IP Library › Granted Patent US 12,455,280
Granted Patent B2
US 12,455,280 · App. 19/014,949 · Granted Oct 28, 2025

Ligand discovery and gene delivery via retroviral surface display

Inventors: Michael Birnbaum (Arlington, MA); Connor Dobson (Somerville, MA)
Assignee: Massachusetts Institute of Technology
G01N33/505C07K14/005C07K14/4748C07K14/5437C07K14/70532C07K14/70539C07K16/2803C12N15/86C07K2317/622C07K2319/02C07K2319/035C07K2319/60C12N2740/15045
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,455,280
App. No.
19/014,949
Granted
Oct 28, 2025
Kind
B2
Abstract

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.

Claims (40)

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 an amino acid substitution at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid substitution(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 T cell or a B 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 VSV-G envelope protein comprises a K47A or K47Q amino acid substitution or an R354A or R354Q amino acid substitution.

4. The lentivirus of claim 1 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution and an R354A or R354Q amino acid substitution.

5. The lentivirus of claim 1 , wherein the extracellular targeting domain comprises a protein, a peptide, or an antibody.

6. The lentivirus of claim 5 , 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.

7. The lentivirus of claim 5 , wherein the ligand comprises a T cell receptor (TCR), a cytokine receptor, a cytokine, a T cell surface marker, CD3, CD19, or CD20.

8. A method of delivering a nucleic acid to a T cell or B cell, the method comprising:

(a) providing a lentivirus comprising: a nucleic acid and 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 an amino acid substitution at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid substitution(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 T cell or a B cell;

(b) contacting the lentivirus with the T cell or B cell,

thereby delivering the nucleic acid to the T cell or B cell.

9. The method of claim 8 , wherein the nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein.

10. The method of claim 8 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution or an R354A or R354Q amino acid substitution.

11. The method of claim 8 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution and an R354A or R354Q amino acid substitution.

12. The method of claim 8 , wherein the extracellular targeting domain comprises a protein, a peptide, or an antibody.

13. The method of claim 12 , 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.

14. The method of claim 12 , wherein the ligand comprises a T cell receptor (TCR), a cytokine receptor, a cytokine, a T cell surface marker, CD3, CD19, or CD20.

15. 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 an amino acid substitution at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid substitution(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.

16. The lentivirus of claim 15 , wherein the nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein.

17. The lentivirus of claim 15 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution or an R354A or R354Q amino acid substitution.

18. The lentivirus of claim 15 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution and an R354A or R354Q amino acid substitution.

19. The lentivirus of claim 15 , 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, a single chain antibody (scFv), or an anti-TCR antibody.

20. The lentivirus of claim 15 , 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.

21. A method of delivering a nucleic acid to a T cell, the method comprising:

(a) providing a lentivirus comprising: a nucleic acid and 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 an amino acid substitution at position 47 (lysine, K) and/or position 354 (arginine, R), wherein the amino acid substitution(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;

(b) contacting the lentivirus with the T cell,

thereby delivering the nucleic acid to the T cell.

22. The method of claim 21 , wherein the nucleic acid encodes an mRNA, a double-stranded DNA, an antisense RNA, a microRNA, a gene of interest, or a protein.

23. The method of claim 21 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution or an R354A or R354Q amino acid substitution.

24. The method of claim 21 , wherein the VSV-G envelope protein comprises a K47A or K47Q amino acid substitution and an R354A or R354Q amino acid substitution.

25. The method of claim 21 , 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, a single chain antibody (scFv), or an anti-TCR antibody.

