MODIFIED ADENO-ASSOCIATED VIRUS (AAV) PARTICLES FOR GENE THERAPY

Abstract

The present invention relates to improved adeno-associated virus (AAV) particles for gene delivery and gene therapy. Provided are adeno associated virus (AAV) particles that comprise a modified capsid. The present invention further relates to methods for producing the improved AAV particles of this invention by removing natural binding sites in adeno associated virus (AAV) capsids and introducing ligand binding sites into said capsid to provide AAVs that transduce only particular cells of interest. An additional aspect of the present invention relates to modified AAV particles for use in the treatment of a disease and methods for treating a disease, comprising administering the modified AAV particles to a subject in need thereof. Yet a further aspect of this invention relates to the AAV particles of this invention for the transfection of cells, for example as a gene delivery tool in basic research.

Claims

1-16. (canceled)

17. An adeno associated virus (rAAV) particle comprising a chemically modified capsid protein, wherein the chemically modified capsid protein comprises one or more of a ligand binding site; the ligand binding site comprising a dibenzocyclooctyne group or an azide group.

18. The rAAV particle of claim 17, further comprising a ligand that is attached to the ligand binding site.

19. The rAAV particle of claim 17, wherein the ligand binding site is attached to a primary amine of the capsid protein.

20. The rAAV particle of claim 17, wherein the ligand binding site comprises a dibenzocyclooctyne group and the ligand comprises an azide group.

21. The rAAV particle of claim 17, wherein the ligand binding site comprises an azide group and the ligand comprises a dibenzocyclooctyne group.

22. The rAAV particle of claim 17, wherein the ligand is attached to the ligand binding site via the product of a Staudinger reaction or a strain-promoted click reaction.

23. The rAAV particle of claim 17, wherein the modified capsid protein is selected from one or more of VP1, VP2 and VP3.

24. The rAAV particle of claim 17, wherein the AAV particle has a higher infectivity rate at lower titers compared to an unmodified AAV particle of the same serotype.

25. The rAAV particle of claim 17, wherein the modified capsid protein is further modified to remove a natural mammalian cell binding site.

26. The rAAV particle of claim 17, wherein the natural mammalian cell binding site is a heparan sulfate proteoglycan binding site.

27. The rAAV particle of claim 17, wherein the AAV particle has a modified tropism compared to an unmodified AAV particle of the same serotype.

28. The rAAV particle of claim 17, wherein the ligand is selected from a protein ligand, a toxin subunit, a lectin, an adhesion factor, an antibody, a peptide, and an enzyme.

29. A method of producing an adeno associated virus (rAAV) particle comprising a chemically modified capsid protein, the method comprising the steps of: attaching at least one ligand binding site to a capsid protein, the ligand binding site comprising an azide group or a dibenzocyclooctyne group; and attaching a ligand to the ligand binding site.

30. The method according to claim 29, wherein the step of attaching at least one ligand binding site to a capsid protein occurs before the step of attaching a ligand to the ligand binding site.

31. The method according to claim 29, wherein the ligand is attached to the ligand binding site via a Staudinger reaction or a strain-promoted click reaction.

32. The method according to claim 29, wherein the ligand binding site is attached to a primary amine of the capsid protein.

33. The method according to claim 29, further comprising a step of modifying the capsid to remove a natural mammalian cell binding site.

34. A pharmaceutical composition, comprising the rAAV particle according to claim 17, and at least one pharmaceutically acceptable carrier and/or diluent.

35. A method for treating a patient having a genetic abnormality, comprising administering the AAV particle according to claim 17, or the pharmaceutical composition according to claim 34 to a subject in need thereof, wherein said disease is one that can be treated by gene therapy.

36. The method of claim 35, wherein the disease is selected form cancer, an inherited monogenic disease, a genetic skin disease, an infectious disease, type I diabetes, and wound healing.

37. A method for transfecting a cell comprising the steps of contacting a cell with a composition comprising the AAV particle according to claim 17.

Description

[0064] The present invention will be further illustrated in the following examples and figures, which are given for illustration purposes only and are not intended to limit the invention in any way. For the purpose of the present invention, all references as cited are hereby incorporated by reference in their entireties.

[0065] FIG. 1 shows a schematic depiction of SNAP-tagged ligands with the BG-modified virus.

[0066] FIG. 2 shows that the ΔHSPG virus particle according to the invention has no more infective activity (dark picture); the construct was tested on sensory neurons in a fluorescent reporter mouse model. The inset shows the phase contrast microscopic image of the cells.

[0067] FIG. 3 shows that the wheat germ agglutinin (WGA) fusion fully reverted the viral transduction efficiency to 100% (fluorescent cells) when tested on sensory neurons in a fluorescent reporter mouse model in analogy to FIG. 2.

[0068] FIG. 4 shows that neurotrophic factors NGF (A), NT3 (B) and BDNF (C) deliver virus to different neuronal populations. The insets show the microscopic image of the cells, the constructs were tested on sensory neurons in a fluorescent reporter mouse model in analogy to FIG. 2.

