2 injected animal and colocalization quantification of Td-tomato and TrkA positive neurons for 1E11 VG of either modified (NGF) or unmodified (Control) VR-IV AAV2 injected intraplanatarly. Doubling the viral dose, 2X VR-IV AAV2 (FIG. 19 - B) increased the percentage of neurons positive for Td-tomato and TrkA neurons to 71.6% in the experimental condition and to 28.4% in the control condition (FIG. 19 - B). It was found that the higher dose led to a higher efficiency of the unmodified viral vector but not of the modified one.

[0387] FIG. 19 - C depicts the confocal acquisition of whole-mount DRGs from VR- IV AAV2 injected animal and colocalization quantification of Td-tomato and TrkA positive neurons for 1.5E11 VG of either modified (NGF) or unmodified (Control) VR-IV AAV injected intraplanatarly. The same results were obtained when the viral concentration was tripled (3X VR-IV AAV2) (FIG. 19 - C). Indeed, also in this case, the percentage of Td- tomato expressing neurons that were also positive for TrkA staining increased approximately 6%, reaching a final value of 77.8% in the experimental condition. In the control condition, it reached 55.2%.

[0388] Considering TrkA+ neurons that were also positive for Td-Tomato, the percentage remains similar across the conditions. Indeed, independently on the viral dose, the value was around 10-16% for the experimental group and 0-1% for the control one.

[0389] All together, these results suggest that the chemogenetic approach allows one to redirect the viral vector to cells expressing the complementary receptor of the ligand of interest, with around 68-76% of transduction in target cells. Furthermore, approximately 10- 16% of TrkA+ neurons were efficiently transduced by VR-IV AAV2. This percentage likely corresponds to the percentage of neurons innervating the injection site in the murine hind paw. Increasing the concentration of the vector can help reaching more cells of interested but it is not directly proportional. On the contrary, it can lead to a higher transduction of off- target cells.

[0390] To further confirm that, once conjugated with NGFR121W, VR4 AAV tropism was successfully redirected to TrkA+ neurons, spinal cord sections of injected adult mice were analysed. In particular, immunofluorescence staining was performed in order to distinguish between lamina I and lamina II, using anti-TrkA antibody and conjugated IB4- AF647, respectively (FIG. 20). FIGs. 20 A and B depict the confocal acquisition of spinalcord from VR-IV AAV2 injected animal. Spinal cord slices (30 um) derived from injected adult mice were stained for TrkA receptor (green) and IB4 (cyan) to assess whether NGF exposed on the viral capsid was able to re-direct AAV tropism to peptidergic neuros expressing TrkA. FIG. 20 - A depicts the confocal acquisition of spinal cord from VR-IV AAV2 injected animal at 4x. FIG. 20 - B depicts the confocal acquisition of spinal cord from VR-IV AAV2 injected animal at lOx. In the experimental condition (NGF), it is possible to visualize the colocalization of Td-Tomato positive fibres and TrkA receptors in lamina I, and IB4 staining in lamina II. Indeed, the two markers highlight two different subpopulation of neurons, peptidergic neurons express TrkA receptor that project to lamina I, whereas non- peptidergic nociceptors express N-acetyl-D-galactosamine end groups, which are recognized by IB4.

[0391] Observing at higher magnification (FIG. 20-B) it was confirmed that, once modified via chemogenetic approach to attach NGFR121W on the capsid surface, VR4 AAV2 acquire target specificity for TrkA+ neurons. Indeed, it is possible to appreciate an overlapping between Td-Tomato signal and the secondary antibody for TrkA. On the other hand, when VR4 AAV2 is not modified (control condition) the viral vector is not able to efficiently transduce non-peptidergic neurons and there is no expression of the reporter gene neither in the neural endings projecting towards the spinal cord.

