Method for preparing surface modified virus capsid

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 making a surface modified viral capsid using an efficient process of (a) reacting (i) a viral capsid comprising a plurality of surface accessible primary amines and (ii) a capsid-reactive linker comprising a terminal tetrafluorophenyl (TFP) ester and a first member of a cross-linking agent reactive pair, to provide a composition comprising a surface functionalized viral capsid; and (b) reacting the composition comprising the surface functionalized viral capsid with a functionalized ligand comprising a second member of the crosslinker reactive pair.

Description

[0001] 1. Background

[0002] Introducing molecules carrying genetic information into cells is a useful tool in modern medicine and basic research. Preferred methods include using virus-derived gene delivery vectors, including adenoviruses, retroviruses, lentiviruses, vaccinia viruses, and adeno-associated viruses. Among these, recombinant adeno-associated virus (AAV) has become the preferred virus for in vivo gene therapy due to its lack of pathogenicity, replication incompetence, and stable expression. More than 100 clinical trials are being conducted using AAV-based capsids (also known as vectors or virions), and two AAV gene therapy products have recently received FDA approval: Voretigene neparvovec-rzyl (LUXTURNA) for the treatment of inherited retinal diseases and onasemnogene abeparvovec-xioi (ZOLGENSMA) for the treatment of spinal muscular atrophy.

[0003] However, most clinical trials using AAV as a transgenic vector have shown its key limitations: (i) its reduced therapeutic index (i.e., high doses of capsid are often required to achieve therapeutic efficacy); (ii) its wide biodistribution; and (iii) its poor efficacy in the presence of pre-existing neutralizing antibodies.

[0004] One limitation of AAVs lies in their broad tropism, which leads to transgene expression in tissues other than those where expression is expected. It is well known that long-term gene transfer requires overcoming host- and vector-related immune challenges.

[0005] To date, most gene therapy applications have used serotype 2 (AAV2). Its popularity is partly explained by its ability to transduce a wide range of postmitotic cells in vivo, such as muscle cells, hepatocytes, or neurons in mammals. This serotype has also been used in clinical trials to transfer genes into muscle and liver for hemophilia B, and to the retina for the treatment of Leber congenital amaurosis.

[0006] However, the use of such vectors still presents complications and limitations. First, high doses of the vector are typically required because in vivo transduction efficacy is often low, leading to increased toxicity. One of the most significant complications is that gene delivery is impaired because 50-90% of the population is seropositive for AAV2 and neutralizing antibodies (NAbs) against AAV2 have been developed. The discovery of naturally occurring AAV isolates (from 1 to 12) in human and animal species, and the genetic engineering of capsids for these AAV serotypes using molecular tools, has yielded promising results in preclinical animal models and Phase I / II clinical trials, facilitating exciting clinical translations in the near future. However, their therapeutic index remains low, meaning that high concentrations still need to be administered, accompanied by adverse reactions. Meanwhile, significant progress has been made in the preparation of clinical-batch AAV capsids over the past decade, and large-scale preparation methods are now available for gene therapy entering the pre-industrial pharmaceutical stage. Nevertheless, current methods will not be able to support the high doses of capsids required for Phase III and commercialization.

[0007] The strategies described above have indeed demonstrated the potential of novel AAV serotypes (and associated genetic variants), but they are not considered satisfactory due to the failure to achieve precise telogenization, enabling selective transduction of target cell types. Further efforts are underway to increase the telogenization of AAV-derived capsids. In fact, to compensate for the lack of specificity in AAV-derived capsids, extremely large amounts of AAV-derived capsids are required to reach the therapeutic threshold, which is undesirable given safety concerns and manufacturing limitations.

[0008] Various attempts have been made to achieve this goal, such as the genetic introduction of target-specific peptide epitopes into the viral surface. Further strategies include using adaptor molecules with two specificities, such as bispecific antibodies, one specifically targeting the viral capsid and the other the receptor, or introducing adaptor domains (protein A, biotin's Z domain) for non-covalent linking of protein ligands.

[0009] For example, in document WO00 / 002654, the altered tropism is primarily used to prevent AAV from binding to the viral receptors on the original target cells. In a specific embodiment, this document also mentions increased affinity relative to the target cells. Still in that document, the antibody fragment is attached to the capsid. According to an alternative embodiment, the other end of the antibody can be conjugated to a ligand to improve affinity relative to the target (preparing a "biantibody").

[0010] In the past, combined biochemical conjugations on AAV capsids have been proposed to improve the selectivity of AAV-derived capsids for target tissues.

[0011] For example, WO2005 / 106046 proposes a method combining genetic engineering and chemical modification of the capsid. The chemical modification, as the second stage of this method, depends on the presence of cysteine ​​residues, which are enriched in the capsid via a genetic pathway in the first step. Therefore, AAV particles can be modified with ligands, polymers, gold nanoparticles, fluorescent molecules, magnetic or biochemically active substances. However, coupling occurs via disulfide bonds, thioester bonds, and / or thioether bonds, as well as via NCS bonds.

[0012] The article "Glycated AAV Vectors: Chemical Redirection of Viral Tissue Tropism" by ED Horowitz et al., *Bioconjugate Chemistry*, 2011, 22, 529-532, describes the problems of cell tropism and other technical challenges. In particular, it describes an orthogonal strategy using capsid glycosylation to generate non-natural amino acid side chains to engineer novel AAV capsids exhibiting tissue tropism for gene therapy applications.

[0013] In WO2015 / 062516, non-natural amino acids (e.g., amino acids containing azide groups) are inserted into the capsid via genetic engineering prior to the coupling step via click chemistry to alter the capsid of AAV and its tropism toward target cells.

[0014] WO 2022 / 101363 describes a method for preparing surface-modified viral capsids; however, the use of the described NHS esters for surface functionalization requires extensive purification, which greatly reduces the titer of the resulting composition.

[0015] Therefore, there is a need to find a way to chemically couple AAV-derived capsids to increase their ability to target specific organs or tissues, particularly through in vivo gene delivery.

[0016] It is also necessary to modify the AAV-derived capsid without requiring engineering modifications to the AAV amino acid capsid sequence.

[0017] Furthermore, there is a need for novel surface-modified AAV-derived capsids that enable improved virus-mediated gene transfer into specific cell types.

[0018] More generally, there is a need for new methods for chemically coupling ligands of arbitrary properties (i.e., multiple chemical moieties) to the surface of AAV-derived capsids, for example, to improve "specific activity" and / or "therapeutic index," thereby allowing for reductions in therapeutic doses. 2. Overview of the Invention

[0020] This disclosure provides novel recombinant adeno-associated virus (rAAV) vector particles, methods for their production, and their therapeutic and / or diagnostic uses.

[0021] In one aspect of this disclosure, a composition is provided comprising a surface-modified viral capsid having a titer of at least 1.0E+10 vg / ml, wherein the surface-modified viral capsid comprises a ligand covalently conjugated to the viral capsid via a linker.

[0022] In some implementations, the surface-modified viral capsid is of formula V:

[0023]

[0024] in:

[0025] For optionally containing nucleic acid cargo;

[0026] SP 1 and SP 2 Independently acting as a bond or spacer group;

[0027] Q represents the cross-linked portion; and

[0028] L is a ligand.

[0029] In another aspect of this disclosure, a method is provided for preparing a composition containing a surface-modified viral capsid of formula V, comprising the following steps:

[0030] (a) Combining the following: (i) a viral capsid comprising a plurality of surface-accessible primary amines and (ii) a capsid-reactive linker comprising a tetrafluorophenyl (TFP) ester and the first member (CRP1) of a crosslinking agent-reactive pair, thereby providing a composition comprising a surface-functionalized viral capsid; and

[0031] (b) Combining the following: (i) a functionalized ligand comprising a second member (CRP2) of a crosslinker-reactive pair and (ii) a composition comprising a surface-functionalized viral capsid; thereby providing a surface-modified viral capsid.

[0032] The preferred features of each aspect of this disclosure, with necessary modifications, are applicable to every other aspect. References cited herein are incorporated to the fullest extent permitted by law. While this disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations may be made herein without departing from the spirit and scope of this disclosure as defined by the appended claims.

[0033] 3. Brief description of several views in the attached drawings

[0034] The foregoing summary of this disclosure and the following detailed description can be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating this disclosure, the drawings show some, but not all, alternative embodiments. However, it should be understood that this disclosure is not limited to the precise arrangements and means shown. These drawings, incorporated in and constituting a part of this specification, help to explain the principles of this disclosure.

[0035] Figure 1 The study showed a significant loss of physical titers of NHS-modified AAV2 after centrifugation, as measured by ddPCR.

[0036] Figures 2a-2b The transduction efficiency of compositions prepared using different clean-up methods after NHS modification of AAV2 is shown, such as the transduction efficiency in HEK293 cells. Figure 2a ) and physical titers measured by ddPCR ( Figure 2b These figures also demonstrate the transduction efficiency of AAV2 with TFP ester modification, as measured by the same method.

[0037] Figure 3a The transduction efficiency of AAV2 and WGA-PEG4-azide in PC12 cells was compared, as demonstrated by the number of PC12 cells expressing the reporter gene EGFP after infection with WGA-conjugated AAV2 modified with TFP-PEG4-DBCO+WGA-PEG4-azide using the following different protocols: Reaction #1 was carried out in 0.1 M sodium bicarbonate in water at pH 8.5, quenched with glycine; Reaction #2 was carried out in 0.1 M sodium bicarbonate in PBS / Pluronic 0.001% / NaCl 200 mM at pH 8.5, quenched with glycine. Cells were imaged 4 days post-AAV infection.

[0038] Figure 3b A comparison of transduction efficiency was described, as demonstrated by the number of PC12 cells expressing the reporter gene EGFP after infection with WGA-conjugated AAV2 cells modified with TFP-PEG4-DBCO+WGA-PEG4-azide using the following different protocols: Reaction #3 was performed in water with 0.1 M sodium bicarbonate at pH 8.5, glycine-free; Reaction #4 was performed in PBS / Pluronic 0.001% / NaCl 200 mM, quenched with glycine. Cells were imaged 4 days post-AAV infection.

[0039] Figure 3cA comparison of transduction efficiency was described, as demonstrated by the number of PC12 cells expressing the reporter gene EGFP after infection with WGA-conjugated AAV2 cells modified with TFP-PEG4-DBCO+WGA-PEG4-azide using the following different protocols: Reaction #5 was performed in water with 0.2 M sodium bicarbonate at pH 8.5 and quenched with glycine; and a control reaction using NHS was performed in PBS / Pluronic 0.001% / NaCl 200 mM without glycine. Cells were imaged 4 days post-AAV infection.

[0040] Figures 4a-4b The comparison of transduction efficiency is shown, as demonstrated by the number of PC12 cells expressing the reporter gene EGFP after infection with different regimens (1-5) using TFP-PEG4-DBCO+WGA-PEG4-azide and with WGA-conjugated AAV2 cells modified with NHS-PEG4-DBCO+WGA-PEG4-azide. Cells were analyzed by cell counting at 7 days post-AAV infection. Figure 4a The mean fluorescence intensity (MFI) of EGFP+ cells normalized with appropriate controls is shown (ΔMFI). Figure 4b The percentage of EGFP+ positive cells normalized by appropriate controls is shown.

[0041] Figure 5 The reduced viral titers of viral fractions prepared by ddPCR with TFP-PEG4-DBCO + NGF-PEG4-azide at different ratios, NHS-PEG9-BG + NGF-SNAP, and wild-type AAV2 were confirmed.

[0042] Figure 6 The transduction efficiency of lumbar DRGs isolated from mice subcutaneously injected with AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-azide at different ratios (TFP3,000 and TFP10,000), AAV2 modified with NHS-PEG4-BG + NGF-SNAP, and wild-type AAV2 (control) is shown. DRGs were collected 3 weeks after in vivo AAV injection, flat-mounted, and imaged using confocal microscopy. The figure shows the number of EGFP+ positive cells.

[0043] Figure 7 The selectivity of TFP-functionalized NGF-AAV in targeting nociceptive neurons was demonstrated by co-staining positive ganglia with an antibody against Trka. Quantification of transducing cells co-localized with the nociceptive neuronal marker Trka in lumbar DRGs 3, 4, and 5 was also shown.

[0044] Figure 8 Transduction was shown in skin collected from the paws of mice subcutaneously injected with wild-type AAV2 and AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-azide (virus:combinant ratio = 1:10,000). Nerve bundles in the dermis were identified by Trka staining (red), and the presence of the virus was identified by the fluorescent protein EGFP (green).

[0045] Figure 9 Transduction of the spinal cord was shown in mice collected from those subcutaneously injected with wild-type AAV2 and AAV2 modified with TFP-PEG4-DBCO + NGF-PEG4-azide (virus:linker ratio = 1:10,000). Viral transduction was identified by the fluorescent protein EGFP (green), and the first and second lamina layers of the dorsal horn were identified by Trka (red) and Ib4 (cyan), respectively.

[0046] Figure 10a Images of TrkA / p75 HEK293 cells transduced with AAV9 or NGF-AAV9 at different multiples of infection (MOI) are shown.

[0047] Figure 10b The fluorescence intensities of AAV9 and NGF-AAV9 were shown; the MOI required for transducing TrkA / p75HEK293 cells was significantly reduced after NGF was conjugated with AAV9.