[0069] FIG. 5 shows cholera toxin B subunit transported virus retrogradely to neuronal cell bodies when injected into the skin. The inset shows the microscopic image of the cells, the construct was tested on sensory neurons in a fluorescent reporter mouse model in analogy to FIG. 2.

[0070] FIG. 6 shows sensory neuron tissue in the trigeminal ganglia three weeks after injection with a virus with NGF ligand IV according to the invention (A), stained with an antibody against TrkA (the receptor for NGF, B). An overlap of at least 80% can be seen (C).

[0071] FIG. 7 shows a staining of the sections from FIG. 6 with antibodies against NF200 and IB4, which mainly mark other neurons (mechanoreceptors (green/grey) and non-peptidergic nociceptors, respectively (blue/dark grey)). The red (light grey) infected cells are mainly different from the green and blue cells.

[0072] FIG. 8 shows that gene delivery is more efficient with liganded viruses. WGA modified construct resulted in a strong increase of delivery (B). A) normal AAV9 variant PHP.S; B) PHP.S variant of A) modified with WGA.

EXAMPLES

[0073] The aim of the experiments as performed in the context of the present invention was to engineer the adeno associated virus (AAV) capsid so that the virus will transduce only cells of interest. This was achieved by removing the natural binding sites for cells in the native AAV capsid protein(s). The modified virus is then suitably (in particular chemically) modified in order to accept a selective controlled ligand attachment. These desired ligands are then covalently attached to the virus, and tested in vitro on cells, and in vivo in mice. Although AAV2 is used, these examples can be readily applied to other AAV capsids as well.

1. Removal of Natural Binding Sites in AAV2

[0074] AAV2 binds to Heparan Sulfate Proteoglycans through arginine 585 and 588. These positions were mutated to alanine to create the deletion ΔHSPG.

[0075] The Plasmid pTAV2-0 contains the entire AAV-2 genome from pAV-2, including both inverted terminal repeats, cloned into the BamHI site of pBluescript II. A sub-plasmid containing a suitable fragment of the AAV-2 was created and used as the template for site-directed mutagenesis reactions. Mutagenesis was performed by using a Stratagene (Amsterdam, The Netherlands) QuikChange site-directed mutagenesis kit according to the manufacturer's protocol. For each mutant, two complementary PCR primers were designed to contain the sequence of the substitution, flanked by 15 to 20 homologous base pairs on each side of the mutation. Mutant plasmids were identified by DNA sequencing. The fragment containing the suitable mutation was then subcloned into a plasmid backbone (e.g. pTAV2-0), containing the rest of the protein, and the complete fragment was sequenced to check for additional PCR mutations.

2. Chemical Modification of ΔHSPG for Accepting Ligands

[0076] Typically, selective attachment of ligands to proteins, e.g. protein labeling, is accomplished by incorporation of bioorthogonal groups into a protein, followed by chemoselective modifications. This approach is also designated as “tag-and-modify”. A variety of bioorthogonal reactions have been developed, which can be classified into: (1) condensation reactions through carbonyls, (2) “click” reactions through azides, (3) inverse electron-demand Diels-Alder cycloadditions (DA.sub.INV) and other cycloaddition reactions, (4) transition metal-catalyzed coupling and decaging reactions, and (5) labeling reactions at cysteine residues.

[0077] Benzylguanine (BG) was subsequently attached to exposed lysine by reacting virus with benzylguanine NHS ester (SNAP tag substrate or BG NHS). For this, using a needle, non-aqueous DMSO was added to the vial with the dry SNAP tag ligand BG-NHS to the desired final concentration (e.g. 20 mM) at room temperature. The protein to be amine-functionalized was diluted in solvent (PBS) to the desired final concentration. The two preparations were mixed and incubated at room temperature for 180 minutes, followed by removal of the unreacted components using a centrifugal 100 Kda MWCO filter unit.

3. Covalent Attachment of Ligands

[0078] There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-Tag® fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O.sup.6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. The SNAP-tag substrate in this case is the guanine leaving group connected to a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag.

[0079] The SNAP-tag protein labeling system enables the specific, covalent attachment of virtually any molecule to a protein of interest (for the present invention, see 4., below).

[0080] Recombinant ligands with C terminal SNAP tags were produced in E. coli or in mammalian cells in suspension culture. For the covalent attachments, SNAP-tagged ligands were then attached to the BG-modified virus (see FIG. 1) by adding saturating concentrations of ligand and incubating at room temperature overnight. Excess non-reacted ligand was removed by passing the reaction through a centrifugal 100 Kda MWCO filter unit.

[0081] For the present invention, the experiments were performed in accordance according with the instructions of the SNAP-Cell® Starter Kit (NEB) containing a mammalian expression plasmid (pSNAP.sub.f) encoding the SNAP-Tag® flanked by restriction sites for cloning a gene of interest, with modifications for the present purpose.