[0392] Analysing the spinal cord sections of the experimental group, projections in lamina I of the ipsilateral side of the injection were positive for Td-Tomato expression, whereas the contralateral region was not, confirming the specificity of the signal. Furthermore, also in this case, cell-specific delivery of the viral cargo was confirmed due to the overlapping of Td-Tomato and TrkA signal (FIG. 21).5.15.12. Example 12. Comparison of sortagged AAV variants to wild-type AAV25.15.12.1 Chemogenetic strategy as alternative to chemical engineering of sortagged viral particles

[0393] The chemogenetic strategy appears to be a potential alternative to the chemical engineering of “sortagged” viral particles, however it is crucial to compare the technology to a wild-type AAV2. Indeed, even though from the protein modelling there was not a significance difference in the 3D structure of the viral protein, and the particle analysis suggested the virus is correctly packed, the insertion of the sortag motif could lead toalteration to the binding with the primary receptor or to the intracellular trafficking.Therefore, a comparison between wild-type AAV2 and “sortagged” AAV2 was performed.5.15.12.2 Results

[0394] FIGs. 22 A-C depict the comparison between WT AAV2 and “sortagged” variants. The three variants were compared to WT AAV2 via chemical modification to investigate whether the “sortagged” AAV efficiency can be comparable to the WT one. For the variants, a series of higher concentration (lx; 3x; lOx; 30x) were used to infect PC-12 cells, whereas the WT concentration remained fixed (lx). FIG. 22 - A depicts the comparison between WT AVV2 and VR-IV AAV2. FIG. 22 - B depicts the comparison between WT AVV2 and VR-VIII AAV2. In the case of VR- VIII vs WT AAV2, 30 times more of the first vector must be used to transduce an equivalent number of PC-12 cells. FIG. 30 - C depicts the comparison between VR-IV AAV2 and VR-VIII AAV2. VR-IV / VR-VIII AAV2 resulted to be 30 times less efficient than WT, therefore also in its case, a higher VG was needed to reach a similar transduction level.

[0395] In particular, 4E9 VG of viral vectors were either modified via chemical modification (lx) or not (control lx) by coupling the WGA ligand in order to boost the transduction level in PC-12 cells (FIG. 22). Focusing on the comparison between WT AAV2 and VR-IV AAV2 (FIG. 22 - A), the controls of both vectors presented a minimal number of positive cells for Td-Tomato, whereas 4E9 VG of modified WT AAV2 and VR-IV AAV2 exhibited strong transduction. However, the infection with 4E9 VG of VR-IV AAV2 did not led to the same transduction level of cell infected with 4E9 VG of WT AAV2. Higher concentrations of VR-IV AAV2 (1.2E10 VG (3x); 4E10 VG (lOx); 1.2E11 VG (30X)) were tested to assess which conditions better resemble WT transduction. As result, it was observed that 3 times more of VR-IV AAV2 was needed in order to obtain a transduction efficiency similar to the WT vectors.

[0396] Increasing further the dose (lOx and 3 Ox) of VR-VIII AAV2 showed an increase Td-Tomato signal, underlying a dose-dependent pattern.

[0397] The same experiment was conducted for both VR-VIII AAV2 (FIG. 22 - B) and VR-IV / VR-VIII AAV2 (FIG. 22 - C). For both of them, the control condition was similar to the WT AAV control, with minimal or no reporter signal. Studying the lx experimental condition instead, a substantial difference between the WT and VR vectors was noted. Indeed, for both VR-VIII and VR-IV / VR-VIII AAV2, 4E9 VG (lx) and 1.2E10 VG(3x) were not sufficient to transduce any cells. Increasing the dose to 10-fold (4E10 VG), led to an increased reporter signal, however, it did not reach the level of WT transduction.

[0398] Approximately 30 times more of “sortagged” viral vectors were needed to obtain a comparable transduction to lx WT condition. These experiments confirmed that the insertion of LPETG motif (SEQ ID NO: 7) in variable regions IV and VIII interfere with the functionality of the vectors, requiring one to increase the dose of vectors to use in order to obtain a transduction level comparable to a WT AAV. Furthermore, engineering of VR-IV seemed to be more susceptible than VR-VIII suggesting that the alteration of the amino acids involved in the primary binding may affect also other mechanisms, such as intracellular trafficking.6. SEQUENCE LISTING7. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0399] While the disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure.