26. The method of claim 21 , 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.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 11, 2025
From: BIRNBAUM, MICHAEL; DOBSON, CONNOR
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 070824/0965 →
Continuity (4)
Continuation 18458657 · Aug 30, 2023
Continuation 16826665 · Mar 23, 2020
Provisional Application 62851889 · May 23, 2019
Related Publication 20250147007A1 · May 8, 2025
References Cited (86)
US 7901671B2 · Leboulch et al. · 2011 [cited by applicant]
US 9994867B2 · Baltimore et al. · 2018 [cited by applicant]
US 12061187B2 · Birnbaum et al. · 2024 [cited by applicant]
US 12061188B2 · Birnbaum et al. · 2024 [cited by applicant]
US 12222347B2 · Birnbaum et al. · 2025 [cited by applicant]
US 20080241929A1 · Naldini et al. · 2008 [cited by applicant]
US 20140017766A1 · Chen et al. · 2014 [cited by applicant]
US 20150031651A1 · Moller et al. · 2015 [cited by applicant]
US 20150182617A1 · Bauche et al. · 2015 [cited by applicant]
US 20150316511A1 · Guo · 2015 [cited by applicant]
US 20160333374A1 · Anastasov et al. · 2016 [cited by applicant]
US 20170051252A1 · Morgan et al. · 2017 [cited by applicant]
US 20170176435A1 · Seidell, III et al. · 2017 [cited by applicant]
US 20170192011A1 · Birnbaum et al. · 2017 [cited by applicant]
US 20170356010A1 · Frost et al. · 2017 [cited by applicant]
US 20180201954A1 · Buchholz et al. · 2018 [cited by applicant]
US 20180362966A1 · Flechtner et al. · 2018 [cited by applicant]
US 20190144885A1 · Costa Fejoz et al. · 2019 [cited by applicant]
US 20200216502A1 · Albertini et al. · 2020 [cited by applicant]
US 20200371088A1 · Birnbaum et al. · 2020 [cited by applicant]
US 20220204946A1 · Antunes Tomas et al. · 2022 [cited by applicant]
US 20240044873A1 · Birnbaum et al. · 2024 [cited by applicant]
CN 1643164A · 2005 [cited by applicant]
EP 1461079B1 · 2011 [cited by applicant]
WO WO2008037458A2 · 2008 [cited by applicant]
WO WO2009013324A1 · 2009 [cited by applicant]
WO WO2012088381A2 · 2012 [cited by applicant]
WO WO2015104376A1 · 2015 [cited by applicant]
WO WO2015112541A2 · 2015 [cited by applicant]
WO WO2015117027A1 · 2015 [cited by applicant]
WO WO2017182585A1 · 2017 [cited by applicant]
WO WO2019005601A1 · 2019 [cited by applicant]
WO WO2019056015A2 · 2019 [cited by applicant]
WO WO2019057974A1 · 2019 [cited by applicant]
WO WO2020236263A1 · 2020 [cited by applicant]
SG11202112344V, Sep. 15, 2023, Singaporean Search Report. [cited by applicant]
PCT/US2020/024175, Jul. 15, 2020, Invitiation to Pay Additional Fees. [cited by applicant]
PCT/US2020/024175, Sep. 14, 2020, International Search Report and Written Opinion. [cited by applicant]
PCT/US2020/024175, Dec. 2, 2021, International Preliminary Report on Patentability. [cited by applicant]
Singaporean Search Report dated Sep. 15, 2023, for Application No. 11202112344V. [cited by applicant]
Invitation to Pay Additional Fees mailed Jul. 15, 2020, for Application No. PCT/US2020/024175. [cited by applicant]
International Search Report and Written Opinion mailed Sep. 14, 2020, for Application No. PCT/US2020/024175. [cited by applicant]
International Preliminary Report on Patentability mailed Dec. 2, 2021, for Application No. PCT/US2020/024175. [cited by applicant]
Altschul et al., Basic local alignment search tool. J Mol Biol. Oct. 5, 1990;215(3):403-10. [cited by applicant]
Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. Sep. 1, 1997;25(17):3389-402. [cited by applicant]
Bentzen et al., Evolution of MHC-based technologies used for detection of antigen-responsive T cells. Cancer Immunol Immunother. Mar. 17, 2017;66:657-66. [cited by applicant]
Bowie et al., Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science. Mar. 16, 1990;247(4948):1306-10. [cited by applicant]
Buchholz et al., Retroviral display and high throughput screening. Comb Chem High Throughput Screen. Feb. 2008;11(2):99-110. [cited by applicant]
