Methods of preparing surface modified viral capsids

Abstract

The present disclosure relates to compositions of surface modified viral capsids having high physical titers. The present disclosure also relates to a method of preparing surface modified viral capsids using an efficient process of (a) reacting (i) a viral capsid comprising a plurality of surface available primary amines and (ii) a capsid-reactive linker comprising a terminal tetrafluorophenyl (TFP) ester and a first member of a crosslinker reactive pair to provide a composition comprising surface functionalized viral capsid; and (b) reacting the composition comprising surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair.

Description

METHODS OF PREPARING SURFACE MODIFIED VIRAL CAPSIDS 1. BACKGROUND

[0001] Introduction of molecules carrying genetic information into cells is a useful tool in modern medicine and in basic research. Preferred methods include the use of gene delivery vehicles derived from viruses, including adenoviruses, retroviruses, lentiviruses, vaccinia viruses, and adeno associated viruses. Among these, recombinant adeno-associated viruses (AAV) have become the preferred viruses for in vivo gene therapy due to lack of pathogenicity, replication incompetence, and stable expression. More than 100 clinical trials are underway using AAV- based capsids (also referred to as vectors or virions), and two AAV gene therapy products have recently been approved by FDA, namely Voretigene neparvovec-rzyl (LUXTURNA) for the treatment of an inherited retinal disease and onasemnogene abeparvovec-xioi (ZOLGENSMA) for the treatment of spinal muscular atrophy.

[0002] However, most clinical trials using AAV as vehicle for transgenes showed its critical limitations: (i) its reduced therapeutic index (i.e. high doses of capsids are usually required to achieve therapeutic efficacy); (ii) its broad biodistribution; (iii) and its poor efficacy in the presence of pre-existing neutralizing antibodies.

[0003] One limitation of AAV lies indeed in their broad tropism, which results in transgene expression in other tissues other than those where transgene expression is desired. It is also well recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer.

[0004] Most of the gene therapy applications to date have used the serotype 2 (AAV2). Transduction of a wide range of post-mitotic cells in vivo such as muscle cells, hepatocytes or neurons in mammals partly explains its popularity. This serotype has also been used for gene transfer to the muscle and liver in clinical trials for Hemophilia B and the retina for treating Leber Congenital Amaurosis.

[0005] However, there are still complications and limitations with the use of this vector. First, high doses of the vector are usually required since transduction efficacy in vivo is generally low, resulting in increased toxicity. One of the most important complications is due to the fact that 50-1 37937 / 59605 / FW / 18328338.1(NAb) against AAV2 that impair gene delivery. The discovery of naturally occurring AAV isolates (from 1 to 12) in humans and animal species and the genetic engineering of the capsid of these AAV serotypes using molecular tools resulted in promising results in preclinical animal models and phase I / II clinical trials, which foster exciting clinical translation in the near future. However, their therapeutic index remains low, which implies that high concentration of them still needs to be administered with concomitant adverse effects. At the same time, manufacturing clinical lots of AAV capsids has considerably progressed in the past decade and large-scale manufacturing methods are now available for the preindustrial pharmaceutical stage in which gene therapy is entering. Nonetheless, if high doses of capsids are required for phase III and commercialization current methods will not be able to support such demand.

[0006] As said strategies, which have indeed demonstrated the potential of novel AAV serotypes (and related genetic variants), were not considered satisfying as not achieving precise tropism, enabling the selective transduction of a target cell type, further efforts to increase tropisms of AAV-derived capsids are currently underway. Indeed, to counterbalance the lack of specificity of the AAV-derived capsids, extremely large amounts of AAV-derived capsids need to be administered to reach a therapeutic threshold, which is not desired for safety concerns as well as manufacturing limitations.

[0007] Various attempts have been pursued for this purpose, such as genetic introduction of peptide epitopes with targeting specificities into the viral surface. Further strategies were using linker molecules with two specificities, such as bispecific antibodies, one specificity being directed to the viral capsid, the other to the receptor, were introducing adaptor domains (Z domain of protein A, biotin) for non-covalent attachment of protein ligands.

[0008] For example, in document WO00 / 002654, the altered tropism is made primarily for preventing the binding of the AAV to virus receptors of the original target cell. In a particular embodiment, the increased affinity vis-a-vis the target cell is also referred in this document. Still in this document, antibody fragments are linked to the capsid. According to an alternative embodiment, the other end of the antibody can be coupled to ligands to improve the affinity vis-a- vis the target (preparation of “diabodies”).

[0009] Combined biological and chemical coupling on the AAV capsid has also been proposed in the past for improving the selectivity of AAV-derived capsids to the target tissue. 2 37937 / 59605 / FW / 18328338.1

[0010] For example, WO2005 / 106046 proposes a method combining genetic engineering and chemical modification of the capsid. The chemical modification that is the second stage of the process relies on the presence of residues cysteines whose capsid was enriched by genetic pathway in a first step. AAV particles may thus be modified with ligands, polymers, gold nanoparticles, fluorescents molecules, magnetic or substances biochemically active substances. However, the coupling is performed via disulfide, thioester and / or thioether bonds and via NCS bonds.

[0011] The article by E. D. Horowitz et al. “Glycated AAV Vectors: Chemical Redirection of Viral Tissue Tropism” Bioconjugate Chemistry, 2011, 22, 529-532 describes the problem of cell tropism among other technical problems. In particular, generation of unnatural amino acid side chains through capsid glycation serves as an orthogonal strategy to engineer AAV capsids displaying novel tissue tropisms for gene therapy applications.

[0012] In WO2015 / 062516, a non-natural amino acid, such as an amino acid comprising an azido, is inserted into the capsid by genetic engineering prior to a coupling step by a click chemical reaction to change the capsid of AAV and its tropism for the target cell.

[0013] WO 2022 / 101363 describes a method of preparing surface modified viral capsids However, the use of the described NHS esters for surface functionalization requires substantial purification that greatly reduces the titer of the resulting composition.

[0014] Therefore, there exists a need for finding a method to modify AAV-derived capsids by chemical coupling, for increasing their ability to target a specific organ or tissue, in particular by an in vivo gene delivery.

[0015] There is also a need to modify the AAV-derived capsids without requiring a step of engineering of the AAV amino acid capsid sequence.

[0016] Furthermore, there is a need for new surface-modified AAV-derived capsids with improved virus-mediated gene transfer into specific cell types.

[0017] More generally, there exists a need for new methods for chemically coupling ligands of any nature on AAV-derived capsid surface, i.e. a variety of chemical moieties, for example to improve the “specific activity” and / or “the therapeutic index” and thereby allowing a decrease in the therapeutic dose. 3 37937 / 59605 / FW / 18328338.12. SUMMARY OF THE DISCLOSURE

[0018] The present disclosure provides novel recombinant Adeno-Associated Virus (rAAV) vector particles, its producing method and its therapeutic and / or diagnostic uses.

[0019] In one aspect of the present disclosure, a composition comprising a surface modified viral capsid having a titer of at least 1.0E+10 vg / ml is provided, wherein the surface modified viral capsid comprises a ligand covalently conjugated to a viral capsid via a linker.

[0020] In some embodiments, the surface modified viral capsid is of Formula V:wherein:a viral capsid optionally comprising nucleic acid cargo; SP1and SP2are individually a bond or a spacer; Q is a crosslinked moiety; and L is the ligand

[0021] In another aspect of the present disclosure, a method of making compositions comprising surface modified viral capsids of Formula V is provided, comprising the steps of: (a) combining (i) a viral capsid comprising a plurality of surface available primary amines and (ii) a capsid-reactive linker comprising a tetrafluorophenyl (TFP) ester and a first member of a crosslinker-reactive pair (CRP1), thereby providing a composition comprising surface functionalized viral capsid; and 4 37937 / 59605 / FW / 18328338.1(b) combining (i) a functionalized ligand comprising a second member of a crosslinker- reactive pair (CRP2) and (ii) the composition comprising surface functionalized viral capsid; thereby providing the surface modified viral capsid.

[0022] Preferred features of each aspect of the present disclosure are as for each of the other aspects mutatis mutandis. The cited documents mentioned herein are incorporated to the fullest extent permitted by law. Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure as defined in the appended claims. 3. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0023] 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.

[0024] FIG. 1 illustrates the significant loss in physical titre of AAV2 after NHS modification and centrifugation as measured by ddPCR.

[0025] FIGs. 2a-2b show transduction efficiency of compositions prepared using different clean- up methods following NHS modification of AAV2 as assessed by transduction efficiency in HEK293 cells (FIG. 2a) and physical titer from ddPCR measurements (FIG. 2b). These figures also demonstrate transduction efficiency for TFP ester modification of AAV2 measured by the same methods.

[0026] FIG. 3a describes a comparison of transduction efficiency as evidenced by the number of PC12 cells that express the reporter gene EGFP upon infection by WGA-conjugated AAV2 modified with TFP-PEG4-DBCO + WGA-PEG4-Azide using different protocols: Reaction #1 conducted in 0.1M Sodium Bicarbonate pH 8.5 in water, quenched with glycine; Reaction #2 conducted in 0.1M Sodium Bicarbonate pH 8.5 in PBS / Pluronic 0.001% / NaCl 200mM, quenched 5 37937 / 59605 / FW / 18328338.1with glycine; of AAV2 and WGA-PEG4-Azide in PC12 cells. Cells were imaged at 4 days after AAV-infection.

[0027] FIG. 3b describes a comparison of transduction efficiency as evidenced by the number of PC12 cells that express the reporter gene EGFP upon infection by WGA-conjugated AAV2 modified with TFP-PEG4-DBCO + WGA-PEG4-Azide using different protocols: Reaction #3 conducted in 0.1M Sodium Bicarbonate pH 8.5 in water, no glycine; Reaction #4 conducted in PBS / Pluronic 0.001% / NaCl 200mM, quenched with glycine. Cells were imaged at 4 days after AAV-infection.

[0028] FIG. 3c describes a comparison of transduction efficiency as evidenced by the number of PC12 cells that express the reporter gene EGFP upon infection by WGA-conjugated AAV2 modified with TFP-PEG4-DBCO + WGA-PEG4-Azide using different protocols: Reaction #5 conducted in 0.2M Sodium Bicarbonate pH 8.5 in water, quenched with glycine; and control reaction with NHS, conducted in PBS / Pluronic 0.001% / NaCl 200mM, no glycine. Cells were imaged at 4 days after AAV-infection.

[0029] FIG. 4a-4b show comparisons of transduction efficiency as evidenced by the number of PC12 cells that express the reporter gene EGFP upon infection by WGA-conjugated AAV2 modified with TFP-PEG4-DBCO + WGA-PEG4-Azide using different protocols (1-5), and NHS- PEG4-DBCO + WGA-PEG4-Azide. Cells were analyzed by cytometry at 7 days after AAV- infection. FIG.4a shows the mean fluorescent intensity (MFI) of EGFP+ cells normalized by the proper control (△MFI). FIG.4b shows the percentage of EGFP+ positive cells normalized by the proper control.

[0030] FIG. 5 confirms viral titer reduction by ddPCR on fractions of virus prepared with TFP- PEG4-DBCO + NGF-PEG4-Azide at different ratios, NHS-PEG9-BG + NGF-SNAP, and wild type AAV2.

[0031] FIG. 6 shows transduction efficiency of lumbar DRG isolated from mice subcutaneously injected with AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-Azide at different ratios (TFP 3,000 and TFP 10,000), NHS-PEG4-BG + NGF-SNAP, and wild type AAV2 (control). DRG were collected at 3 weeks after in vivo AAV-injection, flat mounted and imaged with confocal microscope. The graph shows the number of EGFP+ positive cells. 6 37937 / 59605 / FW / 18328338.1

[0032] FIG. 7 shows the selectivity of TFP functionalized NGF-AAV in targeting nociceptive neurons by co-staining positive ganglia with an antibody against Trka. Quantification of transduced cells that colocalize with nociceptive neuronal marker Trka in lumbar DRG 3, 4, and 5 are shown.

[0033] FIG.8 shows transduction of skin collected from the paw of mice subcutaneously injected with wild type AAV2 and AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-Azide (ratio virus:linker=1:10,000). Nerve bundles in the dermis were identified by Trka staining (red) and the presence of the virus by the fluorescent protein EGFP (green).

[0034] FIG.9 shows transduction of spinal cord collected from mice injected subcutaneously with wild type AAV2 and AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-Azide (ratio virus:linker = 1:10,000). Virus transduction was identified by the fluorescent protein EGFP (green), first and second lamina of the dorsal horn by Trka (red) and Ib4 (cyan), respectively.

[0035] FIG. 10a shows images of TrkA / p75 HEK293 cells transduced with AAV9 or NGF- AAV9 at different Multiplicity of Infections (MOIs).

[0036] FIG. 10b shows the fluorescence intensity of AAV9 and NGF-AAV9; upon conjugation of NGF to the AAV9, the MOI required to transduce TrkA / p75 HEK293 cells is substantially reduced.

[0037] FIG. 11 presents the comparison of transduction efficiency of lumbar DRG isolated from mice subcutaneously injected with AAV2 modified with TFP-PEG4-DBCO at different scales. Numbers above the images indicate the total amount of vector modified at once. All animals were injected with the same dose of modified vector (5E+10 VG).

[0038] FIG.12 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV2 purified with cesium chloride gradient and modified with different virus:linker ratios of DBCO-PEG4-NHS and 50pMol WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0039] FIG.13 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV2 purified with cesium chloride gradient and modified with different virus:linker:ligand ratios of DBCO- PEG4-TFP and WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0040] FIG.14 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV2 purified with affinity chromatography and iodixanol gradient, and modified with different virus:linker 7 37937 / 59605 / FW / 18328338.1ratios of DBCO-PEG4-NHS and WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0041] FIG.15 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV2 purified with affinity purification and iodixanol gradient, and modified with different virus:linker ratios of DBCO-PEG4-TFP and WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0042] FIG. 16 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV5 modified with different virus:linker ratios of DBCO-PEG4-NHS and 50pMol WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0043] FIG. 17 shows transduction efficiencies of PC12 cells by WGA-conjugated AAV5 modified with different virus:linker:ligand ratios of DBCO-PEG4-TFP and WGA-PEG4-Azide. PC12 cells were imaged 5 days after AAV infection.

[0044] FIG. 18 shows transduction efficiencies of PC12 cells by WGA-conjugated Delta-HSPG AAV2 modified with different virus:linker:ligand ratios of DBCO-PEG4-NHS and WGA-PEG4- Azide. PC12 cells were imaged 5 days after AAV infection.