[0400] All literature references, issued patents and patent applications cited within the body of the instant specification, including U.S. Provisional Appl. No. 63 / 514,906 filed July 21, 2023, are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An engineered adeno-associated virus (rAAV) capsid protein, wherein: the capsid protein comprises a functional sequence motif, wherein the functional sequence motif has the sequence:X1X2X3X4X5X6; wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue and wherein X4, X5, and Xe are optional; wherein the functional sequence motif is a substrate for a sortase transamidase enzyme.

2. The engineered rAAV capsid protein of claim 1, wherein the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU.

3. The engineered rAAV capsid protein of claim 2, wherein the enzyme is a sortase A.

4. The engineered rAAV capsid protein of claim 3, wherein the enzyme is heptamutant (SrtA 7M) or a pentamutant (SrtA 5M).

5. The engineered rAAV capsid protein of claim 4, wherein the enzyme is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

6. The engineered rAAV capsid protein of claim 4, wherein the enzyme is SrtA 5M.

7. The engineered rAAV capsid protein of any one of claims 1-6, wherein the functional sequence motif is located within a surface accessible variable region (VR) of the capsid primary sequence.

8. The engineered rAAV capsid protein of claim any one of claims 1-7, wherein the functional sequence motif has a sequence of X1X2X3; wherein Xi, X2, and X3, are each independently selected from any amino acid residue.

9. The engineered rAAV capsid protein of claim 8, wherein the functional sequence motif has a sequence of X1X2X3, and wherein two or more of Xi, X2, and X3, are selected from:Xi is valine (V);X2 is proline (P); and X3 is proline (P).

10. The engineered rAAV capsid protein of claim 8, wherein Xi, X2, and X3, are selected from:Xi is independently proline (P);X2 is independently selected from glycine (G) and glutamic acid (E); and X3 is independently selected from proline (P), and phenylalanine (F).

11. The engineered rAAV capsid protein of claim 10, wherein the functional sequence motif has a sequence selected from: PEF, PGF, or PEP.

12. The engineered rAAV capsid protein of claim any one of claims 1-7, wherein the functional sequence motif has a sequence of X1X2X3X4; wherein Xi, X2, X3, and X4, are each independently selected from any amino acid residue.

13. The engineered rAAV capsid protein of claim any one of claims 1-7, wherein the functional sequence motif has a sequence of X1X2X3X4X5; wherein Xi, X2, X3, X4, X5, are each independently selected from any amino acid residue.

14. The engineered rAAV capsid protein of claim 13, wherein the functional sequence motif has a sequence of X1X2X3X4X5, and wherein:Xi is independently selected from lysine (L), valine (V), isoleucine (I) and asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), glycine (G), and asparagine (N).

15. The engineered rAAV capsid protein of claim 14, wherein two or more of Xi, X2, X3, X4, and X5, are selected from:Xi is independently selected from lysine (L);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A), a glycine (G), and asparagine (N).

16. The engineered rAAV capsid protein of claim 15, wherein three or more of Xi, X2, X3, X4, and X5 are selected from:Xi is independently selected from lysine (L), valine (V), isoleucine (I) and an asparagine (N);X2 is independently proline (P);X3 is independently selected from any amino acid residue;X4 is independently threonine (T); andX5 is independently selected from alanine (A) and glycine (G).

17. The engineered rAAV capsid protein of claim 16, whereinXi is lysine (L);X2 is proline (P);X3 is glutamic acid (E), serine (S), or alanine (A);X4 is threonine (T); andX5 is alanine (A) or glycine (G).

18. The engineered rAAV capsid protein of any one of claims 13-17, wherein the functional sequence motif comprises a sequence selected from: LPX3TG (SEQ ID NO: 3), LPX3TA (SEQ ID NO: 62), LPETG (SEQ ID NO: 7), and LPETA (SEQ ID NO: 63).

19. The engineered rAAV capsid protein of any one of claims 1-7, wherein the functional sequence motif has a sequence of X1X2X3X4X5X6; wherein Xi, X2, X3, X4, X5, and Xe are each independently selected from any amino acid residue.

20. The engineered rAAV capsid protein of claim 19, wherein the functional sequence motif has a sequence of X1X2X3X4X5X6 (SEQ ID NO: 1); wherein two or more of Xi, X2, X3, X4, X5, and Xe are:Xi is independently asparagine (N);X4 is independently threonine (T);X5 is independently asparagine (N); andXe is independently proline (P).

