CONTROLLED MODIFICATION OF ADENO-ASSOCIATED VIRUS (AAV) FOR ENHANCED GENE THERAPY

Abstract

The present invention discloses platforms for chemically modify AAV capsids with control over site and stoichiometry. An AAV packaging system is described that allows the introduction of site-directed natural and unnatural amino acid mutations into any subset of the three capsid proteins. These engineered residues can be subsequently used to chemically functionalize the resulting capsids with precise control over site and stoichiometry. Such controlled modification strategy can be used to attach a wide variety of entities to AAV capsids to engineer its tropism, immunogenicity, etc.

Claims

1. A genetically-modified adeno-associated virus (AAV) wherein the AAV capsid comprises at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2, wherein the variant minor capsid protein is mutated at one, or more, amino acid residue sites to incorporate a natural amino acid or an unnatural amino acid (UAA) relative to the wild-type AAV VP1 or VP2 capsid protein.

2. The genetically-modified adeno-associated virus (AAV) of claim 1, comprising a variant minor capsid protein, wherein the variant capsid coding sequence is mutated at the translation origin of the AAV VP1, or VP2, or VP3 capsid protein open reading frames (ORF) to prevent translation of VP1, VP2 or both VP1 and VP2.

3. The genetically-modified AAV of claim 2, wherein the AAV capsid protein comprises SEQ ID NO:1, or a sequence comprising at least about 80% sequence identity of SEQ ID NO:1.

4. The genetically-modified AAV of claim 3, wherein a stop codon incorporated in the capsid protein(s) and the stop codon is a TAG, TAA or TGA codon.

5. The genetically-modified AAV of claim 1, comprising a variant minor capsid protein, wherein the natural amino acid residue is either a cysteine or a selenocysteine.

6. The genetically-modified AAV of claim 1, comprising a variant minor capsid protein, wherein the unnaturally-occurring amino acid is selected from the group consisting of: phenylalanine analogs; tyrosyl analogs; tryptophanyl analogs; or lysyl analogs.

7. The genetically-modified AAV of claim 6, wherein the analog comprises a formula selected from the group: ##STR00001## .

8. The genetically-modified AAV of claim 7, comprising a variant minor capsid protein, wherein the analogs are selected from the group consisting of: p-benzoylphenylalanine (pBpA); O-methyltyrosine (OMeY); 5-azidotryptophan; 5-propargyloxytryptopha; 5-aminotryptophan; 5-methoxytryptophan; 5-O-allyltryptophan; 5-bromotryptophan; azido-lysine (AzK); C5Az; LCA; Nε-acetyllysine (AcK); cyclopropene amino acid, N.sup.ε-(1-methylcycloprop-2-enecarboxamido)-lysine (CpK); 5-hydroxy-tryptophan (5-HTP);LCAlk; DiaazK and LCKet.

9. The genetically-modified AAV of claim 1, wherein the natural or unnatural amino acid residue of the variant minor capsid protein incorporates a bioconjugation handle.

10. The genetically-modified AAV of claim 9, wherein the AAV capsid comprises 5 to 10 bioconjugation handles per capsid.

11. The genetically-modified AAV of claim 1 , comprising at least one variant minor capsid protein VP1, VP2 or both VP1 and VP2, wherein the AAV is characterized by: a) infectivity of target cells comparable to wild-type AAV; b) is packaged with titers comparable to wild-type AAV; or c) both characteristics a) and b).

12. A genetically-modified infectious adeno-associated virus (AAV) wherein the genetically-modified AAV comprises a variant minor capsid protein VP1, VP2 or both VP1 and VP2 wherein VP1, VP2 or both VP1 and VP2 comprise one, or more mutated amino acid residues.

13. The genetically-modified infectious AAV of claim 12, wherein the variant capsid protein comprises one, or more mutated amino acid residues of VP1, VP2 or both VP1 and VP2 and the mutated amino acid residue site incorporates an unnatural amino acid (UAA).

14. The genetically-modified infectious AAV of claim 13, comprising a variant capsid protein, wherein the unnaturally-occurring amino acid is selected from the group consisting of: phenylalanine analogs; tyrosyl analogs; tryptophanyl analogs; or lysyl analogs.

15. The genetically-modified infectious AAV of claim 14, comprising a variant capsid protein, wherein the analogs are selected from the group consisting of: ##STR00002## .

