Ligand discovery and gene delivery via retroviral surface display
Compositions of retroviruses and methods of using the same for gene delivery, wherein the retroviruses comprise a viral envelope protein comprising at least one mutation that diminishes its native function, a non-viral membrane-bound protein comprising a membrane-bound domain and an extracellular targeting domain.
1. A method of delivering a nucleic acid to a cell, the method comprising:
(i) providing a retrovirus comprising (a) the nucleic acid and (b) a viral envelope that comprises at least two different proteins:
1) a viral envelope protein comprising at least one mutation that diminishes the native viral tropism of the mutated viral envelope protein compared to the non-mutated viral envelope protein, wherein the viral envelope protein is a vesicular stomatitis virus (VSV)-G envelope protein that comprises an amino acid substitution at position K47 and/or an amino acid substitution at position R354, said positions with reference to SEQ ID NO: 13 or a cocal virus G envelope protein that comprises an amino acid substitution at position K64 and/or an amino acid substitution at position R371, said positions with reference to SEQ ID NO: 51; and
2) a non-viral membrane-bound protein comprising a transmembrane domain and an extracellular targeting domain that is capable of binding to a ligand expressed on the surface of the cell; and
(ii) contacting the retrovirus with the cell,
thereby delivering the nucleic acid to the cell.
2. The method of claim 1 , wherein the nucleic acid encodes a gene of interest, optionally wherein the gene of interest encodes a protein.
3. The method of claim 1 , wherein the retrovirus is a lentivirus.
4. The method of claim 1 , wherein:
(a) the at least one mutation of the VSV-G envelope protein is an amino acid substitution at position K47 to alanine (A) or glutamine (Q) and/or position R354 to alanine (A) or glutamine (Q) of SEQ ID NO: 13; or
(b) the at least one mutation of the cocal virus G protein is an amino acid substitution at position K64 to alanine (A) or glutamine (Q) and/or position R371 to alanine (A) or glutamine (Q) of SEQ ID NO: 51.
5. The method of claim 1 , wherein the VSV-G envelope protein comprises the amino acid sequence set forth in SEQ ID NO: 17.
6. The method of claim 1 , wherein the cocal virus G protein comprises the amino acid sequence set forth in SEQ ID NO: 53.
7. The method of claim 1 , wherein the non-viral membrane-bound protein comprises a class I Major Histocompatibility Complex (MHC) protein or class II MHC protein.
8. The method of 7 , wherein the non-viral membrane-bound protein further comprises a peptide displayed by the class I MHC protein or the class II MHC protein.
9. The method of claim 8 , wherein the peptide is an NLV peptide, an SL9 peptide, a GL9 peptide, an NY-ESO1 peptide, or an EBV peptide.
10. The method of claim 1 , wherein the non-viral membrane-bound protein is CD80.
11. The method of claim 1 , wherein the extracellular targeting domain is a protein, a peptide, or an antibody.
12. The method of claim 11 , wherein the protein is selected from the group consisting of: interleukin-2 (IL-2), IL-6, IL-12, IL-13, chemokine ligand 9 (CXCL9), CXCL10, and CXCL 11.
13. The method of claim 11 , wherein the antibody is a full-length antibody, an antibody fragment, a nanobody, or a single chain antibody (scFv).
14. The method of claim 13 , wherein the antibody is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.
15. The method of claim 1 , wherein a linker is positioned between the membrane-bound domain and the extracellular targeting domain.
16. The method of claim 15 , wherein the linker is a rigid linker, a flexible linker, or an oligomerized linker.
17. The method of claim 16 , wherein the linker comprises a PDGFR stalk, a CD8a stalk, an amino acid sequence comprising GAPGAS (SEQ ID NO:5), an amino acid sequence comprising GGGGS (SEQ ID NO:7), an IgG4 hinge, or an amino acid sequence that forms a tetrameric coiled coil.
18. The method of claim 1 , wherein the ligand is expressed on a somatic cell, optionally an antigen-specific cell.
19. The method of claim 1 , wherein the ligand is expressed on a T cell or a B cell.
20. The method of claim 1 , wherein the ligand is:
(a) a T cell receptor (TCR);
(b) a cytokine receptor;
(c) a cytokine;
(d) a cell surface marker of a T cell or a B cell, or
(e) a cell surface marker selected from the group consisting of CD3, CD19, or CD20.