[0045] FIG. 19 shows transduction efficiencies of PC12 cells by WGA-conjugated Delta-HSPG AAV2 modified with different virus:linker:ligand ratios of DBCO-PEG4-TFP and WGA-PEG4- Azide. PC12 cells were imaged 5 days after AAV infection.

[0046] FIG. 20a shows images of PC12 cells transduced with AAV9-WGA chemically modified with NHS-PEG4-DBCO on crude cell extracts (lysates) using different concentrations of ligand during the modification reaction.

[0047] FIG. 20b presents images of PC12 cells transduced with AAV9-WGA chemically modified with TFP-PEG4-DBCO on crude cell extracts (lysates) using different concentrations of ligand during the modification reaction. 4. DETAILED DESCRIPTION 4.1. Definitions

[0048] The term “rAAV” as used herein refers to a recombinant virion comprising a recombinant nucleic acid construct packaged within an AAV capsid. 8 37937 / 59605 / FW / 18328338.1

[0049] The terms “AAV”, “adeno-associated virus”, “AAV virus”, “AAV virion”, “AAV viral particle”, “AAV particle”, “adeno-associated viral vector”, and “AAV vector” are used synonymously herein for rAAV.

[0050] The recombinant nucleic acid construct (synonymously, “recombinant viral genome”) comprises a polynucleotide payload (synonymously, “cargo”) positioned between AAV inverted terminal repeats. The payload can be an expressible polynucleotide or a DNA construct that provides a template for homology directed repair. In various embodiments, the expressible polynucleotide encodes a protein (e.g., a transgene encoding a therapeutic protein), or encodes an miRNA, siRNA, or a guide RNA for gene editing or RNA editing machinery such as CRISPR, ADAR, and ADAT.

[0051] As used herein the term “tropism” refers to preferential infection and / or transduction by a viral capsid of certain cells or tissues. In a preferred embodiment, to modify an AAV capsid’s tropism, the capsids are being given certain features such as certain affinities to receptors on the target cell's surface which they do not possess by nature.

[0052] As used herein viral titer is expressed in scientific notation using a simplified format that is more reliably reproduced by digital text recognition, for example 1.0E+3 vg / m. A person of skill in the art will understand that 1,000 vg / m, 1.0 ✕ 10^3 vg / m and 1.0E+3 vg / m are all equivalent expressions of the same number.

[0053] The term “MOI”, or “Multiplicity of Infection”, as used herein, represents the ratio of the numbers of virus particles to the numbers of the host cells in a given infection medium. A value of MOI = 1 implies that on an average there is a single host cell for a single phage particle. 4.2. Other interpretational conventions

[0054] 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.

[0055] 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 9 37937 / 59605 / FW / 18328338.1example, 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 further noted 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.

[0056] It is appreciated that certain features of the present 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 present 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 present 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 sub-combinations 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.

[0057] 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.

[0058] 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.

[0059] Ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any 10 37937 / 59605 / FW / 18328338.1number, 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, including subranges such as from 11 to 48 or 39 to 41. 4.3. Compositions

[0060] In one aspect, the present disclosure provides compositions comprising a surface modified viral capsid having a titer of at least 1.0E+10 vg / ml, the surface modified viral capsid comprising a ligand covalently conjugated to the viral capsid via a linker.

[0061] The surface modified viral capsid confers improved transduction efficiency, improved cell- type selectivity, or both improved transduction efficiency and improved cell-type selectivity on a recombinant virion of which it is a part, when compared to a recombinant virion comprising a viral capsid having the same primary amino acid sequence but that has not been modified to comprise the targeting ligand.

[0062] Due to the efficiency of production of the surface modified viral capsid described herein, the compositions of the present disclosure have a relatively high physical titer compared to compositions prepared by other methods. In some embodiments, the composition comprising surface modified viral capsid has a physical titer of at least 1E+10 vg / mL, as determined by droplet digital PCR (ddPCR), such as, for example, at least 1.5E+11 vg / ml, at least 2.0E+11 vg / ml, or at least 2.5E+11 vg / ml. In some embodiments, the composition has a physical titer of at least 1.0E+12 vg / ml, at least 1.5E+12 vg / ml, at least 2.0E+12 vg / ml, or at least 2.5E+12 vg / ml. In some embodiments, the composition has a physical titer of at least 1.0 E+13 vg / ml, at least 1.5E+13 vg / ml, at least 2.0E+13 vg / ml, or at least 2.5E+13 vg / ml.

[0063] In some embodiments, the composition comprising surface modified viral capsid has a physical titer from 1.0E+10 vg / ml to 5.0E+13 vg / ml, such as, for example, 1.0E+11 vg / ml to 5.0E+13 vg / ml, from 1.0E+12 vg / ml to 5.0E+13 vg / ml, from 1.0E+13 vg / ml to 5.0E+13 vg / ml, from 2.0E+10 vg / ml to 5.0E+13 vg / ml, from 2.0E+11 vg / ml to 5.0E+13 vg / ml, from 2.0E+12 vg / ml to 5.0E+13 vg / ml, from 2.0E+13 vg / ml to 5.0E+13 vg / ml, from 3.0E+10 vg / ml to 5.0E+13 vg / ml, from 3.0E+11 vg / ml to 5.0E+13 vg / ml, from 3.0E+12 vg / ml to 5.0E+13 vg / ml, from 3.0E+13 vg / ml to 5.0E+13 vg / ml, from 4.0E+10 vg / ml to 5.0E+13 vg / ml, from 4.0E+11 vg / ml to 11 37937 / 59605 / FW / 18328338.15.0E+13 vg / ml, from 4.0E+12 vg / ml to 5.0E+13 vg / ml, from 4.0E+13 vg / ml to 5.0E+13 vg / ml, from 5.0E+10 vg / ml to 5.0E+13 vg / ml, from 5.0E+11 vg / ml to 5.0E+13 vg / ml, or from 5.0E+12 vg / ml to 5.0E+13 vg / ml.

[0064] In some embodiments, the composition comprising surface modified viral capsid has a physical titer from 1.5E+11 vg / ml to 3.0E+12 vg / ml, from 2.0E+11 vg / ml to 3.0E+12 vg / ml, or from 2.5E+11 vg / ml to 3.0E+12 vg / ml.

[0065] The compositions of the present disclosure comprise surface modified viral capsids of Formula V:wherein:a viral capsid that optionally comprises nucleic acid cargo; SP1and SP2are each is individually selected from a bond or a spacer; Q is a crosslinked moiety; and L is the ligand.

[0066] The variable “LCR” refers to the Ligand to Capsid Ratio and corresponds to the number of ligands conjugated to the capsid surface. In some embodiments, LCR is an integer from 10 to 500, such as, for example, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 50 to 500, from 50 to 400, from 50 to 300, from 50 to 200, from 50 to 100, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 500, from 200 to 400, from 200 to 300, from 300 to 500, from 300 to 400, or from 400 to 500. In some embodiments, LCR is an integer from 100 to 200, such as, for example, from 100 to 190, from 100 to 180, from 100 to 170, from 100 to 160, or from 100 to 150.

[0067] In some embodiments, the composition comprises less free ligand (in any form that is not conjugated to the capsid surface including functionalized ligand and also functionalized ligand 12 37937 / 59605 / FW / 18328338.1conjugated to unbound TFP linker) than bound ligand (in the form of surface modified viral capsid). The amount of free ligand in the composition can be measured by gold standard analytics for AAVs such as analytical ultracentrifugation (AUC) and mass photometry. In some embodiments, the composition comprises 20% or less free ligand, such as, for example, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. 4.3.1. Bifunctional Linker

[0068] The bifunctional linker in accordance with the present disclosure comprises two components, i) TFP ester, and ii) a member of a crosslinker reactive pair or a ligand. Optionally, the bifunctional linker further comprises a spacer. In certain embodiments, the bifunctional linker comprises a TFP ester, optionally a spacer, and a member of a crosslinker reactive pair. In certain embodiments, the bifunctional linker comprises an TFP ester, a spacer, and a member of a crosslinker reactive pair.

[0069] 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.

[0070] In some embodiments, the capsid-reactive linker is of the following structure:, wherein SP1is a bond or spacer known in the art; and a is an integer from 1 to 5, from 2 to 4, e.g., 1, 2, 3, 4, or 5. In certain embodiments, a is 4. 13 37937 / 59605 / FW / 18328338.14.3.1.1 Spacers

[0071] In certain of these embodiments, the spacer comprises 10 to 30 non-hydrogen atoms. In further embodiments, the spacer comprises 10 to 30 divalent groups selected from -CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (secondary amine, where R is H or C1-3 alkyl, e.g., methyl). In certain of these embodiments, the spacer comprises from 5-40 methylene groups, from 1-20 ether groups, 1-5 amines, and or 1-4 carbonyl groups. In certain embodiments, the spacer comprises 10-15 methylene groups, 4-6 ether groups, 1-2 amines, and or 3-4 carbonyl groups.

[0072] In some embodiments, the capsid-reactive linker is of the following structure:, wherein: X is -CH2- or -O- groups, b is an integer from 2 to 20, n is an integer from 2 to 20, m is an integer from 2 to 10; and a is an integer from 1 to 5, from 2 to 4, e.g., 1, 2, 3, 4, or 5.

[0073] In some embodiments, the capsid-reactive linker is of the following structure: 14 37937 / 59605 / FW / 18328338.1, wherein: n is an integer from 2 to 20, m is an integer from 2 to 10; and a is an integer from 1 to 5, from 2 to 4, e.g., 1, 2, 3, 4, or 5.

[0074] In some embodiments, the capsid-reactive linker is of the following structure:wherein: n is an integer from 2 to 20, m is an integer from 2 to 10; and a is an integer from 1 to 5, from 2 to 4, e.g., 1, 2, 3, 4, or 5.

[0075] In some embodiments, the capsid-reactive linker is of the following structure:15 37937 / 59605 / FW / 18328338.1

[0076] In some embodiments, n is an integer from 2 to 15.

[0077] In some embodiment, m is selected from 2, 3, and 4.

[0078] In some embodiments, n is from 4 to 12 and m is 2, 3, or 4.

[0079] In some embodiments, the n is 12 and m is 3 and the capsid-reactive linker is referred to as DBCO-PEG12-TFP and has the structure below:.

[0080] In some embodiments, the n is 12 and m is 4 and the capsid-reactive linker is referred to as DBCO-PEG4-TFP and has the structure below:. 4.3.2. Crosslinker Reactive Pair

[0081] In some embodiments, CRP1 comprises a reactive moiety selected from: an azide; alkyne, 1,4-triazole; 1,3-nitrone; cyclooctyne or derivative thereof, e.g.,dibenzylazacyclooctyne (referred to as DBCO herein) or derivative thereof; triazine; tetrazine; strained dienophile; aryl or alkyl phosphine; isocyanide; benzylguanine group, a benzylcytosine group, or a chloroalkane group.

[0082] In some embodiments, CRP1 comprises a cyclooctyne reactive moiety. In some embodiments, CRP1 is selected from OCT, MOFO, DIFO, DIMAC, COMBO, DIBO, DIBAC (DBCO), BARAC, BCN, and TMTH such as those depicted below: 16 37937 / 59605 / FW / 18328338.1

[0083] In some embodiments, CRP1comprises DBCO. In some embodiments, CRP1is of the following formula: 17 37937 / 59605 / FW / 18328338.1wherein m is from 1 to 20. In some embodiments, m is from 1 to 15. In some embodiments, m is from 1 to 10. In some embodiments, m is 4. In certain embodiments, the squiggle bond indicates attachment to SP1. 4.3.3. Linker

[0084] The surface modified viral capsid of the present disclosure comprises a linker of formed via the conjugation of the bifunctional linker with the viral capsid and to the. In some embodiments, the linker is of Formula VI:wherein SP1and SP2are independently a bond or a spacer; and Q comprises a crosslinked moiety.

[0085] In some embodiments, the acyl group of the linker is bound to a surface available primary amine of the viral capsid, thereby forming an amide bond. In certain embodiments, SP2is bound to the ligand. 4.3.3.1 Spacers

[0086] 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). 18 37937 / 59605 / FW / 18328338.1

[0087] In certain of these embodiments, the spacer (SP1, SP2, or both SP1and SP2)comprises 10 to 30 non-hydrogen atoms. In further embodiments, the spacer comprises 10 to 30 divalent groups selected from -CH2- (methylene), -O- (ether), -C(=O)- (carbonyl), and -N(R)- (secondary amine, where R is H or C1-3alkyl, e.g., methyl). In certain of these embodiments, the spacer comprises from 5-40 methylene groups, from 1-20 ether groups, 1-5 amines, and or 1-4 carbonyl groups. In certain embodiments, the spacer comprises 10-15 methylene groups, 4-6 ether groups, 1-2 amines, and or 3-4 carbonyl groups.

[0088] In some embodiments, SP1, SP2, or both SP1and SP2comprise one or more PEGs (i.e., - (−(O−CH2−CH2)n)- or -([PEG]n)-), and SP2is -Y-C(O)-, -Y-C(O)O-, -Y-NHC(O)-, -Y-NHC(S)-, or -Y-C(O), wherein Y is a bond or one or more PEGs.

[0089] In certain embodiments, SP1comprises -([PEG]n)-, wherein n is from 1 to 100, such as, for example, from 1 to 50, from 1 to 25, from 1 to 15, from 1 to 10, from 1 to 5, from 4 to 20, from 4 to 15, from 4 to 12, or from 4 to 10. In some embodiments, n is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0090] In some embodiments, SP1, SP2, or both SP1and SP2comprise from 2 to 20 PEGs, such as, for example, from 2 to 15, from 2 to 10, from 2 to 5, from 5 to 20, from 5 to 15, from 5 to 10, from 10 to 20, from 10 to 15, or from 15 to 20. In some embodiments, SP1comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 PEGs.

[0091] In some embodiments, SP2comprises -([PEG]n)-C(O)-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10. 4.3.4. Crosslinked Moiety – Q

[0092] 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.

[0093] 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. 19 37937 / 59605 / FW / 18328338.1

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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, Q comprises 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.

[0098] 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. 20 37937 / 59605 / FW / 18328338.1

[0099] In some embodiments, Q comprises a structure shown below:

[0100] In some embodiments, Q comprises the reaction product of a cyclooctyne reactive moiety and an azide reactive moiety.

[0101] In some embodiments, Q comprises one of the following structures:

[0102] In some embodiments, Q comprises one of the following structures:wherein m is from 1 to 10, e.g., from 2 to 8, 3 to 7, or 4 to 6. 21 37937 / 59605 / FW / 18328338.14.3.5. Viral Capsids

[0001] In embodiments of the present disclosure, the type of viral capsid is not particularly limited. In some embodiments, the viral capsid is selected from a non-enveloped virus, such as an adenovirus or adeno-associated virus. In some embodiments, the viral capsid is the protein capsid of an enveloped virus, such as retroviruses, lentiviruses, herpes simplex virus, and baculoviruses. Embodiments include non-naturally occurring capsids and includes a biologic or chemical alteration or variation of a naturally occurring capsid protein.