16. The genetically-modified infectious AAV of claim 12, wherein the variant capsid protein is VP1 comprising SEQ ID NO:1, or a sequence comprising at least about 80% sequence identity of SEQ ID NO:1.

17. The genetically-modified infectious AAV of claim 16, wherein the variant VP1 capsid protein is mutated at one, or more locations of the protein at position(s) 263, 454, 456, 587 and 588.

18. The genetically-modified infectious AAV of claim 12, wherein the variant capsid protein comprises one, or more mutated amino acid residues of VP1, VP2 or both VP1 and VP2 and the mutated amino acid residue site incorporates a naturally-occurring amino acid.

19. The variant capsid protein of claim 18, wherein the naturally-occurring amino acid is cysteine or selenocysteine.

20. The genetically-modified AAV of claim 18, wherein the mutated VP1 amino acid sequence comprises SEQ ID NO:1, or a sequence with at least about 80% sequence identity with Seq ID NO:1, wherein the variant VP1 capsid protein is mutated at one, or more locations at position(s) 263, 454, 456, 587 or 588.

21. The genetically-modified infectious AAV of claim 12, comprising the variant capsid protein comprising a mutated amino acid residue, wherein the mutated amino acid is conjugated with a chemical or biological (protein, nucleic acid, lipid, or carbohydrate) entity.

22. The genetically modified infectious AAV comprising a chemical or protein entity of claim 21, wherein the entity is selected from the group consisting of probes, small molecule ligands, peptides, cyclic peptides, nucleotides, polymers, or protein conjugates.

23. The genetically-modified infectious AAV comprising a chemical or protein entity of claim 22, wherein the entity is a cyclic peptide cRGD or polyethylene glycol.

24. A method of producing an infectious genetically-modified AAV, wherein the AAV comprises a variant AAV capsid protein, wherein VP1, VP2 or both VP1 and VP2 comprise one, or more mutated amino acid residue sites, relative to wild-type VP1, VP2 or both VP1 and VP2, the method comprising: a) providing competent host cells in culture; b) transfecting the cultured cells with one, or more plasmids comprising: 1) AAV variant VP3 that does not express VP1, or VP2, or both VP1 and VP2; 2) variant VP1, VP2 or both VP1 and VP2 with a suitable promoter that do not express VP3; 3) additional factors required for AAV expression; c) providing the required unnatural amino acid; and plasmid encoding an engineered aminoacyl-tRNA synthetase/tRNA pair that selectively charge the unnatural amino acid in response to a stop codon; d) culturing the cells under conditions sufficient for expression of the plasmid genes and assembly of the AAV; thereby producing an infectious genetically-modified AAV comprising a variant AAV capsid protein, wherein VP1, VP2, VP3 or both VP1 and VP2 are mutated at one, or more amino acid residue sites relative to the wild-type AVV.

25-34. (canceled)

35. A therapeutic or antigenic composition comprising a and further genetically-modified AAV of claim 1 comprising one, or more therapeutic or antigenic gene constructs.

36. (canceled)

37. A method of treating a disease or condition in a subject, or of eliciting an immune response in a subject, the method comprising administering the therapeutic composition of claim 35 to the subject, wherein the composition comprises a gene construct encoding a protein or peptide in a therapeutic amount capable of decreasing or alleviating the disease or condition, or eliciting and immune response in the subject.

38. (canceled)

39. A kit comprising the genetically-modified AAV of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee. Of the drawings:

[0044] FIG. 1 shows the examples of the structures of the natural and the unnatural amino acids that can be incorporated into the capsid of AAV in a controlled stoichiometry. Specifically shown are UAA analogs comprising the formulas 1-12 (upper line 1 formulas 1-6, left to right and second line formulas 7-12 left to right, including length of carbon chain extensions and substitutions.

[0045] FIG. 2 shows the scheme for introducing mutations (natural or unnatural amino acids) selectively into VP1, or VP2, or VP1 and VP2. Three capsid proteins are expressed from the same open reading frame Cap via the use of alternative splicing and start codon usage. Mutations have been engineered at the translation origin (demonstrated by red crosses) that prevent the expression of VP1, VP2, or VP3 from this ORF. The missing minor capsid protein(s) can then be supplied in trans, driven by a strong promoter such as CMV. Separating the expression of VP1, VP2, or VP1+VP2 from the rest of the capsid proteins makes it possible to selectively mutate these without affecting the other capsid proteins.