21. The engineered rAAV capsid protein of claim 20, wherein the functional sequence motif has a sequence of X1X2X3X4X5X6 (SEQ ID NO: 1); wherein at least three of Xi, X2, X3, X4, X5, and Xe are:Xi is independently asparagine (N);X4 is independently threonine (T);X5 is independently asparagine (N); andXe is independently proline (P).

22. The engineered rAAV capsid protein of claim 21, wherein the functional sequence motif comprises a sequence selected from: NX2X3TNX6 and VPX3X4X5P.

23. The engineered rAAV capsid protein according to any one of claims 1-22, wherein the capsid protein further comprises a nucleophile linker.

24. The engineered rAAV capsid protein according to claim 1, wherein the capsid protein further comprises a ligand.

25. The engineered rAAV capsid protein according to claim 23, wherein the nucleophile linker is selected from GGG-PEG4-DBCO, Amine-PEG4-DBCO, and Amine-DBCO.

26. The engineered rAAV capsid protein according to claim 25, wherein the nucleophile linker is GGG-PEG4-DBCO.

27. The engineered rAAV capsid protein according to claim 25, wherein the nucleophile linker is Amine-PEG4-DBCO.

28. The engineered rAAV capsid protein according to claim 25, wherein the nucleophile linker is Amine-DBCO.

29. The engineered rAAV capsid protein according to claim 24, wherein the ligand is an azide-tagged ligand.

30. The engineered rAAV capsid protein according to claim 29, wherein the azide-tagged ligand is selected from azide-tagged WGA, and azide tagged NGF.

31. The engineered rAAV capsid protein according to claim 30, wherein the azide-tagged ligand is WGA-PEG4-Azide.

32. The engineered rAAV capsid protein according to any one of claims 1-22, wherein the engineered rAAV capsid protein comprises one or more sequence changes that serve to introduce the functional sequence motif, the sequence changes selected from insertions, deletions, or substitutions, compared to a wild type protein.

33. The engineered rAAV capsid protein according to claim 32, comprising at least one insertion mutation wherein the insertion forms the functional sequence motif.

34. The engineered rAAV capsid protein according to claim 33, wherein the insertion mutation is the insertion of an exogenous peptide selected from LPET (SEQ ID NO. 2) and LPETG (SEQ ID NO: 7).

35. The engineered rAAV capsid protein according to any one of claims 1-34, wherein the rAAV capsid protein is selected from one or more of VP1, VP2, and VP3.

36. The engineered rAAV capsid protein according to claim 35, wherein the functional sequence motif is located within a surface loop region selected from: the AB, BC, CD, DE, EF, FG, GH, and HI loops.

37. The engineered rAAV capsid protein according to claim 36, wherein the functional sequence motif is located within a GH surface loop region.

38. The engineered rAAV capsid protein according to any one of claims 1-37, wherein the functional sequence motif is located within one or more variable regions selected from VR1, VR2, VR3, VR4, VR5, VR6, VR7, VR8 and VR9.

39. The engineered rAAV capsid protein according to claim 38, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV or VIII.

40. The engineered rAAV capsid protein according to claim 39, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region VIII.

41. The engineered rAAV capsid protein according to claim 39, wherein the engineered rAAV capsid protein comprises a functional sequence motif within variable region IV.

42. The engineered rAAV capsid protein according to claim 38, wherein the engineered rAAV capsid protein comprises a first functional sequence motif within variable region IV and asecond functional sequence motif within variable region VIII; and wherein the first and second functional sequence motifs are independently selected.

43. The engineered rAAV capsid protein according to claim 42, wherein the first and second functional sequence motifs are the same.

44. The engineered rAAV capsid protein according to any one of claims 1-43, wherein the rAAV capsid protein is selected from an AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 serotype.

45. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV1 capsid protein.

46. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV2 capsid protein.

47. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV4 capsid protein.

48. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV5 capsid protein.

49. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV8 capsid protein.