[0103] In some embodiments, the viral capsid is a capsid of an adeno-associated virus or recombinant adeno-associated virus (rAAV, or AAV used interchangeably herein). Such AAV particles are capable of transducing a wide range of post-mitotic cells in vivo in the mammal, e.g, (including but not limited to) muscle cells, hepatocytes and neurons.

[0104] In some embodiments, the AAV comprises a VP1, VP2, and / or VP3 capsid protein of a naturally occurring AAV serotype. In some embodiments, the AAV comprises one or more of a non-naturally occurring VP1, VP2, and / or VP3 capsid protein. In certain of these embodiments, the non-naturally occurring VP1, VP2, or VP3 capsid protein differs in primary amino acid sequence from naturally occurring capsids. In certain embodiments, the non-naturally occurring capsid includes a biologic or chemical alteration or variation of a naturally occurring AAV capsid protein other than or in addition to a change in the primary amino acid sequence.

[0105] In some embodiments, the capsid of AAV is composed of three overlapping capsid proteins (VP1, VP2, VP3) containing a unique VP1 N-terminus, a VP1 / VP2 common portion and a portion which is common to VP1, VP2 and VP3.

[0106] In certain embodiments, one or more capsid proteins comprise amino acid residues that are naturally occurring, that is the primary sequence corresponds to a wild-type capsid protein. In alternative embodiments, the primary sequence of one or more capsid proteins comprises amino acid residues that are engineered into a wild-type capsid protein sequence. In certain of these embodiments, the engineered amino acids include one or more amino groups present at the surface of the capsid and are involved in the surface functionalization of one or more capsid protein. In certain embodiments, the naturally occurring or engineered amino group that is surface functionalized using the methods of the present disclosure is lysine. 22 37937 / 59605 / FW / 18328338.1

[0107] In various embodiments, the capsid proteins are those of an AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9 naturally occurring AAV serotype. In various embodiments, the capsid protein is selected from capsid proteins disclosed in PCT / US2014 / 060163, USP9695220, PCT / US2016 / 044819, PCT / US2018 / 032166, PCT / US2019 / 031851, and PCT / US2019 / 047546, which are incorporated herein by reference in their entireties.

[0108] In some embodiments, the capsid is of AAV2. In some embodiments, the capsid is of AAV5.

[0109] Also, the adeno-associated virus may be chosen among synthetic serotypes generated by non-natural methods, such as, but not limited to: capsid mutagenesis, peptide insertions into, or deletions from, the capsid sequence, capsid shuffling from various serotypes or ancestral reconstruction.

[0110] The AAVs for use with the present disclosure are produced by any method known in the art, without limitation. For example, the AAV can be produced by several methods including: transient transfection of HEK293 cells, stable cell lines infected with Ad or HSV, mammalian cells infected with Ad or HSV (expressing rep-cap and transgene) or insect cells infected with baculovirus capsids (expressing rep-cap and transgene). AAV produced by any of these methods can be used to produce the surface functionalized and surface modified viral capsid described herein. In certain embodiments, the AAV are produced by transient transfection of HEK293 cells with calcium phosphate-HeBS method with two plasmids: pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus helper genes (E2A, VA RNA, and E4) and pVector ss-CAG-eGFP.

[0111] In some embodiments, the AAV of the present disclosure comprises one or more sequences from extraviral origin, as desired.

[0112] According to a particular embodiment, the AAV comprises one or more wild-type capsid proteins from naturally occurring serotypes.

[0113] According to another particular embodiment, the AAV comprises a genetically modified capsid protein. In certain embodiments, the genetically modified capsid protein is a naturally occurring serotype engineered to comprise one or more genetic modifications (mutation, insertions or deletions). In an alternative embodiment, the AAV capsid is composed of one or more of a 23 37937 / 59605 / FW / 18328338.1synthetic capsid protein. In particular embodiments, the AAV capsid is engineered to modify the natural tropism, e.g., to reduce heparin binding.

[0114] In the framework of the present disclosure, a synthetic capsid includes any combination of capsid proteins from natural, genetically modified and artificially created serotypes (e.g., random mutations, sequence shuffling, in silico design, etc.) that are able to assemble and produce a new AAV that is not known to exist in nature.

[0115] 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 transduce different 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 differentiate between capsid protein motifs that bind specific cell surface receptors for cell attachment. In the context of the present disclosure, an AAV capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 is preferred. However, it should be understood that any other AAV capsid can be used.

[0116] In one embodiment, the adeno associated virus (AAV) particle is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The most commonly used gene transfer systems to date are derivatives of viruses, e.g., adeno- associated virus type 2 (AAV2), AAV9, and AAV8. In particular embodiments, the AAV capsid is selected from AAV2 and AAV9, where the capsid proteins are optionally further engineered to reduce or modify native tropism, e.g., to reduce heparin binding. In some embodiments, the AAV particle is selected from AAV2, AAV5, and AAV9. In some embodiments, the AAV particle is AAV2. In some embodiments, the AAV particle is AAV9. In some embodiments, the AAV particle is AAV5.

[0117] In particular embodiments, the binding site of the AAV capsid that enables binding to heparin sulfate proteoglycans has been removed.

[0118] In particular embodiments, removal of the heparin binding has been engineered by replacing at least one of arginine 585 or arginine 588 of VP1 and / or an analogous arginine in VP2 24 37937 / 59605 / FW / 18328338.1or VP3 with a different amino acid, such as alanine. In some embodiments, at least one of arginine 448 and arginine 451 in VP2 or 383 and 386 in VP3 is altered.

[0119] In particular embodiments, the AAV of the present disclosure is comprised of at least one capsid protein that is mutated from wild-type, e.g., wherein the engineered / mutated protein is selected from wild-type protein is VP1, VP2, and / or VP3. Alternatively, two of the proteins VP1, VP2 and / or VP3 in said capsid are mutated, or all three of the proteins VP1, VP2 and VP3 in said capsid are modified. In particular embodiments, at least one part, e.g., one amino acid, of the at least one of the proteins to be modified in said capsid is mutated (replaced, inserted or deleted). However, it is also possible to mutate multiple parts of the proteins VP1, VP2 and VP3 in said capsid, e.g., multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of parts or amino acids. In particular embodiments, at least one of arginines 484, 487, 585 and 588 and lysine 532 of VP1, and / or an analogous arginine in VP2 or VP3, are replaced with a different amino acid, such as alanine.

[0120] Primary amines exist at the N-terminus of each capsid protein and in the side-chain (epsilon) of lysine (Lys, K) amino acid residues in the capsid protein sequence. These primary amines are surface available and therefore capable of reacting with a TFP ester, e.g., of the capsid-reactive linker (infra) to provide amide bonds. 4.3.6. Ligands

[0121] The ligand for use with the present disclosure is not particularly limited, as long as the ligand is amenable to conjugation as described herein. In some embodiments, the ligand is selected from a protein ligand having a cognate that is located on the surface of mammalian cells, such as receptors. In some of these embodiments, the cognate protein is involved in transduction of the surface modified viral capsid.

[0122] 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 25 37937 / 59605 / FW / 18328338.1(don't eat me signal); IgG proteases such as IdeZ and IdeS; protein based and small molecule adjuvants for vaccination.

[0123] 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.

[0124] According to one embodiment, a cell-type specific ligand is derived from mono- or polysaccharides such as galactose, N-acetylgalactosamine and mannose.

[0125] According to one embodiment, a cell-type specific ligand is derived from vitamins such as folates.

[0126] According to one embodiment, a cell-type specific ligand is derived from small molecules including naproxen, ibuprofen or other known protein-binding molecules.

[0127] 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 some embodiments, the lectin is selected from wheat germ agglutinin (WGA), isolectin B4 (IB4), Maackia amurensis lectin, Lens culinaris lectin, Wisteria floribunda lectin, and Pha-L. In some embodiments, the lectin is WGA. In some embodiments, the lectin is IB4.

[0128] 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. 26 37937 / 59605 / FW / 18328338.14.3.7. Cargo

[0129] In some embodiments, nucleic acid cargo is packaged inside the surface modified capsid of the present disclosure. The nucleic acid cargo can be any kind of nucleic acid molecule usefully transduced into cells by rAAV.

[0130] In some embodiments, the payload or cargo of the rAAV of the present disclosure 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.

[0131] In some embodiments, the payload provides a DNA homology construct for homology directed repair.

[0132] 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.

[0133] In some embodiments, the cargo encodes an immunogenic polypeptide, e.g., for vaccination. The nucleic acid may encode any immunogen of interest known in the art including, but are not limited to, immunogens from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like. Alternatively, the immunogen can be presented in the virus capsid (e.g., incorporated therein) or tethered to the virus capsid (e.g., by covalent modification).

[0134] An immunogenic polypeptide, or immunogen, may be any polypeptide suitable for protecting the subject against a disease, including but not limited to microbial, bacterial, protozoal, 27 37937 / 59605 / FW / 18328338.1parasitic, fungal and viral diseases. For example, the immunogen may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIV matrix / capsid proteins, and the HIV or SIV gag, pol and env genes products). The immunogen may also be an arenavirus immunogen (e.g., Lassa fever virus immunogen, such as the Lassa fever virus nucleocapsid protein gene and the Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as the vaccinia L1 or L8 genes), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as NP and OP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as the human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen such as a S [S1 or S2], M, E, or N protein or an immunogenic fragment thereof). The immunogen may further be a polio immunogen, herpes immunogen (e.g., CMV, EBV, HSV immunogens) mumps immunogen, measles immunogen, rubella immunogen, diphtheria toxin or other diphtheria immunogen, pertussis antigen, hepatitis (e.g., hepatitis A, hepatitis B or hepatitis C) immunogen, or any other vaccine immunogen known in the art.

[0135] In some embodiments, the immunogen may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of the cancer cell. Exemplary cancer and tumor cell antigens are described in S. A. Rosenberg, (1999) Immunity 10:281). Illustrative cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, NY-ESO-1, CDK-4, β- catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124) including MART-1 (Coulie et al., (1991) J. Exp. Med. 180:35), gp100 (Wick et al., (1988) J. Cutan. Pathol.4:201) and MAGE antigen (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), 28 37937 / 59605 / FW / 18328338.1CEA, TRP-1; TRP-2; P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER- 2 / neu gene product (U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5 (endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialyl Tn antigen); c-erbB- 2 proteins; PSA; L-CanAg; estrogen receptor; milk fat globulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem.62:623); mucin antigens (international patent publication WO 90 / 05142); telomerases; nuclear matrix proteins; prostatic acid phosphatase; papilloma virus antigens; and antigens associated with the following cancers: melanomas, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer and others (see, e.g., Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

[0136] In some embodiments, the nucleic acid cargo encodes any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo. For example, the virus vectors may be introduced into cultured cells and the expressed protein product isolated therefrom.

[0137] It will be understood by those skilled in the art that the nucleic acid cargo of interest may be operably associated with appropriate control sequences. For example, the nucleic acid cargo may be operably associated with expression control elements, such as transcription / translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, and the like.

[0138] Those skilled in the art will further appreciate that a variety of promoter / enhancer elements may be used depending on the level and tissue-specific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0139] Promoter / enhancer elements can be native to the target cell or subject to be treated and / or native to the nucleic acid cargo. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer 29 37937 / 59605 / FW / 18328338.1element is a mammalian promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0140] Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the nucleic acid cargo. Inducible promoters / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and / or smooth muscle), neural tissue specific or preferred (including brain-specific), eye (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter / enhancer elements. In one embodiment, a CNS cell-specific or CNS cell- preferred promoter is used. Examples of neuron-specific or preferred promoters include, without limitation, neuronal-specific enolase, synapsin, and MeCP2. Examples of astrocyte-specific or preferred promoters include, without limitation, glial fibrillary acidic protein and S100β. Examples of ependymal cell-specific or preferred promoters include, without limitation, wdr16, Foxj1, and LRP2. Examples of microglia-specific or preferred promoters include, without limitation, F4 / 80, CX3CR1, and CD11b. Examples of oligodendrocyte-specific or preferred promoters include, without limitation, myelin basic protein, cyclic nucleotide phosphodiesterase, proteolipid protein, Gtx, and Sox10. Use of a CNS cell-specific or preferred promoter can increase the specificity achieved by the AAV vector. Other inducible promoter / enhancer elements include hormone- inducible and metal-inducible elements. Exemplary inducible promoters / enhancer elements include, but are not limited to, a Tet on / off element, a RU486-inducible promoter, an ecdysone- inducible promoter, a rapamycin-inducible promoter, and a metallothionein promoter.

[0141] In embodiments wherein the nucleic acid cargo is transcribed and then translated in the target cells, specific initiation signals are generally employed for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0142] 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 30 37937 / 59605 / FW / 18328338.1inserted into a host genome. In certain of these embodiments, the cargo comprises a transgene known to be associated with a genetic disorder.

[0143] 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). 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).

[0144] In some embodiments, the vector is an “empty” capsid particle (i.e., does not contain a vector genome) comprising, consisting of, or consisting essentially of the chimeric AAV capsid proteins of the disclosure. The chimeric AAV capsids of the disclosure can be used as “capsid vehicles,” as has been described in U.S. Pat. No. 5,863,541, Molecules that can be covalently linked, bound to or packaged by the virus capsids and transferred into a cell include DNA, RNA, a lipid, a carbohydrate, a polypeptide, a small organic molecule, or combinations of the same. Further, molecules can be associated with (e.g., “tethered to”) the outside of the virus capsid for transfer of the molecules into host target cells. In one embodiment of the present disclosure the molecule is covalently linked (i.e., conjugated or chemically coupled) to the capsid proteins. Methods of covalently linking molecules are known by those skilled in the art.

[0145] The capsids of the present disclosure also find use in raising antibodies against the novel capsid structures. As a further alternative, an exogenous amino acid sequence may be inserted into the virus capsid for antigen presentation to a cell, e.g., for administration to a subject to produce an immune response to the exogenous amino acid sequence. 31 37937 / 59605 / FW / 18328338.14.4. Method of Making a Surface Functionalized Capsid Composition

[0146] In an aspect of the present disclosure, a method for preparing the compositions described hereinabove comprising surface functionalized viral capsids is provided. The method described herein uses relatively low amounts of capsid-reactive linker (i.e., TFP ester-containing capsid- reactive linker) compared to previous processes that relied on an NHS ester containing capsid- reactive linker. The reduced amount of capsid-reactive linker eliminates the need for a post- reaction purification step, thus preventing viral loss and improved cell transduction compared to surface modified viral capsids made using the NHS ester-containing capsid-reactive linker.