[0046] FIG. 3 shows packaging of AAV2 in HEK293T cells using constructs described in FIG. 2. AAV with variant capsid proteins have comparable yields relative to the original system.

[0047] FIG. 4 shows the infectivity of the packaged, tittered viruses produced in FIG. 3 at constant titer measured by their ability to deliver and express an EGFP reporter gene in HEK293T cells.

[0048] FIG. 5 shows selective unnatural amino acid mutagenesis of individual minor capsid proteins in AAV capsid and their use to chemically attach a fluorophore.

[0049] FIG. 6 shows the number of retargeting ligands attached to the AAV capsid dramatically affects its retargeting efficiency.

[0050] FIGS. 7A-7C show precise labeling of AAV at engineered cysteine residues.

[0051] FIGS. 8A-C show the amino acid sequences for AAV isoform VP1( SEQ ID NO:1). FIGS. 8B and 8C disclose SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0052] FIG. 9 shows the amino acid sequence of AAV capsid protein isoform VP2. (SEQ ID NO:2).

[0053] FIG. 10 shows the amino acid sequence of the AAV capsid protein isoform VP3. (SEQ ID NO:3).

[0054] FIGS. 11A-C shows the selective incorporation of the UAA C5Az either VP1 or VP2 or VP1+VP2Fig.

[0055] FIGS. 12A-C shows the results of LCA incorporated into VP1.

[0056] FIGS. 13A-C shows the results of incorporation of CpK into VP1.

[0057] FIGS. 14A-C shows the results of incorporation of 5HTP into VP1.

[0058] FIGS. 15A-B demonstrates incorporation of several other unnatural amino acids, LCAlk, DiazK and LCKet, into VP1.

[0059] FIGS. 16A-B shows the results of selective PEGylation of AAV at VPI site 454.

[0060] FIG. 17 shows the nucleic acid sequence encoding RC2-VP1-del. (SEQ ID NO:4) The location of the mutation is shown in red.

[0061] FIG. 18 shows the nucleic acid sequence encoding RC2-VP2-del. (SEQ ID NO:5) The location of the mutation is shown in red.

[0062] FIG. 19 shows the nucleic acid sequence encoding RC2-VP12-del. (SEQ ID NOL6) The location of the mutation is shown in red.

[0063] FIG. 20 shows the nucleic acid sequence encoding CMV-VP1-delVP23. (SEQ ID NO:7) The locations of the mutations are shown in red.

[0064] FIG. 21 shows the nucleic acid sequence encoding CMV-VP2-delVP3. (SEQ ID NO:8) The locations of the mutations are shown in red.

[0065] FIG. 22 shows the nucleic acid sequence of CMV-VP1-VP2-delVP3. (SEQ ID NO:9) The locations of the mutations are shown in red.

[0066] FIG. 23A is the plasmid map of pIDTsmart-ITR-GFP-4xEcLtR-LeuRS. FIG. 23B shows the nucleic acid sequence of the plasmid. (SEQ ID NO:10).

[0067] FIG. 24A shows the plasmid map of pIDTsmart-TrpRS-8xWtR-ITR-GFP. FIG. 24B shows the nucleic acid sequence of the plasmid. (SEQ ID NO:11).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0069] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[0070] It will be understood that although terms such as “first” and “second” are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present invention.

[0071] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0072] It should be noted that this invention description is not limited to these specific methodologies, compositions, cell lines/biological systems, or any other standard protocol. The present invention discloses a general platform for producing AAV vectors that can be chemically functionalized using chemo-selective reactions with control over site and stoichiometry of modification, the cell type used for virus packaging, identity of the engineered aminoacyl-tRNA synthetase (aaRS) or tRNA, or UAAs or natural amino acids, the chemical reaction used for introducing the modification, etc., can vary as the technology in this field and in this work advance.