50. The engineered rAAV capsid protein according to claim 44, wherein the rAAV capsid protein is an AAV9 capsid protein.

51. The engineered rAAV capsid protein according to claim 38, wherein the functional sequence motif begins after:(a) S453 in VR-IV of an AAV1 capsid serotype;(b) S452 in VR-IV or R585 VR-VIII of an AAV2 capsid serotype;(c) S576 in VR-VIII of an AAV5 capsid serotype;(d) S453 in VR-IV or S587 VR-VIII of an AAV6 capsid serotype;(e) 454 in VR-IV or A587 VR-VIII of an AAV7 capsid serotype;(f) T454 in VR-IV or Q589 VR-VIII of an AAV8 capsid serotype; and(g) S454 in VR-VIII of an AAV9 capsid serotype.

52. The engineered rAAV capsid protein according to claim 51, wherein the functional sequence motif begins after S452 in VR-IV or R585 VR-VIII of an AAV2 capsid serotype.

53. The engineered rAAV capsid protein according to claim 52, wherein LPET (SEQ ID NO. 2) is inserted after S452 to form the functional sequence motif.

54. The engineered rAAV capsid protein according to any one of claims 1-53, wherein the engineered rAAV capsid protein does not comprise additional mutations when compared to the wildtype capsid protein.

55. A polynucleotide encoding the engineered rAAV capsid protein of any one of claims 1-54.

56. A vector comprising the polynucleotide of claim 55.

57. The vector of claim 56, further comprising a promoter operably linked to the polynucleotide.

58. A host cell comprising the engineered rAAV capsid protein of any one of claims 1-57, the polynucleotide specified in claim 55, or the vector of claim 56 or 57.

59. A recombinant rAAV virion (rAAV) comprising the engineered rAAV capsid protein of any one of claims 1-54.

60. The rAAV virion of claim 59, further comprising an exogenous cargo polynucleotide.

61. The rAAV virion of claim 60, wherein the exogenous cargo polynucleotide comprises a template for homology directed repair.

62. The rAAV virion of claim 61, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.

63. The rAAV virion of claim 61, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

64. The rAAV virion according to any one of claims 59-63, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

65. A surface functionalized rAAV virion according to any one of claims 59-64, wherein the rAAV virion comprises: at least one cross linker reactive moiety covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif, and an exogenous cargo polynucleotide.

66. The surface functionalized rAAV virion according to claim 65, wherein the amino acid is a naturally occurring amino acid.

67. The surface functionalized rAAV virion according to claim 66, wherein the amino acid is a naturally occurring L-a-amino acid.

68. The surface functionalized rAAV virion according to claim 67, wherein the amino acid residue is a lysine.

69. The surface functionalized rAAV virion according to claim 65, wherein the amino acid residue in the viral capsid protein comprises an N terminal amine.

70. The surface functionalized rAAV virion according to claim 65, wherein the amino acid residue in the viral capsid protein is a non-natural amino acid residue.

71. A surface functionalized rAAV virion, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of claims 1-70, and an exogenous cargo polynucleotide, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and at least one cross linker reactive moiety covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.

72. The surface functionalized rAAV virion according to claim 71, wherein the covalent linkage comprises an amide bond.

73. The surface functionalized rAAV virion according to claim 71, wherein the C-terminal amino acid of the N-terminal cleavage fragment is threonine.

74. A surface functionalized rAAV virion, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of claims 1-73, and an exogenous cargo polynucleotide, and wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) an N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment; and optionally at least one cross linker reactive moiety is covalently connected to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

75. The surface functionalized rAAV virion according to any one of claims 65-74, wherein each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEEDD) reaction, a Staudinger ligation, and a [4+1] cycloaddition reaction.

76. The surface functionalized rAAV virion according to claim 75, wherein the crosslinker reactive moiety comprises at least one of an eight membered ring and a triazole ring.

77. The surface functionalized rAAV virion according to claim 75, wherein the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

78. The surface functionalized rAAV virion according to claim 75, wherein the crosslinker reactive moiety is selected from a cyclooctyne and an azide.

79. The surface functionalized rAAV virion according to claim 75, wherein the azide is an azide-tagged ligand.

80. The surface functionalized rAAV virion according to claim 79, wherein the azide-tagged ligand is selected from azide-tagged WGA, and azide tagged NGF.