[0147] The method comprises step (a): combining a viral capsid (I) comprising a plurality of surface available primary amines (p) and a capsid-reactive linker (II) comprising a terminal tetrafluorophenyl (TFP) ester and a terminal first member of a crosslinker reactive pair (CRP1) to provide a composition comprising surface functionalized viral capsid (III), as shown in Scheme 1 below:Scheme 1. Method of making a surface functionalized viral capsid.

[0148] An amide bond is formed from reaction of a primary amine of, for example, a surface available lysine residue of the capsid primary sequence, and the TFP ester-containing capsid- reactive linker as shown above (only one surface lysine amine is shown for clarity).

[0149] A plurality of surface available primary amines (p) will react with a plurality of TFP esters of the capsid-reactive linkers. In some embodiments, p is an integer from 10 to 500, such as, for example, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 50 to 500, from 50 to 400, from 50 to 300, from 50 to 200, from 50 to 100, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 500, from 200 to 400, from 200 to 300, from 300 to 500, from 300 to 400, or from 400 to 500. In some embodiments, p is an integer from 100 to 200, such 32 37937 / 59605 / FW / 18328338.1as, for example, from 100 to 190, from 100 to 180, from 100 to 170, from 100 to 160, or from 100 to 150.

[0150] The variable “q” in the surface functionalized viral capsid (III) reflects the number of amides formed by reaction of the surface available primary amines (p) of the viral capsid (I) with the TFP esters of the capsid-reactive linkers (II). In some embodiments q is the same as p. In some embodiments, q is an integer from 10 to 500, such as, for example, from 10 to 400, from 10 to 300, from 10 to 200, from 10 to 100, from 50 to 500, from 50 to 400, from 50 to 300, from 50 to 200, from 50 to 100, from 100 to 500, from 100 to 400, from 100 to 300, from 100 to 200, from 200 to 500, from 200 to 400, from 200 to 300, from 300 to 500, from 300 to 400, or from 400 to 500. In some embodiments, q is an integer from 100 to 200, such as, for example, from 100 to 190, from 100 to 180, from 100 to 170, from 100 to 160, or from 100 to 150.

[0151] SP1is a bond or one or more PEGs (i.e., -(−(O−CH2−CH2)n)- or -([PEG]n)-). In some embodiments, n is 0. In some embodiments, n is from 1 to 100, such as, for example, from 1 to 50, from 1 to 25, from 1 to 15, from 1 to 10, from 1 to 5, from 4 to 20, from 4 to 15, from 4 to 12, or from 4 to 10.

[0152] In some embodiments, the molar ratio of viral capsid (I) to capsid-reactive linker (II) in reaction (a) is from 1:100 to 1:50,000 such as, for example, from 1:100 to 1:33,000; from 1:100 to 1:10,000; from 1:100 to 1:5,000; from 1:100 to 1:1,000; from 1:100 to 1:500; from 1:100 to 1:250. In some embodiments, the molar ratio of viral capsid (I) to capsid-reactive linker (II) is from 1:500 to 1:10,000.

[0153] In some embodiments, the viral capsid (I) is purified prior to reaction with the capsid- reactive linker (II). It has been found that lower molar ratios of viral capsid to capsid-reactive linker are needed to prepare compositions with strong viral cell transduction when the viral capsid is first purified. Suitable purification methods include, but are not limited to, density gradient centrifugation, chromatography (e.g., ion exchange chromatography or affinity chromatography), and combinations thereof. In some embodiments, the density gradient centrifugation is iodixanol density gradient ultracentrifugation.

[0154] In some embodiments, the viral capsid is used without purification. In some embodiments, the chemical modification is performed directly on crude cell lysates (e.g., unpurified AAV). In some embodiments, the cell lysates (e.g., AAV9, AAV2-VR4) can be 33 37937 / 59605 / FW / 18328338.1effectively modified without purification using, for example, NHS and TFP esters of the capsid- reactive linkers. In some embodiments, AAV9 can be effectively modified without purification using TFP esters of the capsid-reactive linkers. In some embodiments, AAV9 can be effectively modified without purification using NHS esters of the capsid-reactive linkers. In some embodiments, AAV2-VR4 can be effectively modified without purification using TFP esters of the capsid-reactive linkers. In some embodiments, AAV2-VR4 can be effectively modified without purification using NHS esters of the capsid-reactive linkers. In some embodiments, lower concentrations of TFP esters are required compared to NHS.

[0155] In some embodiments, the viral capsid is freeze-thawed prior to surface functionalization.

[0156] In certain embodiments, the reaction is carried out in an aqueous medium. Suitable aqueous mediums include at least one buffering agent. In some embodiments, the buffering agent is selected from N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, and bicarbonate. In some embodiments, the concentration of the at least one buffering agent varies. In some embodiments, the concentration of the at least one buffering agents does not exceed about 0.1M, e.g., from about 0.01 M to about 0.1M or from about 0.05 M to about 0.1M. In some embodiments, the aqueous medium comprises from about 0.01M to about 0.1M sodium bicarbonate.

[0157] In some embodiments, the aqueous medium comprises at least about 200 mM of at least one salt, such as, for example, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 500 mM. In some embodiments, the salt is selected from a chloride salt, a phosphate salt, sulfate salt, and citrate salt. In some embodiments, the salt is selected from a sodium salt, a potassium salt, a calcium salt, and a magnesium salt. In some embodiments, the aqueous medium comprises at least about 200 mM sodium chloride.

[0158] In some embodiments, the ionic strength of the aqueous medium is at least about 150 mM, such as, for example, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 500 mM. 34 37937 / 59605 / FW / 18328338.1

[0159] In some embodiments, the aqueous medium further comprises at least one surfactant. In some embodiments, the surfactant is Pluronic® F68. In some embodiments, the aqueous reaction medium comprises from about 0.001% to about 0.005% Pluronic® F68.

[0160] In some embodiments, the pH of the aqueous medium is from about 6 to about 10, such as, for example, from about 7 to about 10, from about 8 to about 10, from about 9 to about 10, from about 6 to about 9, from about 7 to about 9, from about 8 to about 9, from about 6 to about 8, from about 7 to about 8, or from about 6 to about 7. In some embodiments, the pH of the aqueous medium is from about 8 to about 9.

[0161] In some embodiments, the temperature of the reaction is from about 0 ºC to about 50 ºC, such as, for example, from about 10 ºC to about 40 ºC or from about 20 ºC to about 30 ºC. In some embodiments, the temperature of the reaction is room temperature (ca.23 ºC).

[0162] In some embodiments, the duration of the reaction is from about 5 minutes to about 24 hours, such as, for example, from about 30 minutes to about 24 hours, or from about 1 hour to about 24 hours.

[0163] In some embodiments, following reaction completion, a quenching agent is added in excess to consume unbound linker. In some embodiments, the quenching agent comprises an amine- containing compound. In some embodiments, the quenching agent is selected from glycine and Tris buffer.

[0164] In some embodiments, no purification step is performed following formation of the surface functionalized viral capsid. Exemplary purification methods to be avoided include centrifugation, precipitation, and chromatography. It has been found that, in some embodiments, use of these purification steps results in loss of viral particles.

[0165] In embodiments where no purification is performed, the resulting composition will contain some amounts of starting materials and reagents in addition to surface functionalized viral capsid product.

[0166] The method of the present disclosure further comprises step (b): reacting the composition comprising the surface functionalized viral capsid (III) comprising the first member of a crosslinker reactive pair (CRP1) with a functionalized ligand comprising the second member of 35 37937 / 59605 / FW / 18328338.1the crosslinker reactive pair (CRP2) to provide a composition comprising the surface modified viral capsid (V).

[0167] The first and second crosslinker reactive pairs react to form crosslinked moiety Q (discussed hereinabove), thereby conjugating the viral capsid and ligand, as shown in Scheme 2, below:Scheme 2. Conjugation of a viral capsid and ligand. In scheme 2,is the functionalized ligand, SP2is a bond or spacer, and L is the ligand.

[0168] In some embodiments, the functionalized ligand is purchased from a commercial vendor. In other embodiments, the functionalized ligand is prepared by contacting a ligand comprising a first reactive moiety and a ligand-reactive linker comprising a second reactive moiety and the second member of the crosslinker reactive pair (CRP2).

[0169] In some embodiments, the ligand-reactive linker is selected from TCO-PEGn-NHS; tetrazine-PEGn-NHS; azido-PEGn-NHS; phosphine-NHS; maleimide-PEGn-succinimidyl ester; DBCO-PEGn-TFP ester, and DBCO-PEGn-NHS ester, wherein n is from 1 to 100, preferably from 4 to 10.

[0170] In some embodiments, the ligand-reactive linker is DBCO-PEGn-TFP ester, wherein n is from 1 to 100, preferably from 4 to 10. In some embodiments, the ligand-reactive linker is DBCO-PEGn-NHS ester, wherein n is from 1 to 100, preferably from 4 to 10. In some embodiments, the ligand-reactive linker is DBCO-PEG4-TFP. In some embodiments, the ligand- reactive linker is DBCO-PEG5-TFP. In some embodiments, the ligand-reactive linker is DBCO- PEG6-TFP. In some embodiments, the ligand-reactive linker is DBCO-PEG7-TFP. 36 37937 / 59605 / FW / 18328338.1

[0171] In some embodiments, ligands functionalized for use with the present disclosure are as described in WO2022101363, the entirety of which is incorporated herein by reference.

[0172] In some embodiments, SP2of the functionalized ligand is selected from -Y-C(O)-, -Y- C(O)O-, -Y-NHC(O)-, -Y-NHC(S)-, or -Y-C(O), wherein Y is a bond or one or more PEGs (i.e., -(−(O−CH2−CH2)n)- or -([PEG]n)-). In some embodiments, SP2is -([PEG]n)-C(O)-, wherein n is from 1 to 100, such as, for example, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 4 to 15, or from 4 to 10. In some embodiments, SP2is -([PEG]n)-C(O)-, wherein n is from 1 to 100, preferably from 1 to 10.

[0173] In some embodiments, the functionalized ligand is of formula IV:wherein: is a ligand.

[0174] In such embodiments, surface available primary amines (shown above) are conjugated to SP2.

[0175] In some embodiments, CRP2 comprises a reactive moiety selected from an azide; alkyne; 1,4-triazole; 1,3-nitrone; cyclooctyne or derivative thereof, e.g., dibenzylcyclooctyne or derivative thereof; triazine; tetrazine; strained dienophile; aryl or alkyl phosphine; isocyanide; benzylguanine group, a benzylcytosine group, or a chloroalkane group. In certain of these embodiments, CRP2 comprises an azide. In some embodiments, CRP2 consists of -N3.

[0176] In some embodiments, the functionalized ligand is WGA-[PEG]n-N3(also referred to herein as WGA-[PEG]n-azide), wherein n is from 1 to 20, such as, for example, from 1 to 10, or from 4 to 10. In some embodiments, the functionalized ligand is WGA-PEG4-Azide. In some embodiments, the functionalized ligand is WGA-PEG5-Azide. In some embodiments, the functionalized ligand is WGA-PEG6-Azide. In some embodiments, the functionalized ligand is WGA-PEG7-Azide. 37 37937 / 59605 / FW / 18328338.1

[0177] In some embodiments, the functionalized ligand is NGF -[PEG]n-N3 (also referred to herein as NGF-[PEG]n-azide), wherein n is from 1 to 20, such as, for example, from 1 to 10, or from 4 to 10. In some embodiments, the functionalized ligand is NGF-[PEG]n-azide, wherein n is from 1 to 20, preferably 4 to 10. In some embodiments, the functionalized ligand is NGF-PEG4-Azide. In some embodiments, the functionalized ligand is NGF-PEG5-Azide. In some embodiments, the functionalized ligand is NGF-PEG6-Azide. In some embodiments, the functionalized ligand is NGF-PEG7-Azide.

[0178] In some embodiments, the molar ratio of viral capsid (used in step (a)) to functionalized ligand (used in step (b)) is from 1:100 to 1:50,000 such as, for example, from 1:100 to 1:33,000; from 1:100 to 1:10,000; from 1:100 to 1:5,000; from 1:100 to 1:1,000; from 1:100 to 1:500; from 1:100 to 1:250. In some embodiments, the molar ratio of viral capsid to functionalized ligand is from 1:500 to 1:10,000.

[0179] In some embodiments, the molar ratio viral capsid to capsid-reactive linker in step (a) and the molar ratio of viral capsid to functionalized ligand step (b) are the same, i.e., 1:1. In some embodiments, the molar ratio viral capsid to capsid-reactive linker in step (a) and the molar ratio of viral capsid to functionalized ligand step (b) are different.

[0180] In certain embodiments, the reaction is carried out in an aqueous medium. Suitable aqueous mediums include at least one buffering agent. In some embodiments, the buffering agent is selected from N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, and bicarbonate. The concentration of the at least one buffering agent can vary. In some embodiments, the concentration of the at least one buffering agents does not exceed about 0.1M, e.g., from about 0.01 M to about 0.1M or from about 0.05 M to about 0.1M. In some embodiments, the aqueous medium comprises from about 0.01M to about 0.1M sodium bicarbonate.

[0181] In some embodiments, the aqueous medium comprises at least about 200 mM of at least one salt, such as, for example, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 500 mM. In some embodiments, the salt is selected from a chloride salt, a phosphate salt, sulfate salt, and citrate salt. In some embodiments, the salt is selected from a sodium salt, a potassium salt, a calcium salt, and a magnesium sat. In some embodiments, the aqueous medium comprises at least about 200 mM sodium chloride. 38 37937 / 59605 / FW / 18328338.1

[0182] In some embodiments, the ionic strength of the aqueous medium is at least about 150 mM, such as, for example, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 500 mM.

[0183] In some embodiments, the aqueous medium further comprises at least one surfactant. In some embodiments, the surfactant is Pluronic® F68. In some embodiments, the aqueous reaction medium comprises from about 0.001% to about 0.005% Pluronic® F68.

[0184] In some embodiments, the pH of the aqueous medium is from about 6 to about 10, such as, for example, from about 7 to about 10, from about 8 to about 10, from about 9 to about 10, from about 6 to about 9, from about 7 to about 9, from about 8 to about 9, from about 6 to about 8, from about 7 to about 8, or from about 6 to about 7. In some embodiments, the pH of the aqueous medium is from about 8 to about 9.