Chan et al., Conjugation of lentivirus to paramagnetic particles via nonviral proteins allows efficient concentration and infection of primary acute myeloid leukemia cells. J Virol. Oct. 2005;79(20):13190-4. [cited by applicant]
Chen et al., Human monoclonal antibodies targeting the haemagglutinin glycoprotein can neutralize H7N9 influenza virus. Nat Commun. Mar. 30, 2015;6:6714. [cited by applicant]
Ciré et al., Immunization of mice with lentiviral vectors targeted to MHC class II+ cells is due to preferential transduction of dendritic cells in vivo. PLoS One. Jul. 24, 2014;9(7):e101644. [cited by applicant]
Dobson et al., Antigen identification and high-throughput interaction mapping by reprogramming viral entry. Nat Methods. Apr. 2022;19(4):449-460. doi: 10.1038/s41592-022-01436-z. Epub Apr. 8, 2022. [cited by applicant]
Dreja et al., The effects of N-terminal insertion into VSV-G of an scFv peptide. Virol J. Sep. 2, 2006;3:69. [cited by applicant]
Frank et al., Surface-Engineered Lentiviral Vectors for Selective Gene Transfer into Subtypes of Lymphocytes. Mol Ther Methods Clin Dev. Oct. 17, 2018;12:19-31. [cited by applicant]
Froelich et al., Targeted gene delivery to CD117-expressing cells in vivo with lentiviral vectors co-displaying stem cell factor and a fusogenic molecule. Biotechnol Bioeng. Sep. 1, 2009;104(1):206-15. doi: 10.1002/bit.… [cited by applicant]
Funke et al., Targeted Cell Entry of Lentiviral Vectors. Mol Ther. Aug. 2008;16(8):1427-36. [cited by applicant]
Goyvaerts et al., Targeting of human antigen-presenting cell subsets. J Virol. Oct. 2013;87(20):11304-8. doi: 10.1128/JVI.01498-13. Epub Jul. 17, 2013. [cited by applicant]
Goyvaerts et al., Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells. Gene Ther. Dec. 2012;19(12):1133-40. doi: 10.1038/gt.2011.206. Epub Jan. 12, 2012. [cited by applicant]
Grubaugh et al., Proteins as T cell antigens: methods for high-throughput identification. Vaccine. Aug. 20, 2013;31(37). [cited by applicant]
Guideng et al., T cell antigen discovery via trogocytosis. Nature Methods. Feb. 2019;16(2):183-90. [cited by applicant]
Hastie et al., Understanding and altering cell tropism of vesicular stomatitis virus. Virus Res. Sep. 2013;176(1-2):16-32. doi: 10.1016/j.virusres.2013.06.003. Epub Jun. 22, 2013. [cited by applicant]
Ho et al., Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling. Cell. Mar. 9, 2017;168(6):1041-1052.e18. doi: 10.1016/j.cell.2017.02.011. [cited by applicant]
Höfig et al., Systematic improvement of lentivirus transduction protocols by antibody fragments fused to VSV-G as envelope glycoprotein. Biomaterials. Apr. 2014;35(13):4204-12. doi: 10.1016/j.biomaterials.2014.01.051. E… [cited by applicant]
Joglekar et al., T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nature Methods. Sep. 23, 2019;16(2):191-8. [cited by applicant]
Kameyama et al., Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein. J Virol Methods. Oct. 2008;153(1):49-54. [cited by applicant]
Karlin et al., Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci U S A. Jun. 15, 1993;90(12):5873-7. [cited by applicant]
Karlin et al., Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci U S A. Mar. 1990;87(6):2264-8. [cited by applicant]
Knopp et al., Transient Retrovirus-Based CRISPR/Cas9 All-in-One Particles for Efficient, Targeted Gene Knockout. Mol Ther Nucleic Acids. Dec. 7, 2018;13:256-274. doi: 10.1016/j.omtn.2018.09.006. Epub Sep. 13, 2018. [cited by applicant]
Kussie et al., A single engineered amino acid substitution changes antibody fine specificity. J Immunol. Jan. 1, 1994;152(1):146-52. [cited by applicant]
Lombardo et al., Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. Nov. 2007;25(11):1298-306. doi: 10.1038/nbt1353. Epub Oct. 28, 2007. [cited by applicant]
Milani et al., Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy. EMBO