81. The surface functionalized rAAV virion according to claim 80, wherein the azide-tagged ligand is WGA-PEG4-Azide.

82. The surface functionalized rAAV virion according to claim 78, wherein the cyclooctyne is selected from dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof.

83. The surface functionalized rAAV virion according to claim 82, wherein the cyclooctyne is a DBCO.

84. The surface functionalized rAAV virion according to claim 75, wherein the reaction is an inverse electron demand Diels-Alder (IEEDD) reaction.

85. The surface functionalized rAAV virion according to claim 75, wherein the crosslinker reactive moiety is selected from a transcyclooctene and a tetrazine.

86. A surface modified rAAV virion, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of claims 1-85, and an exogenous cargo polynucleotide.

87. The surface modified rAAV virion according to claim 86, wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form (i) a N-terminal cleavage fragment and (ii) a C-terminal cleavage fragment; and wherein the virion comprises a crosslinked moiety that covalently connects a C-terminal amino acid of the N-terminal cleavage fragment and a targeting ligand.

88. The surface modified rAAV virion according to claim 87, wherein the covalent linkage comprises an amide bond.

89. The surface modified rAAV virion according to claim 86 or 87, wherein the C-terminal amino acid of the N-terminal cleavage fragment is a threonine.

90. The surface modified rAAV virion according to any one of claims 86-88, wherein the crosslinked moiety comprises a product of a reaction selected from: a CuAAC reaction, a SPAAC reaction, a SPANC reaction, an IEEDD reaction, a Staudinger ligation, and a [4+1] cycloaddition reaction.

91. The surface modified rAAV virion according to claim 90, wherein the reaction is selected from: a SPAAC, a SPANC, and a IEEDD reaction.

92. The surface modified rAAV virion according to any one of claims 86-91, wherein the crosslinked moiety comprises a 7 or 8 membered carbocycle comprising from 0-3 heteroatoms selected from O or N.

93. The surface modified rAAV virion according to claim 92, wherein the crosslinked moiety comprises94. The surface modified rAAV virion according to any one of claims 86-93, wherein the functional sequence motif is selected from one of: SEQ ID NOs 1-65.

95. The surface modified rAAV virion according to claim 94, wherein the functional sequence motif is LPETG (SEQ ID NO 7).

96. A surface modified rAAV virion, wherein the virion comprises an exogenous cargo polynucleotide and at least one engineered rAAV capsid protein comprising at least one functional sequence motif LPX3TG (SEQ ID NO 3), wherein X3 is selected from any amino acid residue; wherein the functional sequence motif is located within variable region IV, VIII, orIV and VIII, and (a) wherein the engineered rAAV capsid protein is cleaved within the functional sequence motif to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; wherein the virion comprises a crosslinked moiety that covalently connects the T of the N-terminal cleavage fragment and a targeting ligand wherein the covalent linkage comprises amide bond; and optionally (b) wherein the virion comprises a crosslinked moiety that covalently connects a lysine residue or a N terminal amine of a capsid protein and a targeting ligand.

97. The surface modified rAAV virion according to claim 96, wherein the crosslinked moiety comprises98. The surface modified rAAV virion according to any one of claims 86-97, wherein the targeting ligand is a cell-type specific ligand.

99. The surface modified rAAV virion according to according to claim 98, wherein the targeting ligand is selected from enzymes, peptide mimetics, ligands of endogenous receptors, lipid binding proteins, toxins, bacteriophage peptidases, nucleotides and RNA / DNA based ligands, aptamers, viral vectors, nanoparticles, lipids, and combinations thereof.

100. The surface modified rAAV virion according to any one of claims 86-99, wherein the functional sequence motif attenuates or abrogates binding of the virion to mammalian cell polysaccharides or proteoglycans compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

101. The surface modified rAAV virion according to any one of claims 86-100, characterized by increased infectivity compared to a reference virion having the same capsid protein sequence but without the functional sequence motif.

102. The surface modified rAAV virion according to claim 101, wherein the increased infectivity is with respect to at least one cell or tissue type.