[0185] In some embodiments, the temperature of the reaction is from about 0 ºC to about 50 ºC, such as, for example, from about 10 ºC to about 40 ºC or from about 20 ºC to about 30 ºC. In some embodiments, the temperature of the reaction is room temperature (ca.23 ºC).

[0186] In some embodiments, the duration of the reaction is from about 5 minutes to about 24 hours, such as, for example, from about 30 minutes to about 24 hours, or from about 1 hour to about 24 hours.

[0187] In some embodiments, the surface modified viral capsid is purified. Exemplary purification methods include centrifugation, precipitation, and chromatography. In some embodiments, the purification method is centrifugal ultrafiltration. In some embodiments, the purification method is affinity chromatography.

[0188] In some embodiments, the surface modified viral capsid is bulk modified.

[0189] In some embodiments, the surface modified viral capsid is stable at low temperatures, including -150oC (e.g., -140oC, -130oC, -120oC, -110oC, -100oC, -90oC, -80oC, -70oC, -60oC, - 50oC, -40oC, -30oC, -20oC, or -10oC) for at least 3 weeks (e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, or at least 15 weeks). 39 37937 / 59605 / FW / 18328338.1

[0190] In some embodiments, the surface modified viral capsid is stable at -140oC. In some embodiments, the surface modified viral capsid is stable at -130oC. In some embodiments, the surface modified viral capsid is stable at -120oC. In some embodiments, the surface modified viral capsid is stable at -110oC. In some embodiments, the surface modified viral capsid is stable at -100oC. In some embodiments, the surface modified viral capsid is stable at -90oC. In some embodiments, the surface modified viral capsid is stable at -80oC. In some embodiments, the surface modified viral capsid is stable at -70oC. In some embodiments, the surface modified viral capsid is stable at -60oC. In some embodiments, the surface modified viral capsid is stable at - 50oC. In some embodiments, the surface modified viral capsid is stable at -40oC. In some embodiments, the surface modified viral capsid is stable at -30oC. In some embodiments, the surface modified viral capsid is stable at -20oC. In some embodiments, the surface modified viral capsid is stable at -10oC.

[0191] In some embodiments, the surface modified viral capsid is stable for at least 4 weeks. In some embodiments, the surface modified viral capsid is stable for at least 5 weeks. In some embodiments, the surface modified viral capsid is stable for at least 6 weeks. In some embodiments, the surface modified viral capsid is stable for at least 7 weeks. In some embodiments, the surface modified viral capsid is stable for at least 8 weeks. In some embodiments, the surface modified viral capsid is stable for at least 9 weeks. In some embodiments, the surface modified viral capsid is stable for at least 10 weeks. In some embodiments, the surface modified viral capsid is stable for at least 11 weeks. In some embodiments, the surface modified viral capsid is stable for at least 12 weeks. In some embodiments, the surface modified viral capsid is stable for at least 13 weeks. In some embodiments, the surface modified viral capsid is stable for at least 14 weeks. In some embodiments, the surface modified viral capsid is stable for at least 15 weeks. 4.5. EXAMPLES Example 1. Significant loss in physical titre of AAV2 after NHS modification

[0192] In previous experiments conducted using the NHS ester as the capsid reactive moiety, it was determined that it was necessary to perform two separate clean-up steps to produce a ligand functionalized capsid composition that exhibited strong transduction. First, clean up was required 40 37937 / 59605 / FW / 18328338.1after capsid functionalization with linker, e.g., the NHS-PEGn-DBCO, to remove excess reactive linker since this would effectively quench the functionalized ligand during the crosslinking step and result in contaminating the final product with an excess of ligand crosslinked to the NHS- PEGn-DBCO linker that would compete with AAV capsid transduction.

[0193] While transitioning to in vivo experiments with an NGF modified vector, it was measured for the first time physical AAV titre before and after modification, and unexpectedly observed a 90% loss of AAV particles during the modification process (FIG. 1). This result was surprising because, despite the 90% loss, we were still able to see significant boosting of transduction efficiency when conjugating ligands such as WGA.

[0194] Commercially available wildtype AAV2 with an EGFP cargo as reporter gene of cell transduction was used with a viral titer of 2.7E+12Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM. This AAV2 was modified with NHS-PEG4-DBCO (2.6nMol) in 20ul of PBS / Pluronic 0.001% / NaCl 200mM for 3 hours at room temperature, and then reacted with 55pMol of WGA-PEG4- Azide for 1 hour at room temperature followed by 4 ºC overnight. The surface modified virus was either (1) added directly to PC12 cells or (2) centrifuged in an Amicon 100Kda centrifugal unit one to three times for 1 minute at 10000g.

[0195] As shown in FIG. 1, modification with NHS-PEG4-DBCO resulted in a 90% reduction in AAV2 titre as measured by ddPCR. To investigate this further, WGA conjugated AAV2 to was applied to PC12 cells and transduction efficiency before and after reaction clean-up was monitored. From these experiments it became apparent that loss of virus occurred after a single centrifuge step with a 100Kda centrifugal filter unit. Further centrifugation did not significantly reduce yields. Other clean-up procedures were evaluated including passivation of filter units, precipitation of AAV2, and size exclusion chromatography, with transduction efficiency in HEK293 cells and ddPCR of physical titre as readouts of viral loss.

[0196] In further experiments, wildtype AAV2 was applied directly to HEK293 cells, or one or more of the following clean-up procedures were performed: 1. Centrifugation through a 100Kda Amicon centrifuge unit at 10000g for 1 minute. 41 37937 / 59605 / FW / 18328338.12. Passivation of 100Kda Amicon centrifugation unit by pre-treatment with 3E+9 Vg of wildtype AAV2 carrying a tdTomato reporter for 60 minutes, followed by centrifugation of the modified AAV2 at 10000g for 1 minute. 3. Precipitation of modified AAV2 by incubation in 8% PEG8000 for 1hour at 4 ºC with slow stirring, then for 3 hours at 4 ºC without stirring to allow full precipitation. Then, samples were centrifuged at 2818g for 30 min at 4 ºC, followed by removal of the supernatant and resuspension of the pellet in PBS / Pluronic 0.001% / NaCl 200mM. 4. Size exclusion chromatography by centrifuging modified AAV2 through a column packed with Sepharose 4B resin at 1000g for 2 minutes. Samples were then concentrated to 24ul using a vacuum concentrator at 45 °C.

[0197] Separately, AAV2 (0.0045pMol) was modified with capsid-reactive linker TFP-PEG4- DBCO (45pMol) in PBS / Pluronic 0.001% / NaCl 200mM at room temperature overnight. The reaction was then quenched to remove residual linker with 50mM glycine.

[0198] Samples were analyzed by adding to HEK293 cells, which are permissive to AAV2, and monitoring transduction efficiency using imaging and FACS with a Bio-Rad S3e Cell Sorter, and by performing ddPCR using primers against the ITR region to quantify physical titer.

[0199] As can be seen in FIGs.2a and 2b, all clean-up methods resulted in a substantial reduction in AAV2 recovery, with centrifugal filter units (both untreated and passivated) performing worst, PEG precipitation improved yields slightly, and Sepharose 4B size exclusion marginally increasing recovery further. In contrast, modification of AAV2 with the TFP linker, which did not require a clean-up step, did not lead to a reduction in yield of modified virus as demonstrated by transduction efficiency in HEK293 cells and physical titre as measured by ddPCR. Example 2. Reaction conditions for use with the TFP ester capsid reactive group.

[0200] Commercially available wildtype AAV2 with EGFP cargo as reporter gene of cell transduction was used. The virus titer was 2.7E+12Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0201] 0.0045pMol of AAV (1ul) was reacted with 448pMol of NHS-PEG4-DBCO (ratio of virus:linker was 1:100,000) in 22ul of PBS / Pluronic 0.001% / NaCl 200mM for 3 hours at room temperature. Following this, 55pMol of WGA-PEG4-Azide (ratio of virus:ligand was 1:12,000) 42 37937 / 59605 / FW / 18328338.1was incubated for 2 hours at room temperature, then kept at 4 ºC overnight. The following day, the virus was added to the cells.

[0202] 0.0045pMol of AAV was incubated with a reduced amount of linker i.e., 148pMol of TFP- PEG4-DBCO (ratio virus:linker was1:30,000) in 22ul of final volume, overnight at room temperature. The following day glycine (50mM pH 6.5) was added to quench any unbound, active TFP-PEG4-DBCO. Then, 148pMol of WGA-PEG4-Azide (ratio virus:ligand was 1:30,000) was incubated overnight at room temperature. The following day, the virus was added to the cells. Various reaction parameters were changed including use of (a) reaction buffers (i.e., different preparations of sodium bicarbonate or PBS / Pluronic 0.001% / NaCl 200mM) and (b) and quenching reagent. For each protocol tested, the proper control (unmodified virus) was processed and used for normalizing the data of cytometry. A summary of the protocols evaluated is provided in the table below. Table 1. Summary of the Protocols Reaction Reaction buffer Reaction Quenched time with glycine 1 0.1M Sodium Bicarbonate pH 8.5 in water 2 days Yes 2 0.1M Sodium Bicarbonate pH 8.5 in 2 days Yes PBS / Pluronic 0.001% / NaCl 200mM 3 0.1M Sodium Bicarbonate pH 8.5 in water 2 days No 4 PBS / Pluronic 0.001% / NaCl 200mM 2 days Yes 5 0.2M Sodium Bicarbonate pH 8.5 in water 2 days Yes Control with PBS / Pluronic 0.001% / NaCl 200mM 1 day No NHS In vitro application to PC12 cells

[0203] PC12 cells were maintained at 37 ºC 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 prepared with the NHS linker or TFP linker. Media was then replaced, and cells were maintained at 37 ºC and imaged at 4 days after infection with Zeiss AxioObserver A1 microscope. At 7 days post infection, cells were collected and analysis with Bio-Rad’s S3e Cell Sorter. Results 43 37937 / 59605 / FW / 18328338.1

[0204] From the imaging analysis (FIG. 3), it was clear that reactions 2, 3, and 4 were most efficient. Without wishing to be bound by theory, it is believed that PBS / Pluronic 0.001% / NaCl 200mM may prevent virus aggregation and that higher concentrations of sodium bicarbonate disfavor the conjugation of the TFP linker to the virus.

[0205] For the cytometry analysis, mean fluorescent intensity (MFI) of EGFP was used as the indicator of how much virus can enter each cell (FIG. 4a), and the EGFP+ cells were used to define the percentage of cells transduced by AAV (FIG. 4b). Both MFI and EGFP+ cell percentage were normalized by the controls. Among the protocols with TFP that led to good cell transduction, the conditions associated with reaction 2 were the best. Based on the comparisons of reactions 2-4, we concluded that glycine and 0.1M sodium bicarbonate improved the modification of the virus with TFP linker, thus enabling good cell transduction.

[0206] In addition, a similar amount of WGA liganded-virus entered the cell with either TFP reaction 2 or NHS. However, the percentage of transduced cells was higher with TFP reaction 2 compared to NHS. This data, together with the fact that three times less TFP-PEG4-DBCO linker was used compared to NHS-PEG4-DBCO, suggests that TFP linker chemistry improves the chemical functionalization of AAV, and can be performed at lower molar ratios, thereby circumventing the need for reaction clean-up. Example 3. Comparison of TFP- and NHS-linkers in vitro AAV2 and AAV5

[0207] Both AAV serotypes used were commercially purchased. AAV2 had a titer of 2.7E+12 vg / ml and contained as a cargo an EGFP reporter gene under a CAG promoter, while AAV5 had a titer of 1E+13 vg / ml and contained an EGFP reporter gene under a CMV promoter. NHS-mediated chemical functionalization and coupling of WGA to AAV2 and AAV5

[0208] For NHS-mediated chemical functionalization of AAV2, 1µl (0.0045 pMol) was reacted with DBCO-PEG4-NHS at different molar virus:linker ratios (see table below) in a reaction volume of 20µl PBS (+0.001% pluronic and 200mM NaCl) for 3 hours shaking at room temperature. 44 37937 / 59605 / FW / 18328338.1Table 2 molar ratio virus:linker virus linker 1 3,300 1 10,000 1 33,000 1 100,000 1 330,000

[0209] WGA (0.1nMol) was dissolved in PBS and reacted with a 20-fold molar equivalent of Azide-PEG4-NHS for 3 hours shaking at room temperature. Unreacted Azide groups were removed using a 10 kDa MWCO centrifugal filter. NHS-mediated DBCO modified AAV2 was further incubated with 50pMol WGA-PEG4-Azide for 2 hours shaking at room temperature and then kept at 4°C overnight.

[0210] The NHS-mediated chemical functionalization of 1µl (0.0166 pMol) AAV5 was performed as described above for AAV2 but with distinct virus:linker ratios as indicated in the table below. Table 3 molar ratio virus:linker virus linker 1 150 1 300 1 1,000 1 3000 1 10,000 1 30,000 1 100,000 TFP-mediated chemical functionalization and coupling of WGA to AAV2 and AAV5

[0211] For TFP-mediated chemical modification, 1µl of AAV2 (0.0045 pMol) was reacted with DBCO-PEG4-TFP and WGA-PEG4-Azide at different molar virus:linker:ligand ratios (see table below). 45 37937 / 59605 / FW / 18328338.1Table 4 molar ratio of virus:linker:ligand virus linker ligand 1 100 100 1 500 500 1 1,000 1,000 1 3,300 3,300 1 10,000 10,000 1 33,000 33,000

[0212] The volume of the DBCO-PEG4-NHS linker was minimized (1µl) by preparing dilutions from a 20mM stock. Further 11µl PBS (+0.001% pluronic and 200mM NaCl) and 0.1M sodium bicarbonate buffer (pH 8.3) were added, and the reaction was kept shaking at room temperature overnight. The reaction was stopped by adding 50mM Glycine. WGA (0.1nMol) was dissolved in PBS and reacted with a 20-fold molar equivalent of Azide-PEG4-NHS for 3 hours shaking at room temperature. Unreacted Azide groups were removed using a 10 kDa MWCO centrifugal filter. WGA-PEG4-Azide was added in the indicated molar ratio (see table above) and the reaction was kept shaking at room temperature overnight.