[0073] Previously, incorporation of UAAs into the AAV capsid has been directed to all 60 capsid proteins. Since the three overlapping capsid proteins are expressed from the same open reading frame (ORF) Cap via alternative splicing and start codon usage, it has not been possible to selectively modify a subset of these proteins. However, subsequent chemical modification of all of the 60 capsid proteins perturbs the infectivity of the virus (e.g., FIG. 6, blue trace). While the mechanism of this perturbation is poorly understood, it is reasonable to believe that the molecular processes associated with the complex entry pathway of the virus can be affected by over-modification of the capsid. Additionally, it is desirable to be able to create AAV vectors into which a defined number of chemical modifications can be introduced in a site-specific manner. Indeed, retargeting experiments demonstrate that there is an optimal number of ligands per capsid needed for efficient retargeting (FIG. 6). It is possible to control the degree of modification of an AAV capsid with 60 UAA-handles by controlling the concentration of the modifying reagent or reaction time (FIG. 6). However, this leads to a heterogeneous mixture of capsids in different states of modification. The ability to create homogeneous AAV conjugates where a defined number of modifications are introduced per capsid is critical for gene therapy.

[0074] In the AAV capsid, the three capsid proteins VP1, VP2, and VP3 are incorporated at a roughly 1:1:10 stoichiometry. Consequently, approximately 5 copies of each of the two minor capsid proteins, VP1 and VP2, are present in the capsid, with 10 copies of VP3, for a total of 60 copies of capsid proteins per virus particle. The ability to selectively introduce engineered, modifiable natural or unnatural amino acid residues into these minor capsid proteins provides an avenue to introduce a controlled number of handles per capsid. However, since the AAV capsid genes are all encoded in the same ORF and expressed by alternative splicing and start-codon usage, it is challenging to mutate one capsid protein without affecting the others. The present invention describes a method to separately express the minor capsid proteins (VP1, or VP2, or VP1+VP2) by introducing mutations at the translation origins of VP1 and/or VP2, such that these are not expressed from the native Cap ORF. Next, the missing capsid protein can be expressed in trans from a strong promoter, for example, the CMV promoter as described herein. Expression of the undesired capsid proteins (e.g., VP3) from this second ORF is also similarly eliminated by mutating translation origins (FIG. 2). This platform provides the ability to express any combinations of the three capsid proteins from one ORF and separating the expression of a chosen third from a second ORF, making it possible to selectively engineer any subset of the three capsid proteins.

[0075] Using the platform described herein, it is now possible to selectively introduce unique non-coding codons (such as nonsense, 4-base, or unnatural base-pair-containing codons) into any combination of the three capsid proteins by site-directed mutagenesis. When such a mutant Cap is co-expressed with a suitable engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pair that can decode the unique codon, an UAA residue can be incorporated into these sites.

[0076] In one embodiment of this invention, the UAA AzK was incorporated at residue T454 (VP1 numbering) of VP1 or VP2 or VP1+VP2 (FIG. 3). Using a pyrrolysyl-tRNA synthetase/tRNA pair. The resulting viruses were packaged at an efficiency comparable to the wild-type virus and had comparable infectivity, demonstrating these modifications are well-tolerated (FIG. 3 and FIG. 4). The importance of controlling the number of modifications per capsid was demonstrated by the differential behavior of AAV where the UAA was incorporated at 5 copies per capsid (mutation of either VP1 or VP2 alone), or 10 copies per capsid (mutation of both VP1+VP2), or 60 copies per capsid (all three capsid proteins were mutated), and a retargeting ligand was subsequently attached to these UAA handles (FIG. 6). AAV with 60 attachment handles enabled efficient retargeting at intermediate modification states, but upon full modification, all infectivity was lost. 5 or 10 modifications per capsid were well-tolerated, but 10 retargeting ligands per capsid was needed for efficient retargeting (FIG. 6).

[0077] It should be noted that any other combination of capsid proteins, and any site within these proteins, can be engineered using this strategy. Especially well-suited for mutation is the N-terminus of the capsid proteins as this section of the capsid protein is essentially internal when folded into the functional capsid, whereas the C-terminus of the capsid protein is exposed.

[0078] Additionally, other aaRS/tRNA pairs can be used to (including, but not limited to, bacterial tyrosyl, tryptophanyl, and leucyl-tRNA synthetase/tRNA pairs) and incorporate any other unnatural amino acids (illustrative examples shown in FIG. 1 and described herein). This technology can also be extended to any other natural serotype of AAV as well as engineered and evolved variants of AAV.