103. The surface modified rAAV virion according to claim 102, wherein the cell or tissue type is selected from cardiac, nerve, central nervous system, liver, muscle, lung and retinal.

104. The rAAV virion of claim any one of claims 86-103, wherein the exogenous cargo polynucleotide comprises a template for homology directed repair.

105. The rAAV virion of claim 104, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide.

106. The rAAV virion of claim 105, wherein the expressible polynucleotide encodes a transgene.

107. The rAAV virion of claim 104, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.

108. The rAAV virion of claim 99, wherein the exogenous cargo polynucleotide encodes intracellular antibodies, peptide toxins, optogenetic actuators, pharmacogenetic tools, CRISPR based-editors for precision gene editing, CRISPR-epigenetic tools to regulate gene expression, or suicide genes to induce cell death.

109. The rAAV virion of claim 108, wherein the cargo comprises a gene editing nuclease system.

110. The rAAV virion of claim 109, wherein the gene editing nuclease system comprises a Cas9 orthologue, a gRNA, and a specific DNA to be inserted into a host genome.

111. The rAAV virion of claim 108, wherein the cargo comprises a transgene associated with a genetic disorder.

112. The rAAV virion of claim 108, wherein the exogenous cargo polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

113. A pharmaceutical composition comprising a plurality of rAAV virions according to any one of claims 86-112, the composition further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, excipient or combination thereof.

114. A method of treating a patient having a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the method comprising: administering a therapeutically effective amount of the pharmaceutical composition according to claim 113, wherein the recombinant polynucleotide cargo is capable of treating the disease.

115. A composition for use in treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo, the composition comprising: a therapeutically effective amount of the pharmaceutical composition according to claim 113, wherein the recombinant polynucleotide cargo is capable of treating the disease.

116. Use of the rAAV virion according to any one of claims 86-112, for the manufacture of a medicament for treating a disease treatable by intracellular delivery of a recombinant polynucleotide cargo.

117. A method of preparing a surface functionalized rAAV virion, wherein the virion comprises at least one engineered rAAV capsid protein according to any one of claims 1-73, and an exogenous cargo polynucleotide, the method comprising: contacting the virion with (a) the enzyme and (b) the cross linker moiety, wherebythe functional sequence motif is enzymatically cleaved to form an N-terminal cleavage fragment and a C-terminal cleavage fragment; and at least one cross linker reactive moiety is covalently connected to a C-terminal amino acid of the N-terminal cleavage fragment.

118. The method of claim 117, further comprising the subsequent step of covalently connecting at least one cross linker reactive moiety to an amino acid residue in the engineered rAAV capsid protein, wherein the amino acid residue is not within the functional sequence motif.

119. The method of claim 117 or 118, wherein each crosslinker reactive moiety is independently selected from a crosslinker reactive moiety that participates in a reaction selected from: a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted alkyneazide cycloaddition (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron demand Diels-Alder (IEEDD) reaction, a Staudinger ligation, and a [4+1] cycloaddition reaction.

120. The method of claim 119, wherein the reaction is selected from: a SPAAC, a SPANC, and a IEEDD reaction.

121. The method of any one of claims 117-120, wherein the crosslinker reactive moiety is selected from a cyclooctyne and an azide.

122. The method of claim 121, wherein the azide is an azide-tagged ligand.

123. The method of claim 122, wherein the azide-tagged ligand is selected from azide-taggedWGA and azide tagged NGF.

124. The method of claim 123, wherein the azide-tagged ligand is WGA-PEG4- Azide.

125. The method of claim 121, wherein the cyclooctyne is selected from dibenzylcyclooctyne(DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC), or a derivative thereof.

126. The method of claim 125, wherein the cyclooctyne is a DBCO.

127. The method of any one of claims 117-126, wherein the enzyme is selected from a sortase A, sortase B, archaeosortase A, exosortase A, rhombosortase, and PorU.

128. The method of claim 127, wherein the enzyme is a sortase A.

129. The method of claim 127, wherein the enzyme is heptamutant (SrtA 7M) or a pentamutant (SrtA 5M).

130. The method of claim 129, wherein the enzyme is Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m).

131. The method of claim 129, wherein the enzyme is SrtA 5M.I l l

Images