[0213] The TFP-mediated chemical modification of AAV5 with 1µl (0.0166 pMol) was performed as described above for AAV2 but with distinct virus:linker:ligand ratios as indicated in the table below. Table 5 molar ratio of virus:linker:ligand virus linker ligand 1 500 500 1 1,000 1,000 1 3,000 3,000 1 10,000 10,000 46 37937 / 59605 / FW / 18328338.11 30,000 10,000 1 100,000 10,000 In vitro application to PC12 cells

[0214] PC12 cells were maintained in DMEM / F12 medium (+ 10% horse serum, 5% fetal bovine serum, 15mM HEPES, 2.5mM Glutamax and 100U penicillin / streptomycin) and incubated at 37°C in a humidified atmosphere of 5% CO2. Modified AAVs were added to the cells and incubated overnight. Media was replaced and cells were maintained at 37°C for 5 days. Data acquisition

[0215] Transduction efficiency was determined by imaging PC12 cells with a Nikon A1R confocal microscope. For quantifying efficiency, cells were collected and prepared for flow cytometry. Flow cytometry data were acquired on a S3e Cell sorter from Bio-Rad and analyzed with Flow Jo. Viral genome copy numbers were quantified using Bio-Rad’s Droplet Digital PCR System. Results

[0216] AAV2 modified with DBCO-PEG4-NHS at a virus:linker ratio of 1:100,000 and 2.5uM WGA-PEG4-Azide showed the highest transduction in PC12 cells compared to other virus:linker ratios tested. In contrast, AAV2 modified with DBCO-PEG4-TFP and WGA-PEG4-Azide showed the highest transduction at a virus:linker:ligand ratio of 1:10,000:10,000. TFP modification required less linker to provide the same transduction efficiency as with the NHS chemistry. With another AAV serotype, AAV5, it was observed that a lower amount of virus: linker (1:1000 molar ratio) for TFP-mediated modification than NHS-mediated modification (virus:linker molar ratio of 1:30,000) was required to reach the best transduction efficiency. Example 4. Comparison of TFP- and NHS-linkers in vivo

[0217] Through the series of experiments performed with TFP and NHS linkers in vitro, it was demonstrated that TFP chemistry improved the modification of Adeno-Associated-Virus (AAV) with a ligand of interest, prevented viral loss, and was applicable to more than one serotype. Here, the aim was to verify the superior performance of TFP versus NHS modified AAV2 in vivo in mice. As a ligand, a mutant Nerve Growth Factor ligand (NGFR121W) was chosen that targets a 47 37937 / 59605 / FW / 18328338.1population of nociceptive peptidergic neurons expressing the receptor TrkA / p75 receptor complex. It was further found that AAV2 can be effectively modified using TFP at larger scales. Modified AAV2 was injected subcutaneously in the plantar surface of the hind paw, and transgene expression was analyzed in skin, dorsal root ganglia (DRG) and spinal cord. Methods Chemical functionalization and coupling of NGF to AAV via NHS and TFP chemistry

[0218] For the chemical functionalization we used a wildtype commercially sourced AAV2, with an EGFP reporter gene. The virus titer was 2.7E+12Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0219] The standard functionalization with NHS was performed with 0.2025pMol of AAV (45µl) reacted with 20250pMol of NHS-PEG9-BG (molar ratio virus:linker=1:100,000) in a total volume of 900µl PBS / Pluronic 0.001% / NaCl 200mM for 3 hours at room temperature. After this, the virus was concentrated with an Amicon 100KDa centrifugal filter to remove the excess of unbound linker, and 200pMol of NGF-SNAP was added. The reaction was incubated for 2 hours at room temperature, then kept at 4 ºC overnight. The following day, the virus was concentrated again with an Amicon 100KDa filter to remove the excess of unbound NGF-SNAP.

[0220] For the TFP reaction, we incubated 0.1125pMol of AAV with TFP-PEG4-DBCO at different virus:linker ratios, reported in the table below. The reaction was performed in PBS (+0.001% pluronic and 200mM NaCl) and 0.1M sodium bicarbonate buffer (pH 8.3) at room temperature overnight, quenched with 50mM Glycine, and then incubated with NGF-PEG4- Azide at equimolar ratio with the linker (see table below) for 2 hours at room temperature and then overnight at 4 ºC. Table 6 TFP ratio pMol virus pMol linker pMol ligand virus:linker / ligand (titer 2.7 vg / ml; 25µl) TFP-PEG4-DBCO NGF-PEG4-Azide (in 1µl) 1:100 0.1125 11.25 11.25 1:500 0.1125 56.25 56.25 1:1,000 0.1125 112.5 112.5 1:3,000 0.1125 337.5 337.5 48 37937 / 59605 / FW / 18328338.1TFP ratio pMol virus pMol linker pMol ligand virus:linker / ligand (titer 2.7 vg / ml; 25µl) TFP-PEG4-DBCO NGF-PEG4-Azide (in 1µl) 1:10,000 0.1125 1125 1125

[0221] Bulk modification of AAV9 with NHS and NGF and testing transduction in TrkA / p75 overexpression HEK293 cells

[0222] Scale up was performed for the modification with AAV9 and test transduction in a HEK293 cell line expression TrkA / p75 using NGF as a ligand.

[0223] For the bulk chemical modification, a wildtype commercially sourced AAV9, with an EGFP reporter gene was used. The virus titer was 5.9E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0224] For the NHS reaction, 2832 VG of AAV with NHS-PEG4-DBCO at a virus:linker ratio of 175000 were incubated. The reaction was performed in PBS (+0.001% pluronic and 200mM NaCl) at room temperature overnight, washed with 15ml Amicon falcon 100KDa cutoff for three times, aliquoted and frozen at -80oC. Following, AAV9-DBCO was thawed and incubated with NGF-PEG4-Azide at 3µM for 2 hours at room temperature and then overnight at 4oC. A range of titres (i.e. different MOIs) were added to a HEK293 cell line overexpression TrkA and p75 receptors. As a control, unmodified AAV9 was applied to the cells at the same MOI.

[0225] Bulk modification of AAV2 using TFP and conjugating NGF, testing freeze thaw stability and assaying transduction in vivo in mice

[0226] For the bulk chemical modification, a wildtype commercially sourced AAV2, with an EGFP reporter gene was used. The virus titer was 2.7E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0227] For the TFP reaction, the below titres of AAV with TFP-PEG4-DBCO at a virus:linker ratio of 1:20,000 were incubated. The reaction was performed in PBS (+0.001% pluronic and 200mM NaCl) and 0.1M sodium bicarbonate buffer (pH 8.3) at room temperature overnight, quenched with 50mM Glycine, and then incubated with NGF-PEG4-Azide at 27µM at room temperature overnight. Samples were then either used fresh or frozen as indicated in Table 7. 49 37937 / 59605 / FW / 18328338.1Table 7 Initial volume of Total Vg Volume of 1stVolume of 2ndCondition AAV reaction step reaction step 45 ul 1.22E+11 29.3 ul 74.3 ul Fresh Frozen 1 ml 2.7E+12 650 ul 1.65 ml Frozen 5 ml 1.35E+13 3.25 ml 8.25 ml Frozen 12 ml 3.24E+13 7.8 ml 19.8 ml Frozen ddPCR

[0228] AAV2 modified with NHS and TFP at different ratios was processed for ddPCR using primers targeting the inverted terminal repeats (ITRs) present in the viral genome. In vitro application to PC12 cells

[0229] In order to verify that modified virus was indeed active, we also applied it to PC12 cells in vivo, which express TrkA / p75 endogenously. PC12 cells were maintained at 37 ºC in DMEM / F12 medium containing 5% horse serum 5% fetal bovine serum, and 100 U of penicillin / streptomycin. PC12 cells were incubated with NGF modified AAV2 prepared with NHS and TFP at different ratios. Media was then replaced, and cells were maintained at 37 ºC and imaged at 7 days after infection with Zeiss AxioObserver A1 microscope. Paw injection in mice

[0230] Three hours before virus injection, 60 units of Hyaluronidase were injected to facilitate virus spreading. 5E+10 VG of AAV2 modified with either NHS or TFP at different ratios was injected in the paw of the mice that were anesthetized using isoflurane. The injection was performed using an insulin syringe placed in the center of the paw. The volume of injection was 30µl. Histological analysis

[0231] At 3 weeks after the in vivo injection, the mice were sacrificed, and the lumbar dorsal root ganglia (DRG) were collected. The DRG were fixed overnight in 2% paraformaldehyde (PFA), then washed in PBS, and cleared using ScaleS solution for 2 days at 4C. Following, they were flat mounted and imaged using a Zeiss AxioObserver A1 microscope. 50 37937 / 59605 / FW / 18328338.1

[0232] In other experiments, lumbar DRG were washed in PBS, blocked with 2% donkey / 0.3% Triton / PBS overnight at 4 ºC. Then, they were incubated with 1:200 goat anti-rat Trka antibody in 2% donkey / 0.3% Triton / PBS for 72 hours at 4 ºC. After this, the DRG were washed with 0.3% Triton / PBS for 3 times with intervals of 10-20 minutes. DRG were then incubated with 1:200 donkey anti-goat Ax594 antibody in 2% donkey / 0.3% Triton / PBS for 72 hours at 4 ºC, washed as described before, cleared overnight at 4 ºC, flat mounted, and imaged.

[0233] Skin from the injection site was collected, fixed overnight in 2% paraformaldehyde (PFA), then washed in PBS, and incubated with sucrose 30% / PBS overnight at 4 ºC. It was then embedded in OCT and cut at the cryostat at a thickness of 30 µm. Sections were blocked with 2% donkey / 0.3% Triton / PBS for 2 hours at room temperature. Then, the skin was incubated with 1:200 goat anti-rat Trka and chicken anti-EGFP antibodies in 2% donkey / 0.3% Triton / PBS overnight at 4 ºC. Skin sections were then washed with 0.3% Triton / PBS for 3 times with intervals of 10-20 minutes. Following, the skin was incubated with 1:200 donkey anti-goat Ax594 and donkey anti- chicken Ax488 antibodies in 2% donkey / 0.3% Triton / PBS overnight at 4 ºC, washed as described before, mounted, and imaged.

[0234] Spinal cord was dissected from the lumbar enlargement, fixed overnight in 2% paraformaldehyde (PFA), then washed in PBS, and incubated with sucrose 30% / PBS overnight at 4 ºC. It was then embedded in OCT and cut at the cryostat at a thickness of 30 µm. The sections were blocked with 2% donkey / 0.3%Triton / PBS for 2 hours at room temperature and then incubated with 1:200 goat anti-rat Trka in 2% donkey / 0.3%Triton / PBS overnight at 4 °C. Spinal cord sections were washed with 0.3% Triton / PBS 3 times with intervals of 10-20 minutes and further incubated with 1:200 donkey anti-goat Ax594 and an Isolectin GS-IB4Alexa Fluor™ 647 Conjugate in 2% donkey / 0.3%Triton / PBS overnight at 4 °C. Before imaging, spinal cord sections were washed 3 times as described above and then mounted. Image analysis

[0235] Tissue was imaged with a Nikon A1R confocal microscope and analyzed using ImageJ. Cell counts in DRG were undertaken manually. Object based colocalization was performed using Colocalization Image Creator plugin (1). See Lunde, A., Glover, J.C. A versatile toolbox for semi- automatic cell-by-cell object-based colocalization analysis. Sci Rep 10, 19027 (2020). https: / / doi.org / 10.1038 / s41598-020-75835-7. 51 37937 / 59605 / FW / 18328338.1Results

[0236] The initial experiments confirmed by ddPCR that TFP functionalization did not cause viral loss, while NHS functionalization led to 90% reduction in yield (FIG. 5). Consistently, when the viruses were added to PC12 expressing Trka receptor, NHS did not boost cell transduction compared to the control, probably because of the virus loss, while PC12 expressed EGFP with all ratios tested with TFP.

[0237] Transduction in vivo was tested by injecting viruses subcutaneously in the paw. Wildtype AAV2 was ineffective at transducing DRG neurons (presumably because it is not retrogradely transported from nerve terminals in the skin to the DRG), NHS modified NGF-AAV led to some transduction of neurons, while the highest ratios of TFP functionalized NGF-AAV2, led to efficient transduction of neurons. FIG.6 illustrates quantification of this data, showing the number of positive DRG neurons per ganglion for each condition. NGF-AAV2 capsids modified with higher ratios of TFP are more effective than NHS modified particles, and that the 1:10,000 ratio is the most efficient. Moreover, the data shows increasing transduction efficiency in the L3-L5 ganglia, consistent with the fact that nerves from these ganglia innervate the hindlimb and paw.

[0238] The selectivity of TFP functionalized NGF-AAV in targeting nociceptive neurons by co- staining positive ganglia with an antibody against Trka was further quantified. As shown in FIG. 7, almost all NGF-AAV2 targeted neurons were also positive for TrkA staining across the L3, L4 and L5 ganglia, indicating that targeting to nociceptive neurons is indeed accurate.

[0239] Skin sections at the injection site for transduction by wildtype AAV2 and TFP functionalized NGF-AAV2 (FIG.8) were analyzed. By co-staining sections with a TrkA antibody, nociceptor nerve endings in the skin (red channel) were identified. In samples injected with the wildtype virus, no transduction of nerves was detected. In sections from NGF-AAV2 injected mice, considerable overlap with TrkA positive fibers was observed. In addition, eGFP positive muscle fibres were seen in both conditions, indicating that the natural tropism of AAV2 was not detargeted using this method.

[0240] Sensory innervation of the spinal cord in mice injected with wildtype or TFP functionalized NGF-AAV2 was investigated. The fibers innervating the dorsal horn were eGFP positive only in the spinal sections collected from the animals injected with NGF-AAV2 (FIG.9). Co-staining was further performed to identify the topographical organization of the dorsal horn and understand 52 37937 / 59605 / FW / 18328338.1which neuronal subset contains the eGFP positive fibers. With the marker Trka, the first lamina where the peptidergic nociceptive neurons terminate was visualized, and with the marker Ib4, the second lamina where non peptidergic nociceptive neurons project was identified. The eGFP positive fibers overlaped with Trka, but not with Ib4, demonstrating that the NGF-AAV is targeted to peptidergic nociceptive neurons. Notably, the fibers double positive for Trka and eGFP are mainly localized in the medial region of the first lamina, which is the area that receives input from axons which innervate the paw.

[0241] Scaling up of the modification with AAV9 and testing transduction in a HEK293 cell line expression TrkA / p75 using NGF as a ligand was also investigated. FIG.10a shows the images of TrkA / p75 HEK293 cells transduced with AAV9 or NGF-AAV9 at different MOIs. FIG. 10b shows the analysis of transduction efficiency. It was found that AAV9 can be effectively modified using NHS at larger scales. Upon conjugation of NGF, the MOI required to transduce TrkA / p75 HEK293 cells is substantially reduced.