[0048] Figure 11 This image shows a comparison of the transduction efficiency of lumbar DRGs isolated from mice after subcutaneous injection of AAV2 modified with different sizes of TFP-PEG4-DBCO. The numbers above the images indicate the total amount of modified vector injected at one time. All animals were injected with the same dose of modified vector (5E + 10 VG).

[0049] Figure 12 The transduction efficiency of DBCO-PEG4-NHS and WGA-conjugated AAV2 modified with 50 pMol WGA-PEG4-azide and purified using a cesium chloride gradient and different virus:linker ratios in PC12 cells is shown. PC12 cells were imaged 5 days after AAV infection.

[0050] Figure 13 The transduction efficiency of DBCO-PEG4-TFP and WGA-PEG4-azide-modified WGA-conjugated AAV2, purified with cesium chloride gradients and using different virus:linker:ligand ratios, in PC12 cells is shown. PC12 cells were imaged 5 days post-AAV infection.

[0051] Figure 14The transduction efficiency of AAV2 conjugated to PC12 cells using affinity chromatography and iodixanol gradient purification with different virus:linker ratios in DBCO-PEG4-NHS and WGA-PEG4-azide-modified WGA is shown. PC12 cells were imaged 5 days post-AAV infection.

[0052] Figure 15 The transduction efficiency of AAV2 conjugated to PC12 cells using affinity purification and iodixanol gradient purification with different virus:linker ratios for DBCO-PEG4-TFP and WGA-PEG4-azide-modified WGA is shown. PC12 cells were imaged 5 days post-AAV infection.

[0053] Figure 16 The transduction efficiency of PC12 cells with DBCO-PEG4-NHS and WGA-conjugated AAV5 modified with 50 pM WGA-PEG4-azide at different virus:linker ratios is shown. PC12 cells were imaged 5 days after AAV infection.

[0054] Figure 17 The transduction efficiency of PC12 cells with DBCO-PEG4-TFP and WGA-PEG4-azide-modified WGA-conjugated AAV5 at different virus:linker:ligand ratios is shown. PC12 cells were imaged 5 days post-AAV infection.

[0055] Figure 18 The transduction efficiency of PC12 cells with DBCO-PEG4-NHS and WGA-PEG4-azide-modified WGA-conjugated Delta-HSPG AAV2 with different virus:linker:ligand ratios is shown. PC12 cells were imaged 5 days after AAV infection.

[0056] Figure 19 The transduction efficiency of PC12 cells with DBCO-PEG4-TFP and WGA-PEG4-azide-modified WGA-conjugated Delta-HSPG AAV2 with different virus:linker:ligand ratios is shown. PC12 cells were imaged 5 days after AAV infection.

[0057] Figure 20a Images of AAV9-WGA transduced PC12 cells chemically modified with NHS-PEG4-DBCO on crude cell extracts (lysates) using different concentrations of ligands during the modification reaction are shown.

[0058] Figure 20bImages of AAV9-WGA transduced PC12 cells chemically modified with TFP-PEG4-DBCO on crude cell extracts (lysates) using different concentrations of ligands during the modification reaction are presented.

[0059] 4. Detailed Explanation

[0060] 4.1. Definition

[0061] As used in this article, the term "rAAV" refers to a recombinant virus containing a recombinant nucleic acid construct packaged within an AAV capsid.

[0062] 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 with rAAV in this document.

[0063] A recombinant nucleic acid construct (synonymous, "recombinant viral genome") contains a multinucleotide payload (synonymous, "cargo") located between inverted terminal repeat sequences of AAV. 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 miRNA, siRNA, or guide RNA for gene editing or RNA editing mechanisms such as CRISPR, ADAR, and ADAT.

[0064] As used herein, the term "tropism" refers to the preferential infection and / or transduction of a viral capsid onto certain cells or tissues. In a preferred embodiment, to modify the tropism of the AAV capsid, certain characteristics are endowed to the capsid, such as certain affinity for receptors on the surface of target cells, which they do not inherently possess.

[0065] As used herein, viral titers are expressed in scientific notation using a simplified format that more reliably reproduces the numerical text, such as 1.0E+3 vg / m. Those skilled in the art will understand that 1,000 vg / m, 1.0 x 10^3 vg / m, and 1.0E+3 vg / m are all equivalent expressions of the same number.

[0066] As used herein, the term "MOI" or "Multiplicity of Infection" refers to the ratio of the number of viral particles to the number of host cells in a given infection medium. An MOI of 1 means that, on average, one host cell corresponds to one viral particle.

[0067] 4.2. Other Interpretive Conventions

[0068] Although similar or equivalent methods and materials can be used in the practice or testing of methods and material compositions, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0069] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural indicators. Thus, for example, reference to “antibody or antigen-binding fragment” includes a plurality of such antibody and antigen-binding fragments, and reference to “recombinant adeno-associated virus” includes reference to one or more recombinant adeno-associated viruses and their equivalents known to those skilled in the art, and so on. It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to provide a prior basis for the use of exclusive terms such as “unique,” ​​“only,” or the use of “negative” limitations in the statement of claim elements.

[0070] It should be understood that, for clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of this disclosure described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to this disclosure are specifically covered by this disclosure and disclosed herein, as if each and every combination were individually and explicitly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also specifically covered by this disclosure and disclosed herein, as if each and every such sub-combination were individually and explicitly disclosed herein.

[0071] The publications discussed herein are provided only because they were published prior to the filing date of this application. The publication dates provided may differ from the actual publication dates, which may require independent verification.

[0072] Where a numerical range is provided, it should be understood that the endpoints of the range are included. Furthermore, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range (accurate to one-tenth of the lower limit unit), and any other specified value or intermediate value within the range, is encompassed in this disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered in this disclosure, subject to any specific exclusions of the range. Where the range includes one or two limits, the range excluding one or both of those included limits is also included in this disclosure.

[0073] The ranges described herein should be understood as simplified expressions of all values ​​within that range, including the endpoints. For example, the range 1 to 50 should be understood as including any number, combination of numbers, or subrange from any 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 11 to 48 or 39 to 41.

[0074] 4.3. Composition

[0075] In one aspect, this disclosure provides 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 the viral capsid via a linker.

[0076] When compared to recombinant virions containing a viral capsid with the same primary amino acid sequence but unmodified to contain the target ligand, surface-modified viral capsids confer, as part of the recombinant virion, improved transduction efficiency, improved cell type selectivity, or both improved transduction efficiency and improved cell type selectivity.

[0077] Due to the high production efficiency of the surface-modified viral capsids described herein, the compositions of this disclosure exhibit relatively high physical titers compared to compositions prepared by other methods. In some embodiments, the compositions comprising surface-modified viral capsids have a physical titer of at least 1E+10 vg / mL as determined by droplet digital PCR (ddPCR), 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 compositions have 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 compositions have a physical titer of at least 1.0E+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.

[0078] In some embodiments, the composition comprising a surface-modified viral capsid has a physical titer of 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, 1.0E+12 vg / ml to 5.0E+13 vg / ml, 1.0E+13 vg / ml to 5.0E+13 vg / ml, 2.0E+10 vg / ml to 5.0E+13 vg / ml, 2.0E+11 vg / ml to 5.0E+13 vg / ml, 2.0E+12 vg / ml to 5.0E+13 vg / ml, 2.0E+13 vg / ml to 5.0E+13 vg / ml, 3.0E+10 vg / ml to 5.0E+13 vg / ml, 3.0E+1 ... vg / ml to 5.0E+13 vg / ml, 3.0E+12 vg / ml to 5.0E+13 vg / ml, 3.0E+13 vg / ml to 5.0E+13 vg / ml, 4.0E+10 vg / ml to 5.0E+13 vg / ml, 4.0E+11 vg / ml to 5.0E+13 vg / ml, 4.0E+12 vg / ml to 5.0E+13 vg / ml, 4.0E+13 vg / ml to 5.0E+13 vg / ml, 5.0E+10 vg / ml to 5.0E+13 vg / ml, 5.0E+11 vg / ml to 5.0E+13 vg / ml or 5.0E+12 vg / ml to 5.0E+13 vg / ml.

[0079] In some embodiments, the composition comprising a surface-modified viral capsid has a 1.5E+11 content. vg / ml up to 3.0E+12 vg / ml, 2.0E+11 vg / ml up to 3.0E+12 vg / ml or 2.5E+11 vg / ml up to 3.0E+12 Physical titer in vg / ml.

[0080] The compositions disclosed herein include surface-modified viral capsids of formula V:

[0081] (V)

[0082] in:

[0083] For optionally containing nucleic acid cargo;

[0084] SP 1 and SP 2 Each is independently selected from either the bond or the spacer group;

[0085] Q represents the cross-linked portion; and

[0086] L is the ligand.

[0087] 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, the LCR is an integer from 10 to 500, such as, for example, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400, or 400 to 500. In some implementations, LCR is an integer from 100 to 200, such as, for example, 100 to 190, 100 to 180, 100 to 170, 100 to 160, or 100 to 150.

[0088] In some embodiments, the composition contains less free ligand (in any form not conjugated to the capsid surface, including functionalized ligands and functionalized ligands conjugated to unbound TFP linkers) than the binding ligand (in the form of a surface-modified viral capsid). The amount of free ligand in the composition can be measured by gold-standard analysis of AAV, such as analytical ultracentrifugation (AUC) and mass photometry. In some embodiments, the composition contains 20% or less of 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.

[0089] 4.3.1. Dual-function connector

[0090] The bifunctional linker according to this disclosure comprises two components: i) a TFP ester, and ii) a member or ligand of a crosslinker reactive pair. Optionally, the bifunctional linker further comprises a spacer group. In some embodiments, the bifunctional linker comprises a TFP ester, optionally a spacer group, and a member of a crosslinker reactive pair. In some embodiments, the bifunctional linker comprises a TFP ester, a spacer group, and a member of a crosslinker reactive pair.

[0091] The cross-linking reactive portion of the bifunctional ligand is preferably selected to react selectively with the cross-linking reactive portion of the functionalized ligand of this disclosure.

[0092] In some implementations, the capping-reactive connector has the following structure:

[0093] ,

[0094] SP 1 Bonds or spacers known in the art; and

[0095] a is an integer from 1 to 5, or from 2 to 4, for example, 1, 2, 3, 4, or 5. In some implementations, a is 4.

[0096] 4.3.1.1 Spacer

[0097] In some of these embodiments, the spacer group comprises 10 to 30 non-hydrogen atoms. In further embodiments, the spacer group 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 some of these embodiments, the spacer group comprises 5-40 methylene groups, 1-20 ether groups, 1-5 amine groups, and / or 1-4 carbonyl groups. In some embodiments, the spacer group comprises 10-15 methylene groups, 4-6 ether groups, 1-2 amine groups, and / or 3-4 carbonyl groups.

[0098] In some implementations, the capping-reactive connector has the following structure:

[0099] ,

[0100] in:

[0101] X is a -CH2- or -O- group.

[0102] b is an integer from 2 to 20.

[0103] n is an integer from 2 to 20.

[0104] m is an integer from 2 to 10; and

[0105] a is an integer from 1 to 5, or from 2 to 4, for example, 1, 2, 3, 4, or 5.

[0106] In some implementations, the capping-reactive connector has the following structure:

[0107] ,

[0108] in:

[0109] n is an integer from 2 to 20.

[0110] m is an integer from 2 to 10; and

[0111] a is an integer from 1 to 5, or from 2 to 4, for example, 1, 2, 3, 4, or 5.

[0112] In some implementations, the capping-reactive connector has the following structure:

[0113] ,

[0114] in:

[0115] n is an integer from 2 to 20.

[0116] m is an integer from 2 to 10; and

[0117] a is an integer from 1 to 5, or from 2 to 4, for example, 1, 2, 3, 4, or 5.

[0118] In some implementations, the capping-reactive connector has the following structure:

[0119]

[0120] In some implementations, n is an integer from 2 to 15.

[0121] In some implementations, m is selected from 2, 3, and 4.

[0122] In some implementations, n is 4 to 12 and m is 2, 3 or 4.

[0123] In some implementations, n is 12 and m is 3, and the capsid reactive linker is called DBCO-PEG. 12 -TFP and has the following structure:

[0124] .

[0125] In some implementations, n is 12 and m is 4, and the capping reactive connector is called DBCO-PEG4-TFP and has the following structure:

[0126] .

[0127] 4.3.2. The effect of crosslinking agent reactivity on

[0128] In some embodiments, CRP1 comprises a reactive moiety selected from the following: azide; alkyne; 1,4-triazole; 1,3-nitroketone; cyclooctyne or its derivatives, such as dibenzylazacyclooctyne (referred to herein as DBCO) or its derivatives; triazine; tetraazine; strained dienophile; aryl or alkylphosphine; isocyanate; benzylguanine group, benzylcytosine group or chloroalkane group.