[0079] Cysteine and selenocysteine are natural amino acid residues found in proteins. Because of their low abundance and unique reactivity, these can be used for site-selective bioconjugation reactions. In another embodiment of this invention, engineered surface-exposed cysteine residues can be introduced into the minor capsid proteins (FIGS. 7A and 7B). Even though the same mutation is not well-tolerated when introduced to all 60 capsid proteins, leading to low titer and poor infectivity, robust virus packaging and infectivity was observed when surface exposed cysteine residue was introduced only to VP1 or VP2 at the T454 site. The ability to selectively modify the engineered cysteine residue on the minor capsid protein was also demonstrated (FIG. 7C).

[0080] Because of the complexities associated with the assembly and the cell-entry process, for which the AAV capsid has been optimized through evolution, it frequently resists attempts at engineering the capsid protein through natural/unnatural amino acid mutagenesis, loop insertion, protein fusion etc. However, engineering just the minor capsid proteins is tolerated significantly better as it introduces much less overall perturbation to the capsid overall. This is illustrated by the tolerance of the engineered cysteine residue at the minor capsid protein, but not everywhere (FIG. 7). Thus, this invention provides an opportunity to introduce more aggressive engineering to alter the properties of the AAV capsid through engineering the minor capsid protein. In addition to natural and unnatural amino acid mutagenesis, this invention can also be used to introduce peptide and protein fusions and insertions into the minor capsid proteins that either directly provide a beneficial trait (e.g., binding a certain target), or can be selectively modified through chemical or enzymatic reactions (e.g., biotinylation tag, SNAP or HALO tag, etc.).

[0081] This invention allows the introduction of a defined number of engineered residues (natural or unnatural) per capsid by selectively mutagenizing VP1, or VP2, or VP1 plus VP2. Further control over the number of engineered sites can be achieved by introducing more than one engineered residue into VP1, or VP2, or VP1+VP2. As described herein, various sites of the VP1 capsid protein have been selectively mutated such as 263, 454, 456, 587 and 588 (the numbering corresponds to the amino acid residues of wild-type VPI). The platform can be extended to any packaging platform including, but not limited to, mammalian cells, insect cells, and cell-free translation/packaging systems.

[0082] The invention enables the incorporation of numerous natural and unnatural amino acid residues into AAV with control over site and copy number, with a wide variety of different chemistries which can be used to chemo-selectively attach various entities. In one embodiment of this invention, an azido-containing UAA was introduced selectively into the minor capsid proteins of AAV, followed by their conjugation to a fluorophore or a retargeting ligand using strain-promoted azide-alkyne click reaction (FIG. 5 and FIG. 6). In another embodiment, an engineered cysteine residue in a minor capsid protein was introduced and subsequently conjugated to a fluorophore using cysteine-maleimide coupling reaction. Any other chemo-selective conjugation reaction can be applied for the capsid modification including, but not limited to, inverse-electron demand Diels-Alder reaction between a strained alkene and a tetrazine, or a furan and a maleimide, condensation reaction between an aldehyde/ketone and an amino-oxy/hydrazine groups, chemo-selective rapid azo-coupling reaction (CRACR), oxidative and photocatalyzed coupling reactions, nucleophilic substitution/addition by cysteine or selenocysteine residue to various electrophiles, etc.

[0083] The methods described herein can be used to attach a wide variety of entities including, but not limited to, probes (fluorescent, radioactive, MRI, luminescent, etc.), small molecule ligands, peptides, cyclic peptides, nucleotides (DNA, RNA, LNA, PNA, etc.), polymers (such as PEG), carbohydrates (e.g., sialic acids, etc.), proteins (e.g., enzymes, nanobodies, antibodies, etc.), another AAV of the same or different serotype, etc. Such attachment can provide AAV conjugates that efficiently retargets to a user-defined receptor, thus altering its tissue tropism. Immune-evading AAV can also be created by site specifically attaching groups (such as PEG, peptides, carbohydrates, or other polymers) that passively protect the capsid from the immune system, or ligands that actively bind inhibitory receptors on immune cells to turn off immune response (such as SIGLEC ligands). It can also be used to attach enzymes on AAV capsids to produce capsids with superior infectivity profile, or conjugate two AAVs with same or different serotypes to create novel class of vectors with expanded cargo capacity as well as novel tropism.

[0084] The controlled AAV modification technology descried herein can be used for many applications such as targeting AAV vectors to desired types of cells by attaching retargeting ligands that include, but are not limited to, small molecules, peptides, cyclic peptides, nanobodies, antibodies and antibody fragments; DNA/RNA/PNA aptamers, etc.; optimizing the properties of such conjugates by systematically controlling the attachment site, the number of retargeting groups per capsid, and the chemical properties of the linker; Attenuating the immune response generated by AAV vectors by the controlled attachment of immuno-modulatory entities including, but not limited to, polyethylene glycol and other polymers, carbohydrates (such as sialic acid, etc.), ligands that bind inhibitory receptors on immune cells (such as SIGLEC receptor), etc.