[0242] It was further investigated scaling up of the modification of AAV2 and the stability of modified virus to a freeze thaw cycle and test transduction in vivo in mice. FIG. 11 presents the comparison of transduction efficiency of lumbar DRG isolated from mice subcutaneously injected with AAV2 modified with TFP-PEG4-DBCO at different scales and either used fresh or stored at -80oC. DRG were collected at 3 weeks after in vivo AAV-injection, flat mounted and imaged with confocal microscope. It was found that AAV2 can be effectively modified using TFP at larger scales. Upon conjugation of NGF, the MOI required to transduce TrkA / p75 HEK293 cells is substantially reduced.

[0243] Taken together these data suggest that TFP chemistry allows for the production of surface modified AAV2 more efficiently than NHS chemistry, avoiding viral loss. The TFP protocol can be readily scaled-up and is compatible with in vivo experiments. Importantly, the modification of AAV with NGF via TFP chemistry clearly demonstrated the capacity to target the virus to the population of interest, in this case peptidergic neurons expressing the NGF receptor, TrkA. Example 5. Comparisons of NHS- versus TFP- conjugation to AAVs

[0244] Chemically modifying AAVs with DBCO-PEG(n)-NHS linkers and further conjugation to ligands leads to substantial virus loss due to column-based washing steps during the modification process. To overcome the issue of virus loss, DBCO-PEG(n)-TFP linkers were used, since with this 53 37937 / 59605 / FW / 18328338.1chemistry no washing steps are needed during the modification process. Here, NHS- versus TFP- mediated AAV modification was compared by monitoring transduction efficiency and virus loss. AAV2, AAV5, or Delta-HSPG AAV2 (with mutations in its HSPG binding site) were modified with NHS or TFP linkers, conjugated the ligand Wheat Germ Agglutinin (WGA), and tested for transduction efficiency in PC12 cells. For the serotype AAV2, NHS- and TFP-based chemistries were tested on viruses purified with two methods that lead to a different degree of sample purity. From this data the ratios for virus:linker:ligand conjugation for each chemical modification and serotype were identified.

[0245] Methods

[0246] NHS- and TFP-based chemistries on different AAV serotypes were tested. For one of these serotypes, AAV2, NHS- and TFP-chemistry were tested on viruses purified with two distinct protocols differing in the degree of purity of the final viral product - either using a cesium chloride gradient or via affinity purification followed by isopycnic centrifugation through an iodixanol gradient (which is expected to increase the purity of the AAV vector). AAV5 and Delta-HSPG AAV2 were purified only with affinity chromatography and iodixanol gradient.

[0247] All AAV serotypes used were commercially purchased. One AAV2, purified with cesium chloride gradient, had a titer of 2.7E+12 vg / ml, and contained as a cargo an EGFP reporter gene under a CAG promoter, while the other AAV2, purified via affinity chromatography and iodixanol gradient had a titer of 1E+13 vg / ml and contained an EGFP reporter gene. The AAV5 contained an EGFP with a CMV promoter and had a titer of 1E+13vg / ml. The Delta-HSPG AAV2 had an EGFP with a CAG reporter and a titer of 5.6E+12 vg / ml.

[0248] The table below summarizes the purification and features of the AAVs used. 54 37937 / 59605 / FW / 18328338.1Table 8. Serotype Gradient Further purification step Promoter AAV2 Cesium chloride Nothing CAG AAV2 Iodixanol Affinity Chromatography CMV AAV5 Iodixanol Affinity Chromatography CMV Delta AAV2 Iodixanol Affinity Chromatography CAG

[0249] NHS-mediated chemical modification and coupling of WGA to AAV2, AAV5, and Delta AAV2

[0250] For NHS-mediated chemical modification of AAV2 purified with cesium chloride gradient 1µl (corresponds to 0.0045 pMol) was reacted with DBCO-PEG4-NHS at different molar virus:linker ratios (see table below) in a reaction volume of 20µl PBS (+0.001% pluronic and 200mM NaCl) for 3 hours shaking at room temperature. Table 9. molar ratio virus:linker virus linker 1 3300 1 10000 1 33000 1 100000 1 330000

[0251] WGA (0.1nMol) was dissolved in PBS and reacted with a 20-fold molar equivalent of Azide-PEG4-NHS for 3 hours shaking at room temperature. Unreacted Azide groups were removed using a 10 kDa MWCO centrifugal filter. NHS-mediated DBCO modified AAV2 was further incubated with 50pMol WGA-PEG4-Azide for 2 hours shaking at room temperature and then kept at 4°C overnight.

[0252] The NHS-mediated chemical modification of 1µl (corresponds to 0.0166 pMol) AAV2 purified with affinity chromatography and iodixanol gradient, was performed as described above for AAV2 but with distinct virus:linker ratios as indicated in the table below. 55 37937 / 59605 / FW / 18328338.1Table 10. molar ratio virus:linker virus linker ligand 1 500 500 1 1000 1000 1 3000 3000 1 10000 5000 1 30000 5000 1 100000 5000 1 300000 5000

[0253] The NHS-mediated chemical modification of 1µl (corresponds to 0.0166 pMol) AAV5 was performed as described above for AAV2 but with distinct virus:linker ratios as indicated in the table below. Table 11. molar ratio virus:linker virus linker 1 500 1 1000 1 3000 1 10000 1 30000 1 100000 1 300000 56 37937 / 59605 / FW / 18328338.1

[0254] The NHS-mediated chemical modification of 1µl (corresponds to 0.0093 pMol) Delta AAV2 was performed as described above for AAV2 but with distinct virus:linker ratios as indicated in the table below. Table 12. molar ratio virus:linker virus linker ligand 1 3000 3000 1 10000 6000 1 30000 6000 1 60000 6000

[0255] TFP-mediated chemical modification and coupling of WGA to AAV2, AAV5, and Delta AAV2

[0256] For TFP-mediated chemical modification, 1µl of AAV2 purified with cesium chloride (corresponds to 0.0045 pMol) was reacted with DBCO-PEG4-TFP and WGA-PEG4-Azide at different molar virus:linker:ligand ratios (see table below). Table 13. molar ratio of virus:linker:ligand virus linker ligand 1 100 100 1 500 500 1 1000 1000 1 3300 3300 1 10000 10000 1 33000 33000

[0257] Since TFP is more stable compared to NHS, the volume of the DBCO-PEG4-NHS linker was kept relatively small (1µl) by preparing dilutions from a 20mM stock. Further 11µl PBS (+0.001% pluronic and 200mM NaCl) and 0.1M sodium bicarbonate buffer (pH 8.3) were added, and the reaction was kept shaking at room temperature overnight. The reaction was stopped by adding 50mM Glycine. WGA (0.1nMol) was dissolved in PBS and reacted with a 20-fold molar 57 37937 / 59605 / FW / 18328338.1equivalent of Azide-PEG4-NHS for 3 hours shaking at room temperature. Unreacted Azide groups were removed using a 10 kDa MWCO centrifugal filter. WGA-PEG4-Azide was added in the indicated molar ratio (see table above) and the reaction was kept shaking at room temperature overnight.

[0258] The TFP-mediated chemical modification of 1µl of AAV2, AAV5, and Delta AAV2 purified with affinity chromatography and iodixanol gradient was performed as described above for AAV2 but with distinct virus:linker:ligand ratios as indicated in the tables below. Further, the final volume of the ligand for these three viruses, AAV2, AAV5, and Delta AAV2, would have been too large for the highest ratios, therefore we used a virus:ligand ratio of maximum 1:5000, 1:10000, and 1:6000 respectively. Table 14 (AAV2). molar ratio of virus:linker:ligand virus linker ligand 1 500 500 1 1000 1000 1 3000 3000 1 10000 5000 1 30000 5000 1 100000 5000 58 37937 / 59605 / FW / 18328338.1Table 15 (AAV5). molar ratio of virus:linker:ligand virus linker ligand 1 500 500 1 1000 1000 1 3000 3000 1 10000 10000 1 30000 10000 1 100000 10000 Table 16 (Delta AAV2). molar ratio of virus:linker:ligand virus linker ligand 1 3000 3000 1 10000 6000 1 30000 6000 1 60000 6000

[0259] In vitro application to PC12 cells

[0260] PC12 cells were maintained in DMEM / F12 medium (+ 10% horse serum, 5% fetal bovine serum, 15mM HEPES, 2.5mM Glutamax and 100U penicillin / streptomycin) and incubated at 37°C in a humidified atmosphere of 5% CO2. Modified AAVs were added to the cells and incubated overnight. Media was replaced and cells were maintained at 37°C for 5 days. Data acquisition

[0261] Transduction efficiency was determined by imaging PC12 cells with a Nikon A1R confocal microscope. For quantifying efficiency, cells were collected and prepared for flow cytometry. Flow cytometry data were acquired on a S3e Cell sorter from Bio-Rad and analyzed with Flow Jo. Viral genome copy numbers were quantified using Bio-Rad’s Droplet Digital PCR System. 59 37937 / 59605 / FW / 18328338.1Results

[0262] AAV2 modified with DBCO-PEG4-NHS at a virus:linker ratio of 1:100,000 and 2.5uM WGA-PEG4-Azide showed the highest transduction in PC12 cells compared to other virus:linker ratios tested (FIG. 12). In contrast, AAV2 modified with DBCO-PEG4-TFP and WGA-PEG4- Azide showed the highest transduction at a virus:linker:ligand ratio of 1:10,000:10,000 (FIG.13). This indicates that the TFP modification allows us to use less linker and we still reach the same transduction efficiency as with the NHS chemistry.

[0263] With a higher purity virus, the amount of TFP linker molecules needed to modify the virus and obtain a strong cell transduction was further reduced, as observed with AAV2 purified with affinity chromatography and iodixanol gradient. Indeed, the best ratio virus:TFP linker:ligand ratio was reduced from 1:10,000:10,000 to 1:500:500, when AAV2 was purified this way, instead of only cesium chloride gradient (FIGS. 13 and 15). Also in this case, NHS chemistry required a higher amount of linker (1:100,000) in order to reach the cell transduction shown by TFP (FIGS. 14 and 16). With other AAV serotypes, AAV5 and Delta AAV2, it was observed that with a TFP- mediated modification (FIGS. 17 and 19) the virus:linker ratio can be lowered to 1:1,000 and 1:3,000 respectively, while with NHS-mediated modification (FIGS. 16 and 18) a virus:linker ratio of 1:100,000 and 1:60,000, respectively, was needed to reach the best transduction efficiency. Hereby another advantage of using DBCO-PEG4-TFP linkers due to improvement of AAV modification is demonstrated, in addition to no virus loss during the modification process. Example 6. Incorporation of vector functionalization step into the AAV DSP

[0264] Chemically modifying AAV during AAV purification (i.e., Crude Viral Extract Modification) instead of post-purification (i.e., Original Protocol) was tested, as shown in Scheme 3 below. 60 37937 / 59605 / FW / 18328338.1Scheme 3. Illustrates the vector functionalization steps into the AAV DSP using two protocols. Methods

[0265] Recombinant AAV9 or AAV2 harboring an insertion in the VR4 region and carrying eGFP or tdTomato respectively under a CAG promoter as a cargo were produced in HEK293 cells as described previously. Cells were harvested 3 days post transfection, freeze thawed 3 times and then buffer exchanged into in PBS (+0.001% pluronic and 200mM NaCl) using an 100KDa centrifugal filter. AAV9 was then modified with NHS-PEG4-DBCO at a range of concentrations (3mM, 1mM, 0.3mM, 0.1mM 30µM, 10 µM, 1µM, 0,3µM or 0µM) for 3 hours a room temperature. Samples were again buffer exchanged using a 100KDa centrifugal filter, and then incubated for 2.5uM WGA-Azide overnight. Samples were applied to PC12 cells and transduction efficiency assessed after 5 days. 61 37937 / 59605 / FW / 18328338.1

[0266] For comparison with TFP esters, AAV2-VR4 was modified with TFP-PEG4-DBCO at a range of concentrations (3mM, 1mM, 0.3mM, 0.1mM 30µM, 10 µM, or 0µM) in PBS (+0.001% pluronic and 200mM NaCl) and 0.1M sodium bicarbonate buffer (pH 8.3) at room temperature overnight. Reactions were then quenched with 50mM Glycine, buffer exchanged using a 100KDa centrifugal filter, and then incubated for 2.5uM WGA-Azide overnight. Samples were applied to PC12 cells. Results

[0267] Surprisingly, the modification worked directly on crude cell lysates (i.e. unpurified AAV). FIG. 20A and Fig. 20B present images of PC12 cells transduced with AAV9-WGA chemically modified with NHS-PEG4-DBCO and TFP-PEG4-DBCO, respectively, at the cell lysate stage. As can be seen from FIG.20A and FIG.20B, AAV9 or AAV2-VR4 cell lysates can be effectively modified without purification using both NHS and TFP esters. Lower concentrations of TFP esters are required compared to NHS. 5. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0268] While the present 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 present disclosure.

[0269] All literature references, issued patents and patent applications cited within the body of the instant specification, including U.S. Provisional Appl.63 / 514,909 filed on July 21, 2023, are hereby incorporated by reference in their entirety for all purposes.

[0270] WO 2020 / 225363 and WO 2022 / 101363 are incorporated herein by reference in their entirety for all purposes. 62 37937 / 59605 / FW / 18328338.1

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a surface modified viral capsid having a titer of at least 1.0E+10 vg / ml, the surface modified viral capsid comprising: a ligand covalently conjugated to a viral capsid via a linker.

2. The composition according to claim 1, wherein the linker is of Formula VI:wherein: SP1and SP2are independently a bond or a spacer; and Q is a crosslinked moiety.

3. The composition according to claim 2, wherein the spacer comprises one or more divalent groups selected from: -CH2-, -O-, -C(=O)-, and -N(R)- where R is H or C1-3alkyl.

4. The composition according to any one of claims 1-3, wherein the surface modified capsid is of Formula V:wherein:a viral capsid that optionally comprises nucleic acid cargo; SP1and SP2are independently a bond or a spacer; Q is a crosslinked moiety; and 63 37937 / 59605 / FW / 18328338.1L is the ligand.

5. The composition according to any one of claims 1-4, wherein the composition has a surface modified viral capsid physical titer from 1.0E+10 vg / ml to 5.0E+13 vg / ml.

6. The composition according to any one of claims 1-5, wherein the Ligand to Capsid Ratio (LCR) is from 1 to 480, preferably from 10 to 150 or from 30 to 80.

7. The composition according to any one of claims 1-6, wherein the composition comprises less than 20% free ligand.

8. The composition according to any one of claims 1-7, wherein the viral capsid is an adeno-associated viral capsid (AAV), optionally selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

9. The composition according to claim 8, wherein the AAV is AAV2, AAV5, or AAV9.

10. The composition according to claim 8 or claim 9, wherein the AAV is AAV2.

11. The composition according to claim 8 or claim 9, wherein the AAV is AAV5.

12. The composition according to claim 8 or claim 9, wherein the AAV is AAV9.

13. The composition according to any one of claims 1-12, wherein the capsid comprises at least one protein selected from VP1, VP2, and VP3.