[0129] In some embodiments, CRP1 comprises a cyclooctylene reactive moiety. In some embodiments, CRP1 is selected from OCT, MOFO, DIFO, DIMAC, COMBO, DIBO, DIBAC (DBCO), BARAC, BCN, and TMTH, such as those described below:

[0130]

[0131]

[0132] In some implementations, CRP 1 Includes DBCO. In some implementations, CRP 1 It has the following formula:

[0133]

[0134] Where m is 1 to 20. In some embodiments, m is 1 to 15. In some embodiments, m is 1 to 10. In some embodiments, m is 4. In some embodiments, the wavy line indicates SP. 1 Attachment.

[0135] 4.3.3. Connector

[0136] The surface-modified viral capsid of this disclosure includes a connector formed by attachment of a viral capsid to a bifunctional connector. In some embodiments, the connector is of form VI:

[0137] (VI)

[0138] SP 1 and SP 2 Independently acting as a bond or spacer; and

[0139] Q includes a cross-linked portion.

[0140] In some embodiments, the acyl group of the connector binds to a primary amine accessible on the surface of the viral capsid, thereby forming an amide bond. In some embodiments, SP 2 Binds to ligands.

[0141] 4.3.3.1 Spacer

[0142] The linker optionally further comprises one or more spacer groups. The spacer groups are not particularly limited and can be any spacer group 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).

[0143] In some of these implementations, the spacer base (SP) 1 SP 2 or SP 1 and SP 2 Both contain 10 to 30 non-hydrogen atoms. In a further embodiment, the spacer group contains 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 some of these embodiments, the spacer group contains 5-40 methylene groups, 1-20 ether groups, 1-5 amine groups, and / or 1-4 carbonyl groups. In some embodiments, the spacer group contains 10-15 methylene groups, 4-6 ether groups, 1-2 amine groups, and / or 3-4 carbonyl groups.

[0144] In some implementation schemes, SP 1 SP 2 or SP 1 and SP 2 Both contain one or more PEGs (i.e., -(-(O-CH2-CH2)). n - or -([PEG]) n )-), and SP 2It is -YC(O)-, -YC(O)O-, -Y-NHC(O)-, -Y-NHC(S)- or -YC(O), where Y is a bond or one or more PEGs.

[0145] In some implementations, SP 1 Includes -([PEG]) n )-, where n is 1 to 100, for example 1 to 50, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 4 to 20, 4 to 15, 4 to 12, or 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.

[0146] In some implementation schemes, SP 1 SP 2 Or both contain 2 to 20 PEGs, for example, 2 to 15, 2 to 10, 2 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20. In some embodiments, SP 1 It contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 PEGs.

[0147] In some implementation schemes, SP 2 Contains -([PEG]n)-C(O)-, where n is 1 to 100, such as, for example, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15, or 4 to 10.

[0148] 4.3.4. Crosslinking component – ​​Q

[0149] In embodiments of this disclosure, "crosslinked portion" and "Q" are interchangeable and refer to the reaction product of the crosslinking agent portion as described above.

[0150] In some embodiments, the crosslinking portion comprises a product selected from the following reactions: CuAAC reaction, SPAAC reaction, SPANC reaction, IEEDD reaction, Staudinger linkage, and [4+1] cycloaddition reaction. In some of these embodiments, the reaction is selected from: SPAAC, SPANC, and IEEDD reactions.

[0151] In some embodiments, the crosslinked portion comprises a 7- or 8-membered carbon ring containing 0-3 heteroatoms selected from O or N. In some of these embodiments, the crosslinked portion comprises... .

[0152] In this disclosure, the surface-modified viral capsid includes a portion Q, which is formed by a reaction between reactive portions of a crosslinking agent as described herein.

[0153] In some embodiments, Q comprises a product of the CuAAC reaction. In some embodiments, Q comprises a product of the SPAAC reaction. In some embodiments, Q is a product of the SPANC reaction. In some embodiments, Q comprises a product of the IEEDD reaction. In some embodiments, Q comprises a Staudinger-linked product. In some embodiments, Q comprises a product of a [4+1] cycloaddition reaction. In some embodiments, Q comprises a product of a strain-promoted reaction, such as SPAAC, SPANC, and IEEDD.

[0154] In some embodiments, Q comprises a cyclic group. In some embodiments, Q comprises a bicyclic group. In some embodiments, Q comprises a tricyclic group. In some embodiments, Q comprises a 5-8 membered carbon ring containing 0 to 3 heteroatoms selected from O, S, or N. In some embodiments, Q comprises an 8-membered ring containing 0 to 1 heteroatoms selected from O and N. In some embodiments, Q comprises a 5-membered ring containing 0 to 3 heteroatoms selected from O and N. In some embodiments, Q is a triazole ring. In some embodiments, Q comprises a 6-membered ring containing 0-3 heteroatoms selected from O and N. In some embodiments, Q comprises a 6-membered ring containing 2 N heteroatoms.

[0155] In some embodiments, Q comprises a cyclic group, Q being according to the structure in which Z is a 7- or 8-membered carbon ring containing 0-3 heteroatoms selected from O or N.

[0156]

[0157] In some implementations, Q includes the structure shown below:

[0158]

[0159] In some embodiments, Q comprises the reaction product of the cyclooctylene reactive moiety and the azide reactive moiety.

[0160] In some implementations, Q includes one of the following structures:

[0161] and .

[0162] In some implementations, Q includes one of the following structures:

[0163] and ,

[0164] Where m is 1 to 10, for example, 2 to 8, 3 to 7, or 4 to 6.

[0165] 4.3.5. Viral capsid

[0166] In embodiments of this disclosure, the type of viral capsid is not particularly limited. In some embodiments, the viral capsid is selected from non-enveloped viruses, such as adenoviruses or adeno-associated viruses. In some embodiments, the viral capsid is a protein capsid of an enveloped virus (e.g., retrovirus, lentivirus, herpes simplex virus, and baculovirus). Embodiments include non-naturally occurring capsids and include biological or chemical alterations or variations of naturally occurring capsid proteins.

[0167] In some implementations, the viral capsid is that of adeno-associated virus or recombinant adeno-associated virus (rAAV or AAV, which are used interchangeably herein). Such AAV particles are capable of transducing a wide range of postmitotic cells in mammals, including, but not limited to, muscle cells, hepatocytes, and neurons.

[0168] In some embodiments, AAV comprises the naturally occurring AAV serotype VP1, VP2, and / or VP3 capsid proteins. In some embodiments, AAV comprises one or more of the non-naturally occurring VP1, VP2, and / or VP3 capsid proteins. In some of these embodiments, the non-naturally occurring VP1, VP2, or VP3 capsid proteins differ from the naturally occurring capsid in terms of primary amino acid sequence. In some embodiments, the non-naturally occurring capsid includes biological or chemical alterations or variations of the naturally occurring AAV capsid protein that differ from, or are otherwise modified in terms of, the primary amino acid sequence.

[0169] In some implementations, the capsid of AAV is composed of three overlapping capsid proteins (VP1, VP2, VP3), which contain a unique N-terminus of VP1, a common portion of VP1 / VP2, and a portion shared by VP1, VP2, and VP3.

[0170] In some embodiments, one or more capsid proteins comprise naturally occurring amino acid residues, i.e., a primary sequence corresponding to a wild-type capsid protein. In alternative embodiments, the primary sequence of one or more capsid proteins comprises amino acid residues engineered to correspond to a wild-type capsid protein sequence. In some of these embodiments, the engineered amino acid comprises one or more amino groups present on the capsid surface and participates in the surface functionalization of one or more capsid proteins. In some embodiments, the naturally occurring or engineered amino group surface-functionalized using the methods of this disclosure is lysine.

[0171] In various embodiments, the capsid protein is one of the naturally occurring AAV serotypes, namely AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, or AAV9. In various embodiments, the capsid protein is selected from those 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 entirety.

[0172] In some implementations, the capsid is AAV2. In some implementations, the capsid is AAV5.

[0173] In addition, adeno-associated viruses can be selected from synthetic serotypes generated by non-natural methods, such as, but not limited to: capsid mutagenesis, peptide insertion into or deletion from the capsid sequence, capsid truncation from various serotypes, or ancestral reconstruction.

[0174] The AAV used in this disclosure is produced by any method known in the art, but is not limited thereto. For example, 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 transgenes), or insect cells infected with baculovirus capsids (expressing rep-cap and transgenes). AAV produced by any of these methods can be used to produce the surface-functionalized and surface-modified viral capsids described herein. In some embodiments, AAV is produced by transient transfection of HEK293 cells with two plasmids using the calcium phosphate-HeBS method: pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus accessory genes (E2A, VA RNA, and E4), and pVector ss-CAG-eGFP.

[0175] In some implementations, the AAV disclosed herein may include, as needed, one or more sequences from sources other than viruses.

[0176] According to a specific implementation, AAV contains one or more wild-type capsid proteins derived from naturally occurring serotypes.

[0177] According to another specific embodiment, AAV comprises a genetically modified capsid protein. In some embodiments, the genetically modified capsid protein is a naturally occurring serotype engineered to include one or more genetic modifications (mutations, insertions, or deletions). In alternative embodiments, the AAV capsid comprises one or more synthetic capsid proteins. In a particular embodiment, the AAV capsid is engineered to modify natural tropism, for example, to reduce heparin binding.

[0178] Within the framework of this disclosure, synthetic capsids include any combination of capsid proteins derived from natural, genetically modified, and artificially generated serotypes (e.g., random mutations, sequence shuffling, computer simulation design, etc.) that are capable of assembling and generating novel AAVs that are not known to exist in nature.

[0179] Currently, over 100 AAV serotypes have been identified, each with capsid proteins that bind differently to specific cell surface receptors capable of transducing different cell types. AAV2 was the first serotype cloned into a bacterial plasmid and has since been used as a comparison for identifying other serotypes. The transduction capabilities of twelve serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) for specific cell types have been thoroughly tested, and capsid protein motifs binding to specific cell surface receptors for cell attachment have been differentiated. In the context of this disclosure, AAV capsids selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 are preferred. However, it should be understood that any other AAV capsid may be used.

[0180] In one embodiment, the adeno-associated virus (AAV) particles are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. To date, the most commonly used gene transfer systems are viral derivatives, such as adeno-associated virus type 2 (AAV2), AAV9, and AAV8. In certain embodiments, the AAV capsid is selected from AAV2 and AAV9, wherein the capsid protein is optionally further engineered to reduce or modify natural tropism, for example, to reduce heparin binding. In some embodiments, the AAV particles are 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.

[0181] In a particular implementation, the binding sites of the AAV capsid that can bind to heparan sulfate proteoglycan have been removed.

[0182] In certain embodiments, the removal of heparin binding is engineered by replacing at least one of arginine 585 or arginine 588 in VP1 and / or a similar arginine in VP2 or 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.

[0183] In certain embodiments, the AAV of this disclosure comprises at least one capsid protein mutated from the wild type, for example, wherein the engineered / mutated protein is selected from wild-type proteins VP1, VP2, and / or VP3. Alternatively, two of the proteins VP1, VP2, and / or VP3 in the capsid are mutated, or all three of the proteins VP1, VP2, and VP3 in the capsid are modified. In certain embodiments, at least a portion, for example, one amino acid, of the at least one protein to be modified in the capsid is mutated (replaced, inserted, or deleted). However, multiple portions of the proteins VP1, VP2, and VP3 in the capsid may also be mutated, for example, multiple amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of portions or amino acids. In certain embodiments, at least one of arginine 484, 487, 585, and 588 and lysine 532 in VP1 and / or a similar arginine in VP2 or VP3 is replaced by a different amino acid, such as alanine.

[0184] Primary amines are present at the N-terminus of each capsid protein and in the side chain (ε) of lysine (Lys, K) amino acid residues in the capsid protein sequence. These primary amines are surface-accessible and therefore capable of reacting with TFP esters, such as those with capsid reactive linkers (hereinafter), to provide amide bonds.

[0185] 4.3.6. Ligands

[0186] There are no particular limitations on the ligands used with this disclosure, as long as the ligand is suitable for the conjugation described herein. In some embodiments, the ligand is selected from protein ligands having homologs (e.g., receptors) located on the surface of mammalian cells. In some of these embodiments, the homologous protein is involved in the transduction of surface-modified viral capsids.

[0187] In some embodiments, the ligand is a cell type-specific ligand. In some embodiments, the ligand is selected from peptides, proteins, monosaccharides or polysaccharides, steroid hormones, RGD motif peptides, vitamins, small molecules, or targeting peptides. Antibodies (e.g., single chains) or fragments thereof, and nanobodies or DARPins (designed ankylosing spondylamine repeats), genetically engineered antibody mimics that typically exhibit high specificity and high affinity for target protein binding, are also considered; enzymes such as proteases, glycosidases, lipases, and peptidases; immunoglobulins such as CD47 (Don't Eat Me Signal); IgG proteases such as IdeZ and IdeS; and protein-based adjuvants and small molecule adjuvants for vaccination.

[0188] According to one implementation, the cell type-specific ligand is derived from proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF).

[0189] According to one implementation, the cell type-specific ligand is derived from monosaccharides or polysaccharides, such as galactose, N-acetylgalactosamine, and mannose.

[0190] According to one implementation scheme, cell type-specific ligands are derived from vitamins such as folic acid.

[0191] According to one implementation, the cell type-specific ligand is derived from small molecules, including naproxen, ibuprofen, or other known protein-binding molecules.

