Ligand discovery and gene delivery via retroviral surface display
Compositions of retroviruses and methods of using the same for gene delivery are disclosed, 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 lentivirus comprising:
(a) a nucleic acid;
(b) a viral envelope comprising
(i) a vesicular stomatitis virus (VSV)-G envelope protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 13, wherein the VSV-G envelope protein comprises one or more amino acid mutations at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid mutation(s) diminish the native viral tropism of the VSV-G envelope protein compared to the non-mutated VSV-G envelope protein; and
(ii) a non-viral membrane-bound protein comprising a transmembrane-domain and an extracellular targeting domain that binds to a ligand expressed on the surface of a cell.
2 . The lentivirus of claim 1 , wherein the nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein.
3 . The lentivirus of claim 1 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 or position R354.
4 . The lentivirus of claim 1 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 and position R354.
5 . The lentivirus of claim 1 , wherein the one or more amino acid mutations comprise a K47A or K47Q amino acid substitution and/or an R354A or R354Q amino acid substitution.
6 . The lentivirus of claim 1 , wherein the extracellular targeting domain comprises a protein, a peptide, or an antibody.
7 . The lentivirus of claim 6 , wherein the extracellular targeting domain comprises an interleukin-13 protein domain, a CD80 protein domain, a full-length antibody, an antibody fragment, a nanobody, a single chain antibody (scFv), an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.
8 . The lentivirus of claim 1 , wherein the ligand comprises a T cell receptor (TCR), a cytokine receptor, a cytokine, a T cell surface marker, CD3, CD19, or CD20.
9 . The lentivirus of claim 1 , wherein the cell is a T cell or a B cell.
10 . A method of delivering a nucleic acid to a cell, the method comprising:
(a) providing a lentivirus comprising: a nucleic acid and a viral envelope comprising (i) a VSV-G envelope protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 13, wherein the VSV-G envelope protein comprises one or more amino acid mutations at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid mutation(s) diminish the native viral tropism of the VSV-G envelope protein compared to the non-mutated VSV-G envelope protein; and (ii) a non-viral membrane-bound protein comprising a transmembrane-domain and an extracellular targeting domain that binds to a ligand expressed on the surface of a cell;
(b) contacting the lentivirus with the cell,
thereby delivering the nucleic acid to the cell.
11 . The method of claim 10 , wherein the nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein.
12 . The method of claim 10 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 or position R354.
13 . The method of claim 10 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 and position R354.
14 . The method of claim 10 , wherein the one or more amino acid mutations comprise a K47A or K47Q amino acid substitution and/or an R354A or R354Q amino acid substitution.
15 . The method of claim 10 , wherein the extracellular targeting domain comprises a protein, a peptide, or an antibody.
16 . The method of claim 15 , wherein the extracellular targeting domain comprises an interleukin-13 protein domain, a CD80 protein domain, a full-length antibody, an antibody fragment, a nanobody, an scFv, an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.
17 . The method of claim 10 , wherein the ligand comprises a T cell receptor (TCR), a cytokine receptor, a cytokine, a T cell surface marker, CD3, CD19, or CD20.
18 . The method of claim 10 , wherein the cell is a T cell or a B cell.
19 . A lentivirus, comprising
(a) a nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein;
(b) a viral envelope comprising:
(i) a VSV-G envelope protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 13, wherein the VSV-G envelope protein comprises one or more amino acid mutations at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid mutation(s) diminish the native viral tropism of the VSV-G envelope protein compared to the non-mutated VSV-G envelope protein; and
(ii) a non-viral membrane-bound protein comprising a transmembrane domain and an extracellular targeting domain that comprises an anti-CD3 antibody.
20 . The lentivirus of claim 19 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 or position R354.
21 . The lentivirus of claim 19 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 and position R354.
22 . The lentivirus of claim 19 , wherein the one or more amino acid mutations comprise a K47A or K47Q amino acid substitution and/or an R354A or R354Q amino acid substitution.
23 . The lentivirus of claim 19 , wherein the viral envelope further comprises a non-viral membrane-bound protein comprising a transmembrane domain and an extracellular targeting domain that comprises a full-length antibody, an antibody fragment, a nanobody, an scFv, or an anti-TCR antibody.
24 . The lentivirus of claim 19 , wherein the viral envelope further comprises a non-viral membrane-bound protein that comprises a CD80 protein comprising a CD80 extracellular domain and a transmembrane domain.
25 . A method of delivering a nucleic acid to a T cell, the method comprising:
(a) providing a lentivirus comprising:
(i) a nucleic acid encoding an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein; and a viral envelope comprising
(ii) a VSV-G envelope protein comprising an amino acid sequence at least 95% identical to SEQ ID NO: 13, wherein the VSV-G envelope protein comprises one or more amino acid mutations at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid mutation(s) diminish the native viral tropism of the VSV-G envelope protein compared to the non-mutated VSV-G envelope protein; and
(iii) a non-viral membrane-bound protein comprising a transmembrane domain and an extracellular targeting domain that comprises an anti-CD3 antibody;
(b) contacting the lentivirus with the T cell,
thereby delivering the nucleic acid to the T cell.
26 . The method of claim 25 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 or position R354.
27 . The method of claim 25 , wherein the one or more amino acid mutations comprise an amino acid mutation at position K47 and position R354.
28 . The method of claim 25 , wherein the one or more amino acid mutations comprise a K47A or K47Q amino acid substitution and/or an R354A or R354Q amino acid substitution.
29 . The method of claim 25 , wherein the viral envelope further comprises a non-viral membrane-bound protein comprising a transmembrane domain and an extracellular targeting domain that comprises a full-length antibody, an antibody fragment, a nanobody, an scFv, or an anti-TCR antibody.
30 . The method of claim 25 , wherein the viral envelope further comprises a non-viral membrane-bound protein that comprises a CD80 protein comprising a CD80 extracellular domain and a transmembrane domain.