14. The composition according to any one of claims 1-13, wherein the capsid protein is a wild-type capsid protein.

15. The composition according to any one of claims 1-13, wherein the capsid protein is a genetically modified capsid protein.

16. The composition according to claim 15, wherein the genetic modification is a disruption of the heparin sulfate proteoglycans binding site. 64 37937 / 59605 / FW / 18328338.

117. The composition according to claim 16, wherein at least one of arginine 585 of VP1, arginine 588 of VP1, arginine 488 of VP2, arginine 451 of VP2, arginine 383 of VP3, and arginine 386 of VP3 are replaced with a different amino acid.

18. The composition according to any one of claims 15-17, wherein the capsid is of Delta AAV2.

19. The composition according to any one of claims 1-17, wherein the ligand is selected from a cell-type specific ligand, a polypeptide, a protein, a monosaccharide, a polysaccharide, a steroid hormone, a RGD motif peptide, a vitamin, a small molecule, an antibody, a nanobody, an enzyme, and an immunoglobulin.

20. The composition according to claim 19, wherein the ligand is a protein ligand, a toxin subunit, a lectin, an adhesion factor, an antibody or a single chain variable fragment thereof, a peptide, and a gene editing nuclease.

21. The composition according to claim 20, wherein the lectin is selected from wheat germ agglutinin (WGA), isolectin B4 (IB4), Maackia amurensis lectin, Lens culinaris lectin, Wisteria floribunda lectin, and Pha-L.

22. The composition according to claim 20, wherein the lectin is WGA.

23. The composition according to claim 20, wherein the ligand is a protein, preferably a growth factor or a cytokine.

24. The composition according to claim 23, wherein the protein ligand is a Nerve Growth Factor (NGF).

25. The composition according to any one of claims 1-24, wherein the crosslinked moiety comprises at least one of: an eight membered ring and a triazole ring.

26. The composition according to claim 25, wherein the crosslinked moiety comprises both an eight membered ring and a triazole ring. 65 37937 / 59605 / FW / 18328338.

127. The composition according to any one of claims 1-26, wherein the crosslinked moiety is a product of 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.

28. The composition according to claim 27, wherein the reaction is a strain-promoted alkyne- azide cycloaddition (SPAAC) reaction.

29. The composition according to any one of claims 1-28, wherein the crosslinked moiety comprises:.

30. The composition according to claim 29, wherein crosslinked moiety comprises:

31. The composition according to claim 30, wherein the reaction is an inverse electron demand Diels–Alder (IEEDD) reaction.

32. The composition according to any one of claims 1-31, wherein the viral capsid comprises nucleic acid cargo. 66 37937 / 59605 / FW / 18328338.

133. The composition according to claim 32, wherein the nucleic acid encodes a protein or immunogenic polypeptide.

34. A method of preparing a composition according to any preceding claim, the method comprising the steps of: (a) combining: (i) a viral capsid comprising: a plurality of surface available primary amines; and (ii) a capsid-reactive linker comprising: a tetrafluorophenyl (TFP) ester and a first member of a crosslinker- reactive pair (CRP1); thereby providing a composition comprising surface functionalized viral capsid; and (b) combining: (i) a functionalized ligand comprising a second member of a crosslinker- reactive pair (CRP2); (ii) the composition comprising surface functionalized viral capsid; thereby providing a composition comprising the surface modified viral capsid.

35. The method according to claim 34, wherein the composition comprising surface functionalized viral capsid produced in step (a) is used in step (b) without a purification or isolation step.

36. The method according to claim 34 or 35, wherein the combining of step (b) comprises adding the functionalized ligand directly to the composition comprising surface functionalized viral capsid produced by step (a).

37. The method according to any one of claims 34-36, wherein the capsid-reactive linker is according to Formula (II): 67 37937 / 59605 / FW / 18328338.

1.

38. The method according to any one of claims 34-37, wherein step (a) occurs according to the following Scheme A:, wherein: p is an integer from 10 to 500, and q is an integer from 10 to 500.

39. The method according to any one of claims 34-38, wherein the molar ratio of viral capsid (I) to capsid-reactive linker (II) is from 1:100 to 1:50,000.

40. The method according to claim 39, wherein the molar ratio of viral capsid (I) to capsid- reactive linker (II) is from 1:100 to 1:33,000.

41. The method according to claim 39, wherein the molar ratio of viral capsid (I) to capsid- reactive linker (II) is from 1:100 to 1:10,000.

42. The method according to claim 39, wherein the molar ratio of viral capsid (I) to capsid- reactive linker (II) is from 1:100 to 1:1,000.

43. The method according to any one of claims 34-42, further comprising purifying the viral capsid prior to combining step (a). 68 37937 / 59605 / FW / 18328338.

144. The method according to claim 43, wherein the viral capsid is purified by density gradient centrifugation, chromatography, or a combination thereof.

45. The method according to claim 44, wherein the density gradient centrifugation is iodixanol density gradient ultracentrifugation.

46. The method according to claim 44, wherein the chromatography is affinity chromatography.

47. The method according to any one of claims 34-46, wherein the viral capsid is used in step (a)(i) without prior purification.

48. The method of any one of claims 34-47, wherein SP1is one or more PEGs (i.e., - (−(O−CH2−CH2)n)- or -([PEG]n)-), wherein n is from 2 to 20.

49. The method according to any one of claims 34-48, wherein CRP1 comprises a reactive moiety selected from: an azide; alkyne; 1,4-triazole; 1,3-nitrone; cyclooctyne or derivative thereof, e.g., dibenzylcyclooctyne or derivative thereof; triazine; tetrazine; strained dienophile; aryl or alkyl phosphine; isocyanide; benzylguanine, a benzylcytosine, and a chloroalkane.

50. The method according to claim 49, wherein the CRP1 reactive moiety comprises a cyclooctyne.

51. The method according to claim 50, wherein the cyclooctyne is selected from a dibenzylcyclooctyne analog.

52. The method according to claim 51, wherein the dibenzylcyclooctyne analog is selected from the group: dibenzylcyclooctyne (DIBO), dibenzoazacyclooctyne (DBCO), and biarylazacyclooctynone (BARAC) and functional derivatives thereof.

53. The method according to any one of claims 34-52, wherein CRP1 comprises the following structure: 69 37937 / 59605 / FW / 18328338.1wherein m is an integer from 4-12, and the squiggle bond indicates attachment to the capsid-reactive linker.

54. The method according to any one of claims 34-53, wherein the capsid-reactive linker is of the following formula:, wherein: n is an integer from 2 to 20, and m is an integer from 2 to 10.

55. The method according to claim 54, wherein the capsid-reactive linker is of the following formula: 70 37937 / 59605 / FW / 18328338.

1.

56. The method according to according to any one of claims 34-55, wherein the capsid- reactive linker is DBCO-PEG4-TFP.

57. The method according to any one of claims 34-56, wherein step (a) occurs in an aqueous medium that optionally further comprises at least one buffering agent.

58. The method according to claim 57, wherein the at least one buffering agent is selected from HEPES, MOPS, MES, phosphate, and bicarbonate.

59. The method according to claim 57 or 58, wherein the concentration of at least one buffering agent does not exceed about 0.1 M.

60. The method according to claim 59, wherein the concentration of the at least one buffering agent is from about 0.01 M to about 0.1 M.

61. The method according to any one of claims 57-60, wherein the aqueous medium further comprises at least about 200 mM of at least one salt.

62. The method according to claim 61, wherein the at least one salt is selected from a chloride salt, a phosphate salt, a sulfate salt, a citrate salt, a sodium salt, a potassium salt, a calcium salt, and a magnesium salt.

63. The method according to claim 62, wherein the at least one salt is sodium chloride.

64. The method according to any one of claims 57-63, wherein the ionic strength of the aqueous medium is at least about 150 mM.

65. The method according to any one of claims 57-64, wherein the aqueous medium further comprises at least one surfactant. 71 37937 / 59605 / FW / 18328338.

166. The method according to claim 65, wherein the concentration of the at least one surfactant is from about 0.001% to about 0.005%.

67. The method according to claim 65 or 67, wherein the at least one surfactant comprises Pluronic® F68.

68. The method according to any one of claims 57-68, wherein the pH of the aqueous medium is from about 6 to about 10.

69. The method according to any one of claims 34-68, wherein step (a) occurs at a reaction temperature of from about 0 ºC to about 50 ºC.

70. The method according to any one of claims 34-69, wherein step (a) occurs over a reaction duration of from about 5 minutes to about 24 hours.

71. The method according to any one of claims 34-70, wherein step (a) further comprises the step of adding a quenching agent in excess.

72. The method according to claim 71, wherein the quenching agent comprises an amine- containing compound.

73. The method according to claim 71 or 72, wherein the quenching agent is selected from glycine or Tris buffer.

74. The method according to any one of claims 34-73, wherein step (b) occurs according to the following Scheme B:wherein: 72 37937 / 59605 / FW / 18328338.1is the functionalized ligand, wherein SP2is a bond or spacer and L is the ligand; and r is an integer from 10 to 500.

75. The method according to claim 74, wherein SP1and SP2are independently selected from one or more divalent groups selected from: a bond, -CH2-, -O-, -C(=O)-, and -N(R)- where R is H, or C1-3 alkyl.

76. The method according to claim 75, wherein SP2is -([PEG]n)-C(O)-, wherein n is from 1 to 100, preferably from 1 to 10.

77. The method according to any one of claims 34-76, wherein the functionalized ligand is of the following formula:, wherein Y is a bond or one or more PEGs.

78. The method according to any one of claims 34-77, wherein CRP2 comprises a reactive moiety selected from an azide; alkyne, 1,4-triazole; 1,3-nitrone; cyclooctyne or derivative thereof; dibenzylcyclooctyne or derivative thereof; triazine; tetrazine; strained dienophile; aryl or alkyl phosphine; isocyanide; benzylguanine group, a benzylcytosine group, or a chloroalkane group.

79. The method according to claim 78, wherein CRP2 comprises an azide.

80. The method according to any one of claims 34-79, wherein the functionalized ligand is WGA-[PEG]n-azide, wherein n is from 1 to 20, preferably 4 to 10.

81. The method according to any one of claims 34-80, wherein the functionalized ligand is WGA-PEG4-Azide. 73 37937 / 59605 / FW / 18328338.

182. The method according to any one of claims 34-80, wherein the functionalized ligand is WGA-PEG5-Azide.

83. The method according to any one of claims 34-80, wherein the functionalized ligand is WGA-PEG6-Azide.

84. The method according to any one of claims 34-80, wherein the functionalized ligand is WGA-PEG7-Azide.

85. The method according to any one of claims 34-79, wherein the functionalized ligand is NGF-[PEG]n-azide, wherein n is from 1 to 20, preferably 4 to 10.

86. The method according to any one of claims 34-79 and 85, wherein the functionalized ligand is NGF-PEG4-Azide.

87. The method according to any one of claims 34-79 and 85, wherein the functionalized ligand is NGF-PEG5-Azide.

88. The method according to any one of claims 34-79 and 85, wherein the functionalized ligand is NGF-PEG6-Azide.

89. The method according to any one of claims 34-79 and 85, wherein the functionalized ligand is NGF-PEG7-Azide.

90. The method according to any one of claims 34-89, wherein the molar ratio of viral capsid (I) in step (a) to functionalized ligand in step (b) is from 1:100 to 1:50,000.

91. The method according to claim 90, wherein the molar ratio of viral capsid (I) in step (a) to functionalized ligand in step (b) is from 1:100 to 1:33,000.

92. The method according to claim 90, wherein the molar ratio of viral capsid (I) in step (a) to functionalized ligand in step (b) is from 1:100 to 1:10,000.

93. The method according to claim 90, wherein the molar ratio of viral capsid (I) in step (a) to functionalized ligand in step (b) is from 1:100 to 1:1,000. 74 37937 / 59605 / FW / 18328338.

194. The method according to any one of claims 34-93, wherein the molar ratio of viral capsid to capsid-reactive linker in (a) is the same as the molar ratio of viral capsid to functionalized ligand in (b).

95. The method according to any one of claims 34-94, wherein (a) and (b) occur in the same reaction vessel.

96. The method according to any one of claims 34-95, wherein CRP1 and CRP2 react in (b) to form Q.

97. The method according to claim 96, wherein (i) CRP1 is DBCO and CRP2 is azide or (ii) CRP1 is azide and CRP2 is DBCO.

98. The method according to claim 96 or 97, wherein Q comprises a triazole.

99. The method according to any one of claims 96-98, wherein Q is:.

100. The method according to any one of claims 96-99, wherein Q is:, wherein m is from 1 to 10. 75 37937 / 59605 / FW / 18328338.1101. The method according to any one of claims 34-100, further comprising preparing the functionalized ligand by reacting a ligand comprising a first reactive moiety with a ligand-reactive linker comprising a second reactive moiety and CRP2.

102. The method according to claim 101, wherein the ligand comprises a -NH2 first reactive moiety.

103. The method according to claim 101 or 102, wherein the ligand-reactive linker comprises a second reactive moiety selected from an isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimides anhydride, benzoyl fluoride, and TFP ester.

104. The method according to any one of claims 98-103, wherein the ligand-reactive linker is selected from TCO-PEGn-NHS; tetrazine-PEGn-NHS; azido-PEGn-NHS; phosphine- NHS; maleimide-PEGn-succinimidyl ester; DBCO-PEGn-TFP ester, and DBCO-PEGn- NHS ester, wherein n is from 1 to 100, preferably from 4 to 10.

105. The method according to claim 104, wherein the ligand-reactive linker is DBCO-PEGn- TFP ester, wherein n is from 1 to 100, preferably from 4 to 10.

106. The method according to claim 105, wherein the ligand-reactive linker is DBCO-PEG4- TFP.

107. The method according to claim 105, wherein the ligand-reactive linker is DBCO-PEG5- TFP.

108. The method according to claim 105, wherein the ligand-reactive linker is DBCO-PEG6- TFP.

109. The method according to claim 105, wherein the ligand-reactive linker is DBCO-PEG7- TFP.

110. The method according to any one of claims 34-109, further comprising purifying the composition comprising surface modified viral capsid. 76 37937 / 59605 / FW / 18328338.1111. The method according to any one of claims 34-110, wherein the viral capsid comprises nucleic acid cargo.

112. The method according to claim 111, wherein the nucleic acid encodes a protein or a polypeptide. 77 37937 / 59605 / FW / 18328338.1

Images