[0192] In some embodiments, the ligand is selected from protein ligands, such as growth factors or cytokines; toxin subunits, such as the cholera toxin B subunit; lectins, such as isolectin B4 or wheat germ lectin; adhesion factors, such as milk agglutinin; antibodies or their single-chain variable fragments, such as anti-CD-34 antibodies; more specifically, fragments of recombinant E. coli scFv CD-34 antibody, peptides, such as deltorphin opioid receptor ligands; and gene-editing nucleases, such as Cas9, DARPin such as MP0112. In some embodiments, the lectin is selected from wheat germ lectin (WGA), isolectin B4 (IB4), sophora japonica lectin, lentil lectin, wisteria lectin, and Pha-L. In some embodiments, the lectin is WGA. In some embodiments, the lectin is IB4.

[0193] In other embodiments, the ligand is an oligonucleotide, such as an aptamer, as described in Kelly, L., Maier, KE, Yan, A. et al. A comparative analysis of cell surface targeting aptamers. NatCommun 12, 6275 (2021), which is incorporated herein by reference.

[0194] 4.3.7. Goods

[0195] In some embodiments, the nucleic acid cargo is packaged within a surface-modified capsid of this disclosure. The nucleic acid cargo can be any type of nucleic acid molecule that is usefully transduced into cells via rAAV.

[0196] In some embodiments, the payload or cargo of the rAAV disclosed herein is an expressible polynucleotide. In some embodiments, the expressible polynucleotide encodes a protein (e.g., a therapeutic protein). In some embodiments, the expressible polynucleotide encodes a transgene. In some embodiments, the expressible polynucleotide can be transcribed to provide guide RNA, trans-activating CRISPR RNA (tracrRNA), messenger RNA (mRNA), microRNA (miRNA), or shRNA.

[0197] In some implementations, the payload provides DNA homology constructs for homology-directed repair.

[0198] In some embodiments, the nucleic acid molecule encodes an intracellular antibody (e.g., to neutralize certain proteins within the cell), a nucleic acid molecule encoding a peptide toxin (e.g., to block ion channels in pain pathways), a nucleic acid molecule encoding an optogenetic actuator (e.g., to use light to turn neuronal activity on or off), a nucleic acid molecule encoding a pharmacogenetic tool (e.g., to use chemical ligands that do not interfere with pharmacological effects to turn neuronal signaling on or off), a nucleic acid molecule encoding a CRISPR-based editor for precise gene editing, a nucleic acid molecule encoding a CRISPR-epigenetic tool to regulate gene expression, and / or a nucleic acid molecule encoding a suicide gene to induce cell death.

[0199] In some embodiments, the cargo encodes an immunogenic polypeptide, for example, for use in vaccination. The nucleic acid may encode any immunogen of interest known in the art, including but not limited to immunogens derived from human immunodeficiency virus, influenza virus, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc. Alternatively, the immunogen may be present in (e.g., incorporated therein) or tethered to the viral capsid (e.g., through covalent modification).

[0200] Immunogenic peptides or immunogens can be any peptide suitable for protecting a subject against disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal, and viral diseases. For example, an immunogen can be an orthomyxoviral immunogen (e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentiviral 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 protein, and the HIV or SIV gag, pol, and env gene products). Immunogens can also be arenavirus immunogens (e.g., Lassa virus immunogens, such as the Lassa virus nucleocapsid protein gene and the Lassa envelope glycoprotein gene), poxvirus immunogens (e.g., vaccinia vaccines, such as the vaccinia L1 or L8 gene), flavivirus immunogens (e.g., yellow fever virus immunogens or Japanese encephalitis virus immunogens), filovirus immunogens (e.g., Ebola virus immunogens or Marburg virus immunogens, such as the NP and OP genes), Bunyavirus immunogens (e.g., RVFV, CCHF, and SFS viruses), or coronavirus immunogens (e.g., infectious human coronavirus immunogens, such as the human coronavirus envelope glycoprotein gene, or porcine transmissible gastroenteritis virus immunogens, or avian infectious bronchitis virus immunogens, or severe acute respiratory syndrome (SARS) immunogens such as S [S1 or S2], M, E, or N proteins or their immunogenic fragments). The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, diphtheria toxin or other diphtheria immunogen, pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, or hepatitis C) immunogen, or any other vaccine immunogen known in the art.

[0201] In some implementations, the immunogen can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of cancer cells. Exemplary cancer and tumor cell antigens are described in SA Rosenberg, (1999) Immunity 10:281.Exemplary 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 antigen (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 antigens (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med.178:489); HER-2 / neu gene product (US Patent No. 4,968,603); CA 125; HE4; LK26; FB5 (endothelial sialic acid protein); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialylated Tn antigen); c-erbB-2 protein; PSA; L-CanAg; estrogen receptor; milk lipoglobulin; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigen (international patent publication WO 90 / 05142); telomerase; nuclear matrix protein; prostate acid phosphatase; human papillomavirus antigen; and antigens associated with the following cancers: melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, 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, for example, Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

[0202] In some implementations, the nucleic acid cargo encodes any polypeptide that is desired to be produced in vitro, in vitro, or in vivo within cells. For example, a viral vector can be introduced into cultured cells and the expressed protein product can be isolated therefrom.

[0203] Those skilled in the art will understand that nucleic acid goods of interest can be operatively associated with appropriate control sequences. For example, nucleic acid goods can be operatively associated with expression control elements, such as transcription / translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, etc.

[0204] Those skilled in the art will further understand that a variety of promoter / enhancer elements can be used, depending on the desired level and tissue-specific expression. Promoters / enhancers can be constitutive or inducible, depending on the desired expression pattern. Promoters / enhancers can be natural or foreign, and can be natural or synthetic sequences. Foreign refers to a transcription start region that is absent in a wild-type host where a transcription start region has been introduced.

[0205] The promoter / enhancer element may be inherent to the target cell or the subject to be treated, and / or inherent to the nucleic acid cargo. Typically, the promoter / enhancer element is selected such that it functions in the target cell of interest. In a representative embodiment, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0206] Inducible expression control elements are commonly used in applications where regulation of nucleic acid cargo expression is desired. Inducible promoter / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, and include muscle-specific or preferred (including heart, skeletal muscle, and / or smooth muscle), nerve tissue-specific or preferred (including brain-specific), eye (including retina-specific and corneal-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter / enhancer elements. In one embodiment, CNS cell-specific or CNS cell-preferred promoters are used. Examples of neuron-specific or preferred promoters include, but are not limited to, neuron-specific enolases, synaptic proteins, and MeCP2. Examples of astrocyte-specific or preferred promoters include, but are not limited to, glial fibrillary acidic protein and S100β. Examples of ependymal cell-specific or preferred promoters include, but are not limited to, wdr16, Foxj1, and LRP2. Examples of microglia-specific or preferred promoters include, but are not limited to, F4 / 80, CX3CR1, and CD11b. Examples of oligodendrocyte-specific or preferred promoters include, but are not limited to, myelin basic protein, cyclic nucleotide phosphodiesterase, proteolipoprotein, Gtx, and Sox10. Using CNS cell-specific or preferred promoters can increase the specificity achieved via AAV vectors. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, the Tet on / off element, the RU486 inducible promoter, the ecdysone inducible promoter, the rapamycin inducible promoter, and the metallothionein promoter.

[0207] In embodiments where nucleic acid cargoes are transcribed and then translated in target cells, specific initiation signals are typically employed for the efficient translation of the inserted protein-coding sequence. These exogenous translation control sequences, which may include the ATG start codon and adjacent sequences, can have multiple sources, including both natural and synthetic ones.

[0208] In some embodiments, when the cargo contains a gene-editing nuclease (such as Cas9), the cargo further contains nucleic acid molecules, such as gRNA and / or specific DNA, to be inserted into the host genome. In some of these embodiments, the cargo contains transgenes known to be associated with genetic conditions.

[0209] Technicians are familiar with other gene-editing nucleases besides Cas9, such as Cpf1, TALEN, ZFN, or homing endonucleases. Furthermore, engineering using the DNA-guided Argonaute interference system (DAIS) may be convenient. Argonaute (Ago) proteins are heterologously expressed from polynucleotides introduced into the cell in the presence of at least one exogenous oligonucleotide (DNA guide) that provides cleavage specificity to a preselected locus for the Ago protein. The TALEN and Cas9 systems are described in WO 2013 / 176915 and WO 2014 / 191128, respectively. Zinc finger nucleases (ZFNs) were 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). Cpf1 is a Class 2 CRISPR-Cas system described by Zhang et al. (Cpf1 is a single RNA-guided Endonuclease of a Class 2 CRIPR-Cas System. (2015). Cell; 163:759-771). The argonaute (AGO) gene family was first described by Guo S, Kemphues KJ. (Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser / Thr kinase that isasymmetrically distributed. (1995). Cell;81(4):611-20).

[0210] In some embodiments, the vector is an "empty" capsid particle (i.e., without a vector genome) containing, composed of, or substantially composed of the chimeric AAV capsid protein of this disclosure. The chimeric AAV capsid of this disclosure can be used as a "capsid vector," as described in U.S. Patent No. 5,863,541, and can be covalently linked to, bound to, or packaged by a viral capsid and transferred to cells. Molecules include DNA, RNA, lipids, carbohydrates, polypeptides, small organic molecules, or combinations thereof. Furthermore, the molecule can bind to the exterior of the viral capsid (e.g., "tethered thereto") for transfer of the molecule to a host target cell. In one embodiment of this disclosure, the molecule is covalently linked to (i.e., conjugated or chemically coupled) to the capsid protein. Methods of covalently linking molecules are known to those skilled in the art.

[0211] The capsid disclosed herein can also be used to generate antibodies against novel capsid structures. As a further alternative, exogenous amino acid sequences can be inserted into the viral capsid for antigen presentation to cells, for example, for administration to a subject to generate an immune response against the exogenous amino acid sequence.

[0212] 4.4. Method for preparing surface-functionalized capping compositions

[0213] In this disclosure, a method is provided for preparing the composition described above comprising a surface-functionalized viral capsid. Compared to previous methods relying on NHS-ester-containing capsid-reactive adapters, the method described herein uses a relatively small amount of capsid-reactive adapter (i.e., a TFP-ester-containing capsid-reactive adapter). The reduced amount of capsid-reactive adapter eliminates the need for a post-reaction purification step compared to surface-modified viral capsids prepared using NHS-ester-containing capsid-reactive adapters, thereby preventing viral loss and improving cell transduction.

[0214] The method includes step (a): combining a viral capsid (I) comprising a plurality of surface-accessible primary amines (p) and a capsid-reactive linker (II) comprising a terminal first member (CRP1) of a terminal tetrafluorophenyl (TFP) ester and a crosslinking agent reactive pair to provide a composition comprising a surface-functionalized viral capsid (III), as shown in Scheme 1 below:

[0215]

[0216] Option 1. A method for preparing surface-functionalized viral capsids

[0217] Amide bonds are formed by the reaction of a primary amine, for example, a surface-accessible lysine residue in the primary sequence of the capsid with a capsid-reactive linker containing a TFP ester, as shown above (only one surface lysine amine is shown for clarity).

[0218] Multiple surface-accessible primary amines (p) will react with various TFP esters of the capping-reactive linker. In some embodiments, p is an integer from 10 to 500, such as, for example, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400, or 400 to 500. In some implementations, p is an integer from 100 to 200, such as, for example, 100 to 190, 100 to 180, 100 to 170, 100 to 160, or 100 to 150.

[0219] The variable “q” in the surface-functionalized viral capsid (III) reflects the amount of amide formed by the reaction of the surface-accessible primary amine (p) of the viral capsid (I) with the TFP ester of the capsid-reactive linker (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, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 100 to 500, 100 to 400, 100 to 300, 100 to 200, 200 to 500, 200 to 400, 200 to 300, 300 to 500, 300 to 400, or 400 to 500. In some implementations, q is an integer from 100 to 200, such as, for example, 100 to 190, 100 to 180, 100 to 170, 100 to 160, or 100 to 150.

[0220] SP 1 For a bond or one or more PEGs (i.e., -(-(O-CH2-CH2)). n - or -([PEG]) n In some implementations, n is 0. In some implementations, n is 1 to 100, such as, for example, 1 to 50, 1 to 25, 1 to 15, 1 to 10, 1 to 5, 4 to 20, 4 to 15, 4 to 12, or 4 to 10.

[0221] In some embodiments, the molar ratio of viral capsid (I) to capsid-reactive adapter (II) in reaction (a) is 1:100 to 1:50,000, such as, for example, 1:100 to 1:33,000; 1:100 to 1:10,000; 1:100 to 1:5,000; 1:100 to 1:1,000; 1:100 to 1:500; 1:100 to 1:250. In some embodiments, the molar ratio of viral capsid (I) to capsid-reactive adapter (II) is 1:500 to 1:10,000.

[0222] In some embodiments, the viral capsid (I) is purified prior to reaction with the capsid-reactive adapter (II). It has been found that when the viral capsid is purified first, a lower molar ratio of viral capsid to capsid-reactive adapter is required to prepare compositions with strong viral-cell transduction. 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, density gradient centrifugation is iodixanol density gradient ultracentrifugation.