4. In Vitro and In Vivo Tests

[0082] In the context of the present invention, the above strategy was tested with multiple classes of ligands, namely protein ligands, like growth factors, cytokines etc.; toxin subunits, like cholera toxin B subunit; lectins, such as isolectin B4 or wheat germ agglutinin; adhesion factors, like lactadherin; antibodies, such as anti CD-34 (marker of stem cells); and peptides, such as deltorphin opioid receptor ligand.

[0083] It was shown first that the ΔHSPG virus particle according to the invention had no more infective activity as tested on sensory neurons in a fluorescent reporter mouse model (FIG. 2). The wheat germ agglutinin (WGA, lectin) fusion fully reverted the viral transduction efficiency to 100% when tested on sensory neurons in the same fluorescent reporter mouse model (FIG. 3).

[0084] Then, several factors were tested, the neurotrophic factors NGF, NT3 and BDNF (protein ligands) delivered virus to different specific neuronal populations depending on the factor used in a fluorescent reporter mouse model (FIG. 4). Cholera Toxin B subunit (toxin) specifically directed virus retrogradely to neuronal cell bodies (i.e. cell compartment/part specific) (FIG. 5). In similar tests, lactadherin (adhesion factor) specifically directed virus to macrophages and neurons exposing phosphatidylserine, and deltorphin (peptide) specifically directed virus to neurons expressing the Mu and Delta opioid receptors.

[0085] In the experiments shown in FIG. 6, virus with the NGF ligand IV was injected into the trigeminal ganglia, then sensory neuron tissue was taken and analysed three weeks later. The sectioned tissue was stained with an antibody against TrkA (the receptor for NGF), and a very good overlap was found. The TrkA antibody is not perfect, so an 80% overlap is extremely relevant.

[0086] In the experiments shown in FIG. 7, the sections from FIG. 6 were stained with antibodies against NF200 and IB4, which label other neurons (mechanoreceptors and non-peptidergic nociceptors, respectively). Again, these markers are not perfect but it can be seen that the green and blue cells are different from the red infected cells.

[0087] As a negative control, virally introducing the IL31 ligand into an IL31 receptor knockout mouse does not lead to an infection.

[0088] In summary, all ligand-labeled viruses successfully and specifically transduced only those cells expressing the respective receptor, both when applied in vitro to cultured cells, and when injected in vivo in mice, i.e. can be injected systemically or locally and selectively target different populations of cells.

Additional In Vivo Tests Using Liganded-AA

A) Targeting TrkA+ Nociceptors

[0089] In this example, TrkA+ nociceptors in the peripheral nervous system were targeted with liganded AAV. NGF.sup.R121W ligand which binds to but doesn't activate TrkA was conjugated to ΔHSPG-AAV2 as described above with a tdTomato cargo. The construct was injected into mice subcutaneously, intra-nerve, retro-orbital or intraperitoneal. After three weeks, fluorescence was detected and quantified by using a TrkA antibody.

[0090] It was found that for the retro-orbital application 80% of TrkA.sup.+ cells were infected by NGF-AAV. 83% of NGF-AAV infected cells were TrkA.sup.+. It was also found that the different routes of administration did not differ significantly in their highly effective outcomes.

B) Targeting IL31RA+ Itch Receptors

[0091] In this example, IL31RA was targeted with liganded AAV. IL31.sup.K134A ligand that binds to but doesn't activate IL31RA was conjugated to ΔHSPG-AAV2 as described above with a tdTomato cargo. The construct was injected into wildtype and IL31RA knockout mice. After three weeks, fluorescence was detected by using a keratin 14 antibody. It was found that targeted cells were basically completely positive for K14. Important in IL31RA knockout mice, no fluorescence was detected.

C) Targeting Using AAV with Isolectin B4

[0092] In this example, Isolectin B4 (IB4) was conjugated to ΔHSPG-AAV2 as described above with a tdTomato cargo. IB4 can be used as a marker for vasculature, non-peptidergic nociceptors, and/or microglia. The construct was injected subcutaneously, intra-nerval, or intraspinally. After three weeks, fluorescence was detected. It was found that targeted cells were basically completely positive, irrespective of the route of administration.

D) Targeting Using AAV with Wheat Germ Agglutinin

[0093] In this example, Wheat Germ Agglutinin (WGA) was conjugated to ΔHSPG-AAV2 as described above with a tdTomato cargo. WGA binds to N-acetylglucosamine and the membrane of most neurons and is used as a (transsynaptic) tracer. The construct was injected in mice i.v. in P1 neonates, or intracortical in adult mice. After three weeks, fluorescence was detected.

[0094] It was found that gene delivery is more efficient with liganded viruses (see FIG. 8). Cultured DRG neurons were infected with AAV9 variant PHP.S (1E+9 VG), and it was found that the above WGA modified construct resulted in a strong increase of delivery (see FIG. 8B).