[0085] In particular the genetically-modified AAV of the present invention can be used as an enhance vector for gene therapy, Using techniques described herein, the AAV can be genetically-modified to specifically target/direct delivery of a therapeutic or antigenic gene construct to the target cell with enhanced/increased efficacy over non-modified AAV vectors. The cell can be cultured in vitro or in vivo or ex vivo delivery to a subject in need thereof. The term “subject” as used herein can include any animal subject, and in particular includes a mammalian subject such as a human. The human subject can be treated for medical purposes using the AAV gene vector described herein, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. The methods and compositions of the present invention may be used to treat any type of cancerous tumor or cancer cells. Additionally, the genetically-modified AAV of the present invention can be used in a vaccine composition wherein a nucleic acid sequence encoding an antigenic agent such as a protein or peptide is delivered to the targeted cell along with additional components such as adjuvants wherein an immune response is elicited in the subject.

[0086] The gene construct delivered by the genetically- modified AAV is therapeutically effective. A “therapeutically effective” amount as used herein refers to an amount sufficient to have the desired biological effect to produce the desired effect on the underlying disease state (for example, an amount sufficient to inhibit tumor growth in a subject, produce an immune response to an antigen or to inhibit autoimmune disease) in at least a sub-population of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Determination of therapeutically effective amounts of the constructs/agents used in this invention, can be readily made by one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.

[0087] The technology can also be used for conjugating two distinct AAV vectors, of the same or different serotypes, using a bifunctional linker to increase the overall size of the genetic cargo delivered per cell. Such novel conjugates between two different AAV vectors also will have unique tropism, and for conjugating external payloads (such as proteins, small molecules, nucleic acids, probes, etc.) onto the virus capsid to be delivered into cells in vitro and in vivo for research or therapeutic purposes that work independently, or in conjunction with the genetic cargo inside the AAV capsid.

[0088] Finally, this technology can be used for the investigation of the entry pathway of AAV capsids into mammalian cells by incorporating groups such as fluorescent probes, photo-crosslinkers, and affinity handles (such as biotin).

[0089] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments and examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0090] Incorporation of UAAs into the capsid of AAV requires efficient expression of its capsid protein(s), including the desired UAA modification in a competent cell (e.g., mammalian cell) serving as the host for virus amplification. As described earlier, the site of UAA incorporation can be specified by a stop codon (such as TAG), and the UAA of interest can be delivered by an engineered tRNA/aminoacyl-tRNA synthetase pair, with a cognate anticodon. Consequently, the production of UAA-modified viruses must involve the simultaneous expression of genetic components necessary for virus amplification, as well as those necessary for the amplification of the virus.

[0091] To incorporate an UAA into the capsid proteins of AAV, the replacement of several surface exposed endogenous amino acids residues was targeted on the AAV capsid (guided by a crystal structure of the virus particle, see, e.g., Xie, et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy” PNAS Aug. 6, 2002: vol. 99: no. 16, p. 10407). Since such capsid mutations can potentially perturb viral infectivity, regions that are known to tolerate alterations were targeted, such as those on the threefold proximal spike. The replacement of essential arginine residues was also targeted in the heparan sulfate receptor binding region, which disrupts native tropism and will facilitate re-targeting. This allows “erasing” the native host cell preference of AAV and rewriting it with targeting agents with precise labeling methodology. For example, positions targeted can reside in the domain conserved among all of the three AAV capsid proteins, leading to their replacement with UAAin all of them. AAV can be produced by transfecting e.g., HEK293T cells with plasmids containing the required elements in the presence of the UAA azido-lysine (“AzK”). In a specific example a Methanosarcina barkeri-derive pyrrolysyl-tRNA synthetase and an M. mazeii derived pyrrolysyl tRNA (TAG suppressor) was used to incorporate the AzK amino acid in response to the stop codon, TAG at T454.

[0092] The plasmid maps and sequences used in the examples described below are found in FIGS. 17-24.