[0223] In some embodiments, the capsid virus is used without purification. In some embodiments, chemical modification is performed directly on crude cell lysates (e.g., unpurified AAV). In some embodiments, cell lysates (e.g., AAV9, AAV2-VR4) can be efficiently modified without purification using, for example, NHS and TFP esters of capsid-reactive linkers. In some embodiments, AAV9 can be efficiently modified without purification using TFP esters of capsid-reactive linkers. In some embodiments, AAV9 can be efficiently modified without purification using NHS esters of capsid-reactive linkers. In some embodiments, AAV2-VR4 can be efficiently modified without purification using TFP esters of capsid-reactive linkers. In some embodiments, AAV2-VR4 can be efficiently modified without purification using NHS esters of capsid-reactive linkers. In some embodiments, a lower concentration of TFP ester is required compared to NHS.

[0224] In some implementations, the viral capsid is freeze-thawed before surface functionalization.

[0225] In some embodiments, the reaction is carried out in an aqueous medium. Suitable aqueous media include at least one buffer. In some embodiments, the buffer is selected from N-[2-hydroxyethyl]-piperazine-N'-[2-ethylsulfonic acid] (HEPES), MOPS, MES, phosphates, and bicarbonates. In some embodiments, the concentration of the at least one buffer varies. In some embodiments, the concentration of the at least one buffer does not exceed about 0.1 M, for example, about 0.01 M to about 0.1 M or about 0.05 M to about 0.1 M. In some embodiments, the aqueous medium comprises about 0.01 M to about 0.1 M sodium bicarbonate.

[0226] 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 chlorides, phosphates, sulfates, and citrates. In some embodiments, the salt is selected from sodium, potassium, calcium, and magnesium salts. In some embodiments, the aqueous medium comprises at least about 200 mM sodium chloride.

[0227] 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.

[0228] 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 about 0.001% to about 0.005% Pluronic® F68.

[0229] In some embodiments, the pH of the aqueous medium is about 6 to about 10, for example, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 6 to about 8, about 7 to about 8, or about 6 to about 7. In some embodiments, the pH of the aqueous medium is about 8 to about 9.

[0230] In some embodiments, the reaction temperature is from about 0°C to about 50°C, such as, for example, from about 10°C to about 40°C or from about 20°C to about 30°C. In some embodiments, the reaction temperature is room temperature (ca. 23°C).

[0231] In some implementations, 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.

[0232] In some embodiments, an excess of quencher is added after the reaction is complete to consume unbound linkers. In some embodiments, the quencher comprises an amine-containing compound. In some embodiments, the quencher is selected from glycine and Tris buffer.

[0233] In some embodiments, no purification step is performed after the 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, the use of these purification steps results in the loss of viral particles.

[0234] In embodiments where purification is not performed, the resulting composition will contain a certain amount of starting materials and reagents, in addition to the surface-functionalized viral capsid product.

[0235] The method of this disclosure further includes step (b): reacting a composition comprising a surface-functionalized viral capsid (III) containing a first member (CRP1) with a crosslinking agent reactive pair with a functionalized ligand containing a second member (CRP2) with a crosslinking agent reactive pair to provide a composition comprising a surface-modified viral capsid (V).

[0236] The first and second crosslinking agents react to form a crosslinked portion Q (discussed above), thereby conjugating the viral capsid and ligand, as shown in Scheme 2 below:

[0237]

[0238] Option 2. Conjugation of viral capsid and ligands.

[0239] In Option 2, As a functionalized ligand, SP 2 It is a bond or spacer group, and L is a ligand.

[0240] In some embodiments, the functionalized ligand is purchased from a commercial supplier. In other embodiments, the functionalized ligand is prepared by contacting a ligand comprising a first reactive moiety with a ligand-reactive junction comprising a second member (CRP2) comprising a second reactive moiety and a crosslinker reactive pair.

[0241] In some embodiments, the ligand-reactive linker is selected from TCO-PEGn-NHS; tetrazine-PEGn-NHS; azide-PEGn-NHS; phosphine-NHS; maleimide-PEGn-succinimide ester; DBCO-PEGn-TFP ester and DBCO-PEGn-NHS ester, wherein n is 1 to 100, preferably 4 to 10.

[0242] In some embodiments, the ligand-reactive linker is a DBCO-PEGn-TFP ester, wherein n is 1 to 100, preferably 4 to 10. In some embodiments, the ligand-reactive linker is a DBCO-PEGn-NHS ester, wherein n is 1 to 100, preferably 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.

[0243] In some embodiments, the functionalized ligands used in this disclosure are as described in WO2022101363, the entire contents of which are incorporated herein by reference.

[0244] In some implementations, the SP of the functionalized ligand 2 Selected from -YC(O)-, -YC(O)O-, -Y-NHC(O)-, -Y-NHC(S)- or -YC(O), where Y is a bond or one or more PEGs (i.e., -(-(O-CH2-CH2)). n - or -([PEG]) n In some implementations, SP 2 Yes -([PEG]) n )-C(O)-, where n is 1 to 100, such as, for example, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 4 to 15, or 4 to 10. In some implementations, SP 2 -([PEG]) n )-C(O)-, where n is 1 to 100, preferably 1 to 10.

[0245] In some implementations, the functionalized ligand is of formula IV:

[0246] (IV),

[0247] in:

[0248] It is a ligand.

[0249] In such embodiments, the surface-accessible primary amine (as shown above) and SP 2 Adhesion.

[0250] In some embodiments, CRP2 comprises a reactive moiety selected from: azides; alkynes; 1,4-triazoles; 1,3-nitroketones; cyclooctyne or derivatives thereof, such as dibenzylcyclooctyne or derivatives thereof; triazines; tetraazines; strained dienophiles; aryl or alkylphosphines; isocyanates; benzylguanine, benzylcytosine, or chloroalkane groups. In some of these embodiments, CRP2 comprises an azide. In some embodiments, CRP2 is composed of -N3.

[0251] In some implementations, the functionalized ligand is WGA-[PEG]. n -N3 (also referred to as WGA-[PEG] in this article) n -Azide), where n is 1 to 20, for example, 1 to 10, or 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.

[0252] In some implementations, the functionalized ligand is NGF-[PEG]. n -N3 (also referred to as NGF-[PEG] in this article) n -Azide), where n is 1 to 20, e.g., 1 to 10, or 4 to 10. In some embodiments, the functionalized ligand is NGF-[PEG]n-azide, where n is 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.

[0253] In some embodiments, the molar ratio of the viral capsid (used in step (a)) to the functionalized ligand (used in step (b)) is 1:100 to 1:50,000, such as, for example, 1:100 to 1:33,000; 1:100 to 1:10,000; 1:100 to 1:5,000; 1:100 to 1:1,000; 1:100 to 1:500; 1:100 to 1:250. In some embodiments, the molar ratio of the viral capsid to the functionalized ligand is 1:500 to 1:10,000.

[0254] In some embodiments, the molar ratio of viral capsid to capsid-reactive linker in step (a) and the molar ratio of viral capsid to functionalized ligand in step (b) are the same, i.e., 1:1. In some embodiments, the molar ratio of viral capsid to capsid-reactive linker in step (a) and the molar ratio of viral capsid to functionalized ligand in step (b) are different.

[0255] In some embodiments, the reaction is carried out in an aqueous medium. Suitable aqueous media include at least one buffer. In some embodiments, the buffer is selected from N-[2-hydroxyethyl]-piperazine-N'-[2-ethylsulfonic acid] (HEPES), MOPS, MES, phosphates, and bicarbonates. The concentration of at least one buffer can vary. In some embodiments, the concentration of at least one buffer does not exceed about 0.1 M, for example, about 0.01 M to about 0.1 M or about 0.05 M to about 0.1 M. In some embodiments, the aqueous medium comprises about 0.01 M to about 0.1 M sodium bicarbonate.

[0256] 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 chlorides, phosphates, sulfates, and citrates. In some embodiments, the salt is selected from sodium, potassium, calcium, and magnesium salts. In some embodiments, the aqueous medium comprises at least about 200 mM sodium chloride.

[0257] 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.

[0258] 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 about 0.001% to about 0.005% Pluronic® F68.

[0259] In some embodiments, the pH of the aqueous medium is about 6 to about 10, for example, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 6 to about 9, about 7 to about 9, about 8 to about 9, about 6 to about 8, about 7 to about 8, or about 6 to about 7. In some embodiments, the pH of the aqueous medium is about 8 to about 9.

[0260] In some embodiments, the reaction temperature is from about 0°C to about 50°C, such as, for example, from about 10°C to about 40°C or from about 20°C to about 30°C. In some embodiments, the reaction temperature is room temperature (ca. 23°C).

[0261] In some implementations, 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.

[0262] 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.

[0263] In some implementations, the surface-modified viral capsid is modified in batches.

[0264] In some implementations, the surface-modified viral capsid is stable at low temperatures, including -150°C (e.g., -140°C, -130°C, -120°C, -110°C, -100°C, -90°C, -80°C, -70°C, -60°C, -50°C, -40°C, -30°C, -20°C, or -10°C), 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).

[0265] In some embodiments, the surface-modified viral capsid is stable at -140°C. In some embodiments, the surface-modified viral capsid is stable at -130°C. In some embodiments, the surface-modified viral capsid is stable at -120°C. In some embodiments, the surface-modified viral capsid is stable at -110°C. In some embodiments, the surface-modified viral capsid is stable at -100°C. In some embodiments, the surface-modified viral capsid is stable at -90°C. In some embodiments, the surface-modified viral capsid is stable at -80°C. In some embodiments, the surface-modified viral capsid is stable at -70°C. In some embodiments, the surface-modified viral capsid is stable at -60°C. In some embodiments, the surface-modified viral capsid is stable at -50°C. In some embodiments, the surface-modified viral capsid is stable at -40°C. In some embodiments, the surface-modified viral capsid is stable at -30°C. In some embodiments, the surface-modified viral capsid is stable at -20°C. In some embodiments, the surface-modified viral capsid is stable at -10°C.

[0266] 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.

[0267] 4.5. Examples

[0268] Example 1. Significant loss of physical titer of AAV2 after NHS modification

[0269] In previous experiments using NHS esters as the reactive portion of the capping layer, it was determined that two separate cleanup steps were required to produce ligand-functionalized capping compositions exhibiting strong transduction. First, using a linker (e.g., NHS-PEG) n Following capsid functionalization with -DBCO, a cleaning process is required to remove excess reactive linkers. This effectively quenches the functionalized ligands during the crosslinking step and prevents the final product from being contaminated with NHS-PEG. n Excessive ligand contamination from DBCO junction crosslinking will compete with AAV capsid transduction.

[0270] When switching to in vivo experiments using NGF-modified vectors, the physical AAV titers before and after modification were measured for the first time, and an unexpected 90% loss of AAV particles was observed during the modification process. Figure 1 This result is surprising; despite a 90% loss, we were still able to see a significant improvement in transduction efficiency when conjugated with ligands such as WGA.

[0271] Commercially available wild-type AAV2 with EGFP cargo as a reporter gene for cell transduction was used at a viral titer of 2.7E+12V g / ml in PBS / Pluronic 0.001% / NaCl 200mM. The AAV2 was modified with 20 μL of NHS-PEG4-DBCO (2.6 nMol) in PBS / Pluronic 0.001% / NaCl 200mM at room temperature for 3 hours, followed by reaction with 55 pMol of WGA-PEG4-azide at room temperature for 1 hour, and then overnight at 4°C. The surface-modified virus was either (1) directly added to PC12 cells or (2) centrifuged 1 to 3 times at 10000g for 1 minute each time in an Amicon 100kDa centrifuge unit.

[0272] like Figure 1 As shown, modification with NHS-PEG4-DBCO resulted in a 90% reduction in AAV2 titer as measured by ddPCR. To further investigate this, WGA-conjugated AAV2 was applied to PC12 cells, and transduction efficiency was monitored before and after reaction cleanup. These experiments showed that virus loss occurred after a single centrifugation step using a 100 kDa centrifugation filter unit. Further centrifugation did not significantly reduce the yield. Other cleanup procedures, including filter unit passivation, AAV2 precipitation, and size exclusion chromatography, were evaluated, with ddPCR of transduction efficiency and physical titer in HEK293 cells serving as readings for virus loss.

[0273] In further experiments, wild-type AAV2 was applied directly to HEK293 cells, or one or more of the following cleanup procedures were performed:

[0274] 1. Centrifuge at 10000g for 1 minute using a 100kDa Amicon centrifuge unit.

[0275] 2. 100 kDa Amicon centrifuge units were passivated by pretreatment with wild-type AAV2 carrying the tdTomato reporter gene for 60 min using 3E+9 Vg, and then the modified AAV2 was centrifuged at 10000 g for 1 min.

[0276] 3. The modified AAV2 was precipitated by incubating in 8% PEG8000 at 4°C with slow stirring for 1 hour, followed by maintaining at 4°C for 3 hours (without stirring) to allow the virus to fully precipitate. Then, the sample was centrifuged at 2818g for 30 minutes at 4°C, the supernatant was removed, and the precipitate was resuspended in PBS / Pluronic 0.001% / NaCl 200mM.

[0277] 4. Size exclusion chromatography was performed by centrifuging the modified AAV2 at 1000g through a column packed with Sepharose 4B resin for 2 minutes. The sample was then concentrated to 24 μL using a vacuum concentrator at 45°C.

[0278] Specifically, AAV2 (0.0045 pMol) was used to modify the capsid-reactive linker TFP-PEG4-DBCO (45 pMol) in PBS / Pluronic 0.001% / NaCl 200 mM overnight at room temperature. The reaction was then quenched with 50 mM glycine to remove residual linkers.

[0279] The samples were analyzed by adding them to HEK293 cells that are permissive to AAV2 and monitoring transduction efficiency using imaging and FACS with a Bio-Rad S3e cell sorter, as well as by quantifying the physical titers of the samples using ddPCR with primers targeting the ITR region.

[0280] As in Figure 2a and 2b As can be seen, all cleanup methods led to a significant decrease in AAV2 recovery, with centrifugation filter units (untreated and passivated) performing the worst. PEG precipitation slightly improved the yield, and Sepharose 4B size exclusion further slightly improved the recovery. In contrast, modifying AAV2 with TFP adapters (which does not require a cleanup step) did not lead to a decrease in the yield of the modified virus, as confirmed by transduction efficiency in HEK293 cells and physical titers measured by ddPCR.

[0281] Example 2. Reaction conditions for use with the reactive groups of the TFP ester coating.

[0282] Commercially available wild-type AAV2 with EGFP cargo as the reporter gene for cell transduction was used. The viral titer was 2.7E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0283] 0.0045 pmol of AAV (1 μL) and 448 pmol of NHS-PEG4-DBCO (virus:linker ratio 1:100,000) were reacted in 22 μL of PBS / Pluronic 0.001% / NaCl 200 mM at room temperature for 3 hours. Subsequently, 55 pmol of WGA-PEG4-azide (virus:ligand ratio 1:12,000) was incubated at room temperature for 2 hours, and then kept at 4°C overnight. The next day, the virus was added to the cells.

[0284] 0.0045 pmol of AAV was incubated overnight at room temperature with a reduced amount of adapter, i.e., 148 pmol of TFP-PEG4-DBCO (virus: adapter ratio of 1:30,000), in a final volume of 22 μL. The next day, glycine (50 mM, pH 6.5) was added to quench any unbound, active TFP-PEG4-DBCO. Then, 148 pmol of WGA-PEG4-azide (virus: ligand ratio of 1:30,000) was incubated overnight at room temperature. The next day, the virus was added to the cells. Various reaction parameters were varied, including the use of (a) reaction buffers (i.e., different preparations of sodium bicarbonate or PBS / Pluronic 0.001% / NaCl 200 mM) and (b) quenching reagents. For each protocol tested, appropriate controls (unmodified virus) were processed and data were used for normalized cell counting. A summary of the evaluation protocols is provided in the table below.

[0285] Table 1. Summary of the plans

[0286]

[0287] In vitro application of PC12 cells

[0288] PC12 cells were maintained at 37°C in DMEM / F12 medium containing 5% horse serum, 5% fetal bovine serum, and 100 U penicillin / streptomycin. PC12 cells were then incubated with WGA-modified AAV2 cells prepared using NHS or TFP adapters. The medium was then changed, and the cells were maintained at 37°C and imaged using a Zeiss AxioObserver A1 microscope 4 days post-infection. Cells were collected 7 days post-infection and analyzed using a Bio-Rad S3e cell sorter.

[0289] result

[0290] Based on imaging analysis (Figure 3), it is clear that reactions 2, 3, and 4 are the most efficient. Without being bound by theory, it is believed that PBS / Pluronic 0.001% / NaCl 200mM can prevent viral aggregation, and that higher concentrations of sodium bicarbonate are detrimental to the binding of TFP adapters to the virus.

[0291] For cell counting analysis, the mean fluorescence intensity (MFI) of EGFP is used as an indicator of how much virus can enter each cell. Figure 4a ), and used EGFP+ cells to define the percentage of cells transduced by AAV ( Figure 4bThe cell percentages for both MFI and EGFP+ were normalized using controls. Among the protocols with TFP leading to good cell transduction, the conditions associated with reaction 2 were optimal. Based on the comparison of reactions 2–4, we conclude that glycine and 0.1 M sodium bicarbonate improve the modification of the virus-TFP linker, thereby achieving good cell transduction.

[0292] Furthermore, similar amounts of WGA ligand-virus entered cells using either TFP reaction 2 or NHS. However, TFP reaction 2 resulted in a higher percentage of transduced cells compared to NHS. This data, along with the fact that three times fewer TFP-PEG4-DBCO linkers were used compared to NHS-PEG4-DBCO, suggests that TFP linker chemistry improves the chemical functionalization of AAV and can be carried out at lower molar ratios, thus avoiding the need for reaction cleanup.

[0293] Example 3. In vitro comparison of TFP and NHS connectors

[0294] AAV2 and AAV5

[0295] Both AAV serotypes used were commercially available. AAV2 had a titer of 2.7E+12 vg / ml and contained the commercially available EGFP reporter gene under the CAG promoter, while AAV5 had a titer of 1E+13 vg / ml and contained the EGFP reporter gene under the CMV promoter.

[0296] NHS-mediated chemical functionalization and coupling of WGA with AAV2 and AAV5

[0297] For NHS-mediated chemical functionalization of AAV2, 1 μL (0.0045 pMol) of DBCO-PEG4-NHS was reacted with different virus:linker molar ratios (see table below) in a reaction volume of 20 μL PBS (+0.001% pluronic and 200 mM NaCl) and shaken at room temperature for 3 hours.

[0298] Table 2

[0299]

[0300] WGA (0.1 nMol) was dissolved in PBS and reacted with 20 molar equivalents of azide-PEG4-NHS at room temperature with shaking for 3 hours. Unreacted azide groups were removed using a 10 kDa MWCO centrifugal filter. NHS-mediated DBCO-modified AAV2 was further incubated with 50 pMol of WGA-PEG4-azide at room temperature with shaking for 2 hours, and then kept overnight at 4°C.

[0301] 1 µl (0.0166 pMol) of NHS-mediated chemical functionalization of AAV5 was performed as described above for AAV2, but the virus:linker ratio differed, as shown in the table below.

[0302] Table 3

[0303]

[0304] TFP-mediated chemical functionalization and coupling of WG with AAV2 and AAV5

[0305] For TFP-mediated chemical modification, 1 μL 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).

[0306] Table 4

[0307]

[0308] To minimize the volume of the DBCO-PEG4-NHS linker, a dilution (1 µl) was prepared from the 20 mM stock solution. Then, 11 µl of PBS (+0.001% pluronic and 200 mM NaCl) and 0.1 M sodium bicarbonate buffer (pH 8.3) were added, and the reaction was incubated overnight at room temperature with shaking. The reaction was terminated by adding 50 mM glycine. WGA (0.1 nMol) was dissolved in PBS and reacted with 20 molar equivalents of azide-PEG4-NHS at room temperature with shaking for 3 hours. Unreacted azide groups were removed using a 10 kDa MWCO centrifuge filter. WGA-PEG4-azide was added at the specified molar ratio (see table above), and the reaction was incubated overnight at room temperature with shaking.

[0309] TFP-mediated chemical modification of 1 µl (0.0166 pMol) of AAV5 was performed as described above for AAV2, but the virus:adaptor:ligand ratio was different, as shown in the table below.

[0310] Table 5

[0311]

[0312] In vitro application of PC12 cells

[0313] PC12 cells were maintained in DMEM / F12 medium (+10% horse serum, 5% fetal bovine serum, 15 mM HEPES, 2.5 mM Glutamax, and 100 U penicillin / streptomycin) and incubated at 37°C in a humidified atmosphere of 5% CO2. Modified AAV was added to the cells and incubated overnight. The medium was changed and the cells were maintained at 37°C for 5 days.

[0314] Data collection

[0315] Transduction efficiency was determined by imaging PC12 cells using a Nikon A1R confocal microscope. To quantify efficiency, cells were collected and prepared for flow cytometry. Flow cytometry data were acquired on a Bio-Rad S3e cell sorter and analyzed using FlowJo. Viral genome copy number was quantified using a Bio-Rad droplet digital PCR system.

[0316] result

[0317] Compared to other virus:adaptor ratios tested, AAV2 modified with DBCO-PEG4-NHS at a virus:adaptor ratio of 1:100,000 and 2.5 uM WGA-PEG4-azide showed the highest transduction in PC12 cells. In contrast, AAV2 modified with DBCO-PEG4-TFP and WGA-PEG4-azide showed the highest transduction at a virus:adaptor:ligand ratio of 1:10,000:10,000. TFP modification requires fewer adaptors to provide the same transduction efficiency as NHS chemistry. For the other AAV serotype, AAV5, a lower amount of virus:adaptor (1:1000 molar ratio) was observed for TFP-mediated modification to achieve optimal transduction efficiency than for NHS-mediated modification (1:30,000 virus:adaptor molar ratio).

[0318] Example 4. In vivo comparison of TFP and NHS connectors

[0319] A series of in vitro experiments using TFP and NHS linkers demonstrated that TFP chemistry improved the modification rate of adeno-associated virus (AAV) with the ligand of interest, prevented viral loss, and was applicable to more than one serotype. The aim here was to verify the superior performance of TFP relative to NHS-modified AAV2 in mice in vivo. A mutant nerve growth factor ligand (NGF) was selected. R121WAs a ligand, it targets a population of nociceptive peptidergic neurons expressing the TrkA / p75 receptor complex. Furthermore, it was found that AAV2 can be modified more efficiently on a larger scale using TFP. Modified AAV2 was subcutaneously injected into the plantar surface of the hind paw, and transgene expression in the skin, dorsal root ganglion (DRG), and spinal cord was analyzed.

[0320] method

[0321] Chemical functionalization via NHS and TFP methods and coupling of NGF and AAV

[0322] For chemical functionalization, we used commercially available wild-type AAV2 containing the EGFP reporter gene. The viral titer was 2.7E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0323] The standard functionalization reaction using NHS was performed as follows: 0.2025 pmol of AAV (45 µl) was reacted with 20250 pmol of NHS-PEG9-BG (virus:linker molar ratio = 1:100,000) in 900 µl of PBS / Pluronic 0.001% / NaCl 200 mM at room temperature for 3 hours. Afterward, the virus was concentrated using an Amicon 100 kDa centrifuge to remove excess unbound linker, and 200 pmol of NGF-SNAP was added. The reaction was incubated at room temperature for 2 hours, then maintained overnight at 4°C. The next day, the virus was again concentrated using an Amicon 100 kDa filter to remove excess unbound NGF-SNAP.

[0324] For the TFP reaction, we incubated 0.1125 pmol of AAV with TFP-PEG4-DBCO at different virus:linker ratios, as reported in the table below. The reaction was carried out overnight at room temperature in PBS (+0.001% pluronic and 200 mM NaCl) and 0.1 M sodium bicarbonate buffer (pH 8.3), quenched with 50 mM glycine, and then incubated with NGF-PEG4-azide at an equimolar ratio to linker (see table below) for 2 hours at room temperature, followed by overnight incubation at 4°C.

[0325] Table 6

[0326]

[0327] Batch modification of AAV9 using NHS and NGF and testing transduction in TrkA / p75-overexpressing HEK293 cells.

[0328] The modification of AAV9 was scaled up, and transduction was tested in HEK293 cell lines expressing TrkA / p75 using NGF as a ligand.

[0329] For bulk chemical modification, commercially available wild-type AAV9 containing the EGFP reporter gene was used. The viral titer was 5.9E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0330] For the NHS reaction, 2832 VG of AAV was incubated with NHS-PEG4-DBCO at a virus:adaptor ratio of 175,000. The reaction was carried out overnight at room temperature in PBS (+0.001% pluronic and 200 mM NaCl), washed three times with 15 ml Amiconfalcon 100 kDa cutoff, aliquoted, and frozen at -80 °C. Subsequently, AAV9-DBCO was thawed and incubated with 3 µM NGF-PEG4-azide at room temperature for 2 h, followed by overnight incubation at 4 °C. A series of titers (i.e., different MOIs) were added to HEK293 cell lines overexpressing TrkA and p75 receptors. As a control, unmodified AAV9 was applied to cells at the same MOI.

[0331] AAV2 was modified in bulk using TFP and conjugated with NGF. Freeze-thaw stability was tested and transduction was determined in vivo in mice.

[0332] For bulk chemical modification, commercially available wild-type AAV2 containing the EGFP reporter gene was used. The viral titer was 2.7E+12 Vg / ml in PBS / Pluronic 0.001% / NaCl 200mM.

[0333] For the TFP reaction, AAV titers were incubated with TFP-PEG4-DBCO at a virus:adaptor ratio of 1:20,000. The reaction was carried out overnight at room temperature in PBS (+0.001% pluronic and 200 mM NaCl) and 0.1 M sodium bicarbonate buffer (pH 8.3), quenched with 50 mM glycine, and then incubated overnight at room temperature with 27 µM NGF-PEG4-azide. Samples were then used fresh or frozen as shown in Table 7.

[0334] Table 7

[0335]

[0336] ddPCR

[0337] AAV2 modified with NHS and TFP at different ratios was subjected to ddPCR using primers targeting the inverted terminal repeats (ITRs) present in the viral genome.

[0338] In vitro application of PC12 cells

[0339] To verify that the modified virus was indeed active, we also applied it in vivo to PC12 cells endogenously expressing TrkA / p75. PC12 cells were maintained at 37°C in DMEM / F12 medium containing 5% horse serum, 5% fetal bovine serum, and 100 U penicillin / streptomycin. PC12 cells were then incubated with NGF-modified AAV2 prepared with NHS and TFP at different ratios. The medium was then changed, and the cells were maintained at 37°C and imaged using a Zeiss AxioObserver A1 microscope 7 days post-infection.

[0340] Mouse paw pad injection

[0341] Three hours prior to viral injection, 60 units of hyaluronidase were injected to promote viral spread. AAV2 modified with NHS or TFP at different ratios (5E+10 VG) was injected into the paw of mice anesthetized with isoflurane. Injection was performed using an insulin syringe placed in the center of the paw. The injection volume was 30 µl.

[0342] Histological analysis

[0343] Three weeks after in vivo injection, mice were sacrificed and lumbar dorsal root ganglia (DRGs) were collected. The DRGs were fixed overnight in 2% paraformaldehyde (PFA), washed in PBS, and cleared at 4°C for 2 days using ScaleS solution. They were then mounted and imaged using a Zeiss AxioObserver A1 microscope.

[0344] In other experiments, lumbar DRGs were washed in PBS and blocked overnight at 4°C with 2% donkey / 0.3% Triton / PBS. They were then incubated at 4°C for 72 hours with 2% donkey / 0.3% Triton / PBS containing 1:200 goat anti-rat Trka antibody. Subsequently, the DRGs were washed three times with 0.3% Triton / PBS at 10-20 minute intervals. The DRGs were then incubated at 4°C for 72 hours with 2% donkey / 0.3% Triton / PBS containing 1:200 donkey anti-goat Ax594 antibody, washed as described above, cleared overnight at 4°C, mounted, and imaged.

[0345] Skin samples were collected from the injection site, fixed overnight in 2% paraformaldehyde (PFA), washed in PBS, and incubated overnight at 4°C with 30% sucrose / PBS. The samples were then embedded in OCT and cut to a thickness of 30 μm in a cryostat. Sections were blocked with 2% donkey / 0.3% Triton / PBS at room temperature for 2 hours. The skin was then incubated overnight at 4°C with 2% donkey / 0.3% Triton / PBS containing 1:200 goat anti-rat Trka and chicken anti-EGFP antibodies. The skin sections were then washed three times with 0.3% Triton / PBS at 10–20 min intervals. Subsequently, the skin was incubated overnight at 4°C with 2% donkey / 0.3% Triton / PBS containing 1:200 donkey anti-goat Ax594 and donkey anti-chicken Ax488 antibodies, washed as described above, mounted, and imaged.

[0346] The spinal cord was isolated from the lumbar enlargement, fixed overnight in 2% paraformaldehyde (PFA), washed in PBS, and incubated overnight at 4°C with 30% sucrose / PBS. It was then embedded in OCT and cut to a thickness of 30 μm in a cryostat. Sections were blocked with 2% donkey / 0.3% Triton / PBS at room temperature for 2 hours, then incubated overnight at 4°C with 2% donkey / 0.3% Triton / PBS containing 1:200 goat anti-rat Trka. The spinal cord sections were washed three times with 0.3% Triton / PBS at 10–20 min intervals and further incubated overnight at 4°C with 2% donkey / 0.3% Triton / PBS containing 1:200 donkey anti-goat Ax594 and the isohemagglutinin GS-IB4 Alexa Fluor™ 647 conjugate. Before imaging, the spinal cord sections were washed three times as described above and then mounted.

[0347] Image analysis

[0348] Tissue imaging was performed using a Nikon A1R confocal microscope and analyzed using ImageJ. Cell counting in the DRG was done manually. Object-based colocalization was performed using the colocalization image creator plugin (1). See Lunde, A., Glover, JC 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.

[0349] result

[0350] Initial experiments using ddPCR confirmed that TFP functionalization did not lead to viral loss, while NHS functionalization resulted in a 90% reduction in yield. Figure 5 Consistently, when the virus was added to PC12 expressing the Trka receptor, NHS did not enhance cell transduction compared to the control, possibly due to viral loss, while PC12 expressed EGFP at all ratios tested using TFP.

[0351] In vivo transduction was tested by subcutaneous injection of the virus into the paw. Wild-type AAV2 was ineffective in transducing DRG neurons (presumably because it does not retrogradely transport from nerve endings in the skin to the DRG), NHS-modified NGF-AAV resulted in some neuronal transduction, while the highest rate of TFP-functionalized NGF-AAV2 resulted in effective neuronal transduction. Figure 6 The data are quantified, showing the number of positive DRG neurons per ganglion in each case. NGF-AAV2 capsids modified with TFP at higher ratios were more efficient than NHS-modified particles, with a 1:10,000 ratio being the most efficient. Furthermore, the data showed a progressively increasing transduction efficiency in the L3–L5 ganglia, consistent with the fact that nerves from these ganglia innervate the hindlimb and claw.

[0352] The selectivity of TFP-functionalized NGF-AAV in targeting nociceptive neurons was further quantified by co-staining positive ganglia with an antibody targeting Trka. Figure 7 As shown, almost all NGF-AAV2-targeted neurons were also positive for TrkA staining between the L3, L4, and L5 ganglia, indicating that the targeting of nociceptive neurons was indeed accurate.

[0353] Analysis of the transduction of wild-type AAV2 and TFP-functionalized NGF-AAV2 in skin sections at the injection site ( Figure 8 Nociceptive nerve endings in the skin were identified by co-staining sections with TrkA antibody (red channel). No nerve transduction was detected in samples injected with wild-type virus. Significant overlap with TrkA-positive fibers was observed in sections from mice injected with NGF-AAV2. Additionally, eGFP-positive myofibrils were observed under both conditions, indicating that this method did not detarget the natural tendency of AAV2.

[0354] The sensory innervation of the spinal cord in mice injected with wild-type or TFP-functionalized NGF-AAV2 was investigated. Fibers innervating the dorsal horn were eGFP-positive only in spinal cord sections collected from animals injected with NGF-AAV2. Figure 9Further co-staining was performed to identify the topological organization of the dorsal horn and to determine which neuronal subsets contained eGFP-positive fibers. Using the marker Trka, the first layer of peptidergic nociceptive neuronal termination was visualized, and using the marker Ib4, the second layer of projections from non-peptidotropic nociceptive neurons was identified. eGFP-positive fibers overlapped with Trka but not with Ib4, demonstrating that NGF-AAV targets peptidergic nociceptive neurons. Notably, Trka and eGFP double-positive fibers were predominantly located in the middle region of the first layer, which receives axonal input from the innervation claw.

[0355] We also investigated scaling up the modification of AAV9 and tested transduction in HEK293 cell lines expressing TrkA / p75 using NGF as a ligand. Figure 10a Images of TrkA / p75HEK293 cells transduced with AAV9 or NGF-AAV9 under different MOIs are shown. Figure 10b Analysis of transduction efficiency was presented. It was found that AAV9 can be effectively modified on a larger scale using NHS. The MOI required for transducing TrkA / p75 HEK293 cells was significantly reduced after NGF conjugation.

[0356] Further investigations were conducted on the amplification of AAV2 modification and the stability of the modified virus to freeze-thaw cycles, as well as its transduction assays in mice. Figure 11 This study compares the transduction efficiency of lumbar DRGs isolated from mice after subcutaneous injection of AAV2 modified with TFP-PEG4-DBCO at different scales, either freshly used or stored at -80°C. DRGs were collected 3 weeks after in vivo AAV injection, mounted, and imaged using confocal microscopy. It was found that TFP can be used to efficiently modify AAV2 at a larger scale. Following NGF conjugation, the MOI required for transducing TrkA / p75 HEK293 cells was significantly reduced.

[0357] In summary, these data demonstrate that TFP chemistry allows for more efficient generation of surface-modified AAV2 than NHS chemistry, avoiding viral loss. The TFP protocol can be easily scaled up and is compatible with in vivo experiments. Importantly, the NGF-modified AAV via TFP chemistry clearly demonstrates the ability to target the virus to populations of interest (in this case, peptidotropic neurons expressing the NGF receptor TrkA).

[0358] Example 5. Comparison of NHS and TFP conjugations to AAV

[0359] Use DBCO-PEG (n)-NHS linker chemical modification of AAV followed by ligand conjugation resulted in significant virus loss due to column-based washing steps during the modification process. To overcome this virus loss problem, DBCO-PEG was used. (n) -TFP adapters are used because this chemical method does not require a washing step during the modification process. Here, NHS- and TFP-mediated AAV modification are compared by monitoring transduction efficiency and virus loss. AAV2, AAV5, or Delta-HSPG AAV2 (with a mutation in its HSPG binding site) are modified with NHS or TFP adapters, conjugated with wheat germ lectin (WGA), and transduction efficiency in PC12 cells is tested. For serotype AAV2, the NHS- and TFP-based chemical methods are tested on viruses purified using two methods that result in different levels of sample purity. Based on this data, the virus:adaptor:ligand conjugation ratio for each chemical modification and serotype is identified.

[0360] method

[0361] NHS- and TFP-based chemical methods were tested on different AAV serotypes. For one of these serotypes, AAV2, NHS and TFP chemical methods were tested on viruses purified using two different protocols that differed in the degree of purity of the final viral product—either using a cesium chloride gradient or via affinity purification followed by isodense centrifugation with an iodixanol gradient (which was expected to increase the purity of the AAV vector). AAV5 and Delta-HSPG AAV2 were purified using only affinity chromatography and an iodixanol gradient.

[0362] All AAV serotypes used were commercially available. One AAV2, purified using a cesium chloride gradient, had a titer of 2.7E+12 vg / ml and contained the commercially available EGFP reporter gene under the CAG promoter, while another AAV2, purified via affinity chromatography and iodixanol gradient, had a titer of 1E+13 vg / ml and contained the EGFP reporter gene. AAV5 contained EGFP with the CMV promoter and had a titer of 1E+13 vg / ml. Delta-HSPG AAV2 contained EGFP with the CAG reporter gene and had a titer of 5.6E+12 vg / ml.

[0363] The table below summarizes the purification and characterization of the AAVs used.

[0364] Table 8.

[0365]

[0366] NHS-mediated chemical modification and coupling of WGA with AAV2, AAV5 and Delta AAV2

[0367] For NHS-mediated chemical modification, AAV2 purified with 1 µl of cesium chloride (corresponding to 0.0045 pMol) was reacted with DBCO-PEG4-NHS at different virus:linker molar ratios (see table below) in a reaction volume of 20 μL PBS (+0.001% pluronic and 200 mM NaCl) and shaken at room temperature for 3 hours.

[0368] Table 9.

[0369]

[0370] WGA (0.1 nMol) was dissolved in PBS and reacted with 20 molar equivalents of azide-PEG4-NHS at room temperature with shaking for 3 hours. Unreacted azide groups were removed using a 10 kDa MWCO centrifugal filter. NHS-mediated DBCO-modified AAV2 was further incubated with 50 pMol of WGA-PEG4-azide at room temperature with shaking for 2 hours, and then kept overnight at 4°C.

[0371] 1 µl (corresponding to 0.0166 pMol) of AAV2 purified by affinity chromatography and iodixanol gradient was subjected to NHS-mediated chemical modification as described above for AAV2, but the virus:linker ratio was different, as shown in the table below.

[0372] Table 10.

[0373]

[0374] 1 µl (corresponding to 0.0166 pMol) of NHS-mediated chemical modification of AAV5 was performed as described above for AAV2, but the virus:linker ratio differed, as shown in the table below.

[0375] Table 11.

[0376]

[0377] The NHS-mediated chemical modification of 1 µl (corresponding to 0.0093 pMol) Delta AAV2 was carried out as described above for AAV2, but the virus:linker ratio was different, as shown in the table below.

[0378] Table 12.

[0379]

[0380] TFP-mediated chemical modification and coupling of WGA with AAV2, AAV5 and Delta AAV2

[0381] For TFP-mediated chemical modification, 1 µl of AAV2 purified with cesium chloride (corresponding to 0.0045 pMol) was reacted with DBCO-PEG4-TFP and WGA-PEG4-azide at different virus:linker:ligand molar ratios (see table below).

[0382] Table 13.

[0383]

[0384] Because TFP is more stable than NHS, a dilution was prepared from a 20 mM stock solution to keep the DBCO-PEG4-NHS linker volume relatively small (1 µl). Then, 11 µl of PBS (+0.001% pluronic and 200 mM NaCl) and 0.1 M sodium bicarbonate buffer (pH 8.3) were added, and the reaction was incubated overnight at room temperature with shaking. The reaction was terminated by adding 50 mM glycine. WGA (0.1 nMol) was dissolved in PBS and reacted with 20 molar equivalents of azide-PEG4-NHS at room temperature with shaking for 3 hours. Unreacted azide groups were removed using a 10 kDa MWCO centrifuge filter. WGA-PEG4-azide was added at the specified molar ratio (see table above), and the reaction was incubated overnight at room temperature with shaking.