Example 1: Packaging of AAV2

[0093] FIG. 3 shows packaging of AAV2 in HEK293T cells using constructs described in FIG. 2 to comparable yields relative to the original system. The qPCR titer of the original (WT) AAV2 is shown and the titer for the rest are shown relative to the WT. ΔVP1 and ΔVP2 represents AAV2 packaged using Cap genes from which the expression of VP1 and VP2, respectively, were eliminated. The virus still assembles efficiently in the absence of the minor capsid proteins. ΔVP1-CMV-VP1 and ΔVP2-CMV-VP2 represents AAV2 packaged using Cap genes from which the expression of VP1 and VP2, respectively, were eliminated and the respective proteins were expressed in trans from a CMV promoter. In these systems, the T454 residue in VP1 or VP2 were mutated to TAG and suppressed using a pyrrolysyl-tRNA synthetase/tRNA pair to incorporate an UAA(AzK), which are represented by ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK, respectively. Packaging yield of the virus in the presence or the absence of the UAA added to the media is shown.

Example 2: Infectivity of the Packaged Virus

[0094] FIG. 4 shows the infectivity of the packaged, tittered viruses produced in FIG. 3 at constant titer measured by their ability to deliver and express an EGFP reporter gene in HEK293T cells. While ΔVP1 and ΔVP2 viruses package well, these show significantly attenuated infectivity. Supplying VP1 and VP2 in trans ΔVP1-CMV-VP1and ΔVP2-CMV-VP2) rescues the infectivity. ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK, viruses produced in the presence of the UAA show robust infectivity, while those in the absence does not. It should be noted that in the absence of UAA, the TAG mutants of the minor capsid proteins will fail to express.

Example 3: Selective Fluorophore Labeling of Mutated AAV

[0095] FIG. 5 shows selective unnatural amino acid mutagenesis of individual minor capsid proteins in AAV capsid and their use to chemically attach a fluorophore. Following purification, different recombinant AAV2 preparations were labeled with a cyclooctynefluorophore, which selectively labels the azide group present in the UAA AzK. The top panel shows SDS-PAGE analysis of the AAV2 preparations, the bottom panel shows the fluorescence image of the same gel. As expected, the wild-type AAV2 does not show labeling, T454AzK (AzK in all 60 capsid proteins) show labeling of all the capsid proteins, while ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK show selective labeling of VP1 and VP2, respectively, thus demonstrating our ability to selectively label distinct minor capsid proteins.

Example 4: Retargeting Efficiency of Mutated AAV

[0096] FIG. 6 shows the number of retargeting ligands attached to the AAV capsid dramatically affects its retargeting efficiency. Attaching cRGD ligands onto detargeted AAV2 capsids (where binding of the natural primary receptor, heparin sulfate proteoglycan receptor HSPG, was ablated by mutating key residues R588 and R587 to Ala; designated by amino acid residue location of VP1) enables it to selectively bind and infect cancer cell-lines such as SK-OV-3 that overexpress the αVβ3 integrin receptor. These graphs show the infectivity of the shown detargeted AAV2 mutants as they were incubated with cyclooctyne-cRGD over time, as it progressively functionalizes the AzK side chains with cRGD. For 454AA (60 AzK per capsid), the infectivity toward SK-OV-3 cells first go up and then come back down, suggesting an optimal number of cRGD per capsid needed for efficient retargeting. Over-modification of the capsid at later times leads to a loss of infectivity, likely by perturbing viral entry processes. For VP1-454AA and VP2-454AA (5 AzK each per capsid), infectivity goes up and reaches a plateau upon prolonged incubation, suggesting that attachment of 5 cRGD per capsid is not detrimental to AAV2 infectivity. However, the maximal infectivity reached for these mutants are low, suggesting that 5 cRGDs per capsid may not be sufficient for efficient retargeting. VP1+2-454AA (10 AzK per capsid) behaved just like VP1-454AA and VP2-454AA, but the infectivity upon prolonged incubation reaches levels similar to the optimal infectivity observed with T454-AA at the optimal degree of modification.