[0385] TFP-mediated chemical modification of 1 µl of AAV2, AAV5, and Delta AAV2 purified by affinity chromatography and iodixanol gradient was performed as described above for AAV2, but the virus:linker:ligand ratios differed, as shown in the table below. Furthermore, for these three viral strains (AAV2, AAV5, and Delta AAV2), the final ligand volume would be too large at the highest ratio; therefore, we used maximum virus:ligand ratios of 1:5000, 1:10000, and 1:6000, respectively.

[0386] Table 14 (AAV2).

[0387]

[0388] Table 15 (AAV5).

[0389]

[0390] Table 16 (Delta AAV2).

[0391]

[0392] In vitro application of PC12 cells

[0393] PC12 cells were maintained in DMEM / F12 medium (+10% horse serum, 5% fetal bovine serum, 15 mM HEPES, 2.5 mM Glutamax, and 100 U penicillin / streptomycin) and incubated at 37°C in a humidified atmosphere of 5% CO2. Modified AAV was added to the cells and incubated overnight. The medium was then changed, and the cells were maintained at 37°C for 5 days.

[0394] Data collection

[0395] Transduction efficiency was determined by imaging PC12 cells using a Nikon A1R confocal microscope. To quantify efficiency, cells were collected and prepared for flow cytometry. Flow cytometry data were acquired on a Bio-Rad S3e cell sorter and analyzed using FlowJo. Viral genome copy number was quantified using a Bio-Rad droplet digital PCR system.

[0396] result

[0397] Compared to other virus:adaptor ratios tested, AAV2 modified with DBCO-PEG4-NHS at a virus:adaptor ratio of 1:100,000 and 2.5 uM WGA-PEG4-azide showed the highest transduction in PC12 cells. Figure 12 In contrast, AAV2 modified with DBCO-PEG4-TFP and WGA-PEG4-azide at a virus:linker:ligand ratio of 1:10,000:10,000 showed the highest transduction. Figure 13 This indicates that TFP modification allows us to use fewer linkers while still achieving the same transduction efficiency as NHS chemistry.

[0398] For higher purity viruses, the amount of TFP adapter molecules required for virus modification and strong cell transduction is further reduced, as observed in AAV2 purified by affinity chromatography and iodixanol gradient. In fact, when AAV2 is purified in this manner, rather than solely by cesium chloride gradient, the optimal virus:TFP adapter:ligand ratio decreases from 1:10,000:10,000 to 1:500:500. Figure 13 and 15 In the same scenario, NHS chemistry requires even higher quantities of adapters (1:100,000) to achieve the cell transduction indicated by TFP. Figure 14 and 16 For other AAV serotypes, AAV5 and Delta AAV2, TFP-mediated modification was observed. Figure 17 and 19This can reduce the virus:connector ratio to 1:1,000 and 1:3,000, respectively, while using NHS-mediated modification ( Figure 16 and 18 Virus:connector ratios of 1:100,000 and 1:60,000, respectively, are required to achieve optimal transduction efficiency. This demonstrates that, in addition to no virus loss during the modification process, using the DBCO-PEG4-TFP connector offers another advantage in improving AAV modification.

[0399] Example 6. Incorporating the carrier functionalization step into the AAV DSP

[0400] Chemical modification of AAV was tested during AAV purification (i.e., modification of the crude viral extract) rather than after purification (i.e., the original scheme), as shown in Scheme 3 below.

[0401]

[0402] Option 3 illustrates two approaches to incorporating the carrier functionalization step into the AAV DSP.

[0403] method

[0404] As previously described, recombinant AAV9 or AAV2 was generated in HEK293 cells, containing inserted eGFP or tdTomato, respectively, under the CAG promoter, in the VR4 region as cargo. Cells were harvested 3 days post-transfection, subjected to three freeze-thaw cycles, and then buffer was replaced with PBS (+0.001% pluronic acid and 200 mM NaCl) using a 100 kDa centrifuge filter. AAV9 was then modified with a series of concentrations (3 mM, 1 mM, 0.3 mM, 0.1 mM, 30 μM, 10 μM, 1 μM, 0, 3 μM, or 0 μM) of NHS-PEG4-DBCO for 3 hours at room temperature. The sample was again buffer-replaced using a 100 kDa centrifuge filter and then incubated overnight with 2.5 μM WGA-azide. The sample was applied to PC12 cells, and transduction efficiency was assessed after 5 days.

[0405] To compare with TFP ester, AAV2-VR4 was modified overnight at room temperature in PBS (+0.001% pluronic and 200mM NaCl) with a series of concentrations (3mM, 1mM, 0.3mM, 0.1mM, 30 μM, 10 μM, or 0 μM) of TFP-PEG4-DBCO in room temperature. The reaction was then quenched with 50mM glycine, buffer was replaced using a 100kDa centrifuge filter, and the mixture was incubated overnight with 2.5 μM WGA-azide. The samples were then applied to PC12 cells.

[0406] result

[0407] Surprisingly, the modification directly affects the crude cell lysate (i.e., unpurified AAV). Figures 20A and 20B show images of PC12 cells transduced with AAV9-WGA chemically modified with NHS-PEG4-DBCO and TFP-PEG4-DBCO at the cell lysate stage, respectively. Figures 20A and 20B demonstrate that AAV9 or AAV2-VR4 cell lysates can be effectively modified using both NHS and TFP esters without purification. A lower concentration of TFP ester is required compared to NHS.

[0408] 5. Equivalents and incorporation by reference

[0409] Although this disclosure has been clearly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of this disclosure.

[0410] All references, published patents and patent applications cited in the text of this specification, including U.S. Provisional Application 63 / 514,909 filed July 21, 2023, are incorporated herein by reference in their entirety for all purposes.

[0411] WO 2020 / 225363 and WO 2022 / 101363 are incorporated herein by reference in their entirety for all purposes.

Claims

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: The ligand covalently binds to the viral capsid via the linker.

2. The composition according to claim 1, wherein the connector is of formula VI: (VI) in: SP 1 and SP 2 Independently acting as a bond or spacer; and Q represents the cross-linked portion.

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

4. The composition according to any one of claims 1-3, wherein the surface-modified coating is of formula V: (V) in: For optionally containing nucleic acid cargo; SP 1 and SP 2 Independently acting as a bond or spacer group; Q represents the cross-linked portion; and L 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 of 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-capsule 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 ligands.

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

9. The composition of 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 of claim 15, wherein the genetic modification is the disruption of the heparin sulfate proteoglycan binding site.

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

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

19. The composition according to any one of claims 1-17, wherein the ligand is selected from cell type-specific ligands, polypeptides, proteins, monosaccharides, polysaccharides, steroid hormones, RGD motif peptides, vitamins, small molecules, antibodies, nanobodies, enzymes, and immunoglobulins.

20. The composition of 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, or a gene-editing nuclease.

21. The composition according to claim 20, wherein the lectin is selected from wheat germ lectin (WGA), iso-lectin B4 (IB4), Sophora japonica lectin, hyacinth bean lectin, wisteria vine lectin, and Pha-L.

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

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

24. The composition of claim 23, wherein the protein ligand is nerve growth factor (NGF).

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

26. The composition of claim 25, wherein the crosslinking portion comprises both an eight-membered ring and a triazole ring.

27. The composition according to any one of claims 1-26, wherein the crosslinking portion is a product selected from the following reactions: Cu(I)-catalyzed azido-alkynyl cycloaddition (CuAAC), strain-promoted alkynyl-azido cycloaddition (SPAAC), strain-promoted alkynyl-nitroketone cycloaddition (SPANC), reverse electron-demanding Diels-Alder (IEEDD) reaction, and Staudinger linkage and [4+1] cycloaddition reactions.

28. The composition of claim 27, wherein the reaction is a strain-promoted alkyne-azidocycloaddition (SPAAC) reaction.

29. The composition according to any one of claims 1-28, wherein the crosslinking portion comprises: 。 30. The composition of claim 29, wherein the crosslinking portion comprises: ； Where m ranges from 1 to 10.

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

32. The composition according to any one of claims 1-31, wherein the viral capsid comprises nucleic acid cargo.

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

34. A method for preparing a composition according to any one of the preceding claims, the method comprising the following steps: (a) Combine the following: (i) The viral capsid, comprising: multiple surface-accessible primary amines; and (ii) a capillary reactive connector comprising: Tetrafluorophenyl (TFP) ester and the first member of the crosslinking agent-reactive pair (CRP1); Thus, a composition comprising a surface-functionalized viral capsid is provided; and (b) Combine the following: (i) Functionalized ligands containing a second member (CRP2) of a crosslinker-reactive pair; (ii) the composition comprising a surface-functionalized viral capsid; This provides a composition comprising the surface-modified viral capsid.

35. The method of claim 34, wherein the composition comprising a surface-functionalized viral capsid produced in step (a) is used in step (b) without purification or separation steps.

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

37. The method according to any one of claims 34-36, wherein the capping-reactive connector is according to formula (II): 。 38. The method according to any one of claims 34-37, wherein step (a) occurs according to scheme A: ， in: p is an integer from 10 to 500, and q is an integer between 10 and 500.

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

40. The method of claim 39, wherein the molar ratio of viral capsid (I) to capsid-reactive adapter (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 adapter (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 adapter (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 the combination step (a).

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

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

46. ​​The method of claim 44, wherein the chromatography is affinity chromatography.

47. The composition 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 SP 1 For one or more PEGs (i.e., -(-(O-CH2-CH2)). n - or -([PEG]) n )-), where n is between 2 and 20.

49. The method according to any one of claims 34-48, wherein CRP1 comprises a reactive moiety selected from: azides; alkynes; 1,4-triazoles; 1,3-nitroketones; cyclooctyne or derivatives thereof, for example, dibenzylcyclooctyne or derivatives thereof; triazines; tetrazines; strained dienophiles; aryl or alkylphosphines; isocyanates; benzylguanine, benzylcytosine, and chloroalkanes.

50. The method of claim 49, wherein the CRP1 reactive portion comprises cyclooctylene.

51. The method of claim 50, wherein the cyclooctyne is selected from dibenzylcyclooctyne analogs.

52. The method according to claim 51, wherein the dibenzylcyclooctyne analogue is selected from the group consisting of dibenzylcyclooctyne (DIBO), dibenzozacyclooctyne (DBCO), and biarylazacyclooctyneone (BARAC) and their functional derivatives.

53. The method according to any one of claims 34-52, wherein CRP1 comprises the following structure: Where m is an integer from 4 to 12, and the wavy line indicates attachment to the cap-reactive connector.

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

55. The method of claim 54, wherein the capping-reactive connector has the following formula: 。 56. The method according to any one of claims 34-55, wherein the capping-reactive connector is DBCO-PEG4-TFP.

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

58. The method of claim 57, wherein the at least one buffer is selected from HEPES, MOPS, MES, phosphates and bicarbonates.

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

60. The method of claim 59, wherein the concentration of the at least one buffer 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 one salt of at least about 200 mM.

62. The method according to claim 61, wherein the at least one salt is selected from chloride salts, phosphates, sulfates, citrates, sodium salts, potassium salts, calcium salts, and magnesium salts.

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.

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

67. The method of 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 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 about 0°C to about 50°C.

70. The method according to any one of claims 34-69, wherein step (a) occurs over a reaction duration of 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 an excess of quenching agent.

72. The method of claim 71, wherein the quencher comprises an amine-containing compound.

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

74. The method according to any one of claims 34-73, wherein step (b) occurs according to scheme B: in: For functionalized ligands, SP 2 The group is a bond or spacer and L is a ligand; and r is an integer from 10 to 500.

75. The method of claim 74, wherein SP 1 and SP 2 Independently selected from one or more divalent groups selected from the following: bond, CH2-, -O-, -C(=O)- and -N(R)-, wherein R is H or C1-3 alkyl.

76. The method of claim 75, wherein SP 2 -([PEG]) n )-C(O)-, where n is 1 to 100, preferably 1 to 10.

77. The method according to any one of claims 34-76, wherein the functionalized ligand has the following formula: ， Where Y represents 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: azide; alkyne; 1,4-triazole; 1,3-nitroketone; cyclooctyne or a derivative thereof; dibenzylcyclooctyne or a derivative thereof; triazine; tetraazine; strained dienophile; aryl or alkylphosphine; isocyanate; benzylguanine group, benzylcytosine group or chloroalkane group.

79. The method of 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 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.

82. 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 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 the viral capsid (I) in step (a) to the functionalized ligand in step (b) is 1:100 to 1:50,000.

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

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

94. 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 of claim 96, wherein (i) CRP1 is DBCO and CRP2 is an azide or (ii) CRP1 is an azide and CRP2 is DBCO.

98. The method according to claim 96 or 97, wherein Q comprises 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: ， Where m ranges from 1 to 10.

101. The method according to any one of claims 34-100, further comprising preparing the functionalized ligand by reacting a ligand comprising a first reactive portion with a ligand-reactive linker comprising a second reactive portion and CRP2.

102. The method of 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 portion selected from: isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imine esters, carbodiimide anhydrides, benzoyl fluorides, and TFP esters.

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

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

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

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

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

109. The method of 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 a surface-modified viral capsid.

111. The method according to any one of claims 34-110, wherein the viral capsid comprises nucleic acid cargo.

112. The method of claim 111, wherein the nucleic acid encodes a protein or polypeptide.

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