Example 5: Precise Labeling of Engineered Cysteine Residues of AAV

[0097] FIGS. 7A-7C show precise labeling of AAV at engineered cysteine residues. (A) Packaging efficiency (qPCR) of AAV produced using wild-type Cap (WT), T454C mutant of Cap (T454C at all three capsid proteins), VP1-T454C (T454C mutant of the trans-substituted VP1), VP2-T454C (T454C mutant of the trans-substituted VP2). (B) Infectivity of these viruses measured by their ability to deliver and express an EGFP gene in HEK293T cells (FACS titer). (C) Selective labeling of the engineered 454-cysteine residue on VP1 by fluorescein-maleimide on VP1-454C virus. FAM-fluorescence image shows the result of the labeling reaction on WT and VP1-454C virus, whereas SYPRO stains all proteins.

Example 6: Selective Incorporation of the UAA Into Either VP1 or VP2 or VP1+VP2

[0098] As shown in FIGS. 11A-C, C5Az was incorporated into either VP1 or VP2 or VP1 +VP2 by co-transfecting the HEK293T cells with a plasmid (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS) encoding an engineered E. coli leucyl-tRNA synthetase (EcLeuRS) and tRNA pair that charge C5Az, as well as plasmids encoding [RC2-VPI-del + CMV-VP1-delVP23] or [RC2-VP2-del + CMV-VP2-delVP3] or [RC2-VP12-del + CMV-VP1-VP2-delVP3], respectively. The 454 position (VP1 numbering) in the desired protein was replaced with a TAG stop codon. A) Structure of C5Az, B) qPCR titers of the various preparations shown relative to wild-type AAV2 production, in the presence or absence of C5Az. C) Selective fluorescent labeling of VP1, using DBCO-rhodamine, on AAV2-VP1-454-C5Az.

Example 7: Selective Incorporation of CpK Into VP1

[0099] As shown in FIG. 13 A-C, CpK. was incorporated into VP1 by co-transfecting the HEK293T cells with a plasmid (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS) encoding an engineered E. coli leucyl-tRNA synthetase (EcLeuRS) and tRNA pair that charge CpK, as well as plasmids encoding [RC2-VP1-del + CMV-VPl-delVJ>23]. Various sites (as indicated; VP1 numbering) in VP1 was replaced with a TAG stop codon. A) Structure of CpK, B) Relative titers of the various preparations shown relative to wild-type AAV2 production, in the presence or absence of LCA. C) Selective fluorescent labeling of VP1, using tetrazine-FITC, on AAV preparations incorporating LCA into site 456 of VP1.

Example 8: Selective Incorporation of 5-HTP Into VP1

[0100] As shown in FIGS. 14A-C, 5HTP was incorporated into VP1 by co-transfecting the HEK293T cells with a plasmid (pIDTsmart-TrpRS-8xWtR-ITR-GFP) encoding an engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS) and tRNA pair that charge 5HTP, as well as plasmids encoding [RC2-VP1-del + CMV-VP1-delVP23]. Various sites (as indicated; VP1 numbering) in VP1 was replaced with a TGA stop codon. A) Structure of 5HTP, B) Relative titers of the various preparations shown relative to wild-type AAV2 production, in the presence or absence of 5HTP. C) Selective fluorescent labeling of VP1, using fluorescein amine and ferricyanide, on AAV preparations incorporating 5HTP into site 454 of VP1.

Example 9: Incorporation UAAs Into VPI

[0101] As shown in FIGS. 15A-B, sseveral other unnatural amino acids were incorporated into VP1 by co-transfecting the HEK293T cells with a plasmid (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS) encoding an engineered E. coli leticyl-tRNA synthetase (EcLeuRS) and tRNA pair that charge these unnatural amino acids, as well as plasmids encoding [RC2-VP1-del + CMV-VP1-delVP23]. Site 454 (VP1 numbering) in VP1 was replaced with a TAG stop codon. A) Structure of the unnatural amino acids, B) Relative titers of the various mutant AAV2 preparations shown relative to wild-type AAV2 production, in the presence or absence of the indicated unnatural amino acids.

Example 10: Modification of the LCA Site of AAV2-VP1-454-LCA

[0102] As shown in FIGS. 16A-B, AAV2-VP1-454-LCA, was selectively modified at the LCA site with 20 kDa polyethylene glycol (PEG) polymer using the corresponding PEG-DBCO conjugate. SDS-PAGE analysis shows selective labeling of VP1. Wild-type AAV2, AAV2-VP1-454-LCA, and PEG-modified AAV2-VP1-454-LCA show similar infectivity. Equal genome copies of each virus were added too HEK293 cells and the expression of the encoded luciferase reporter was monitored by a standard luciferase assay.

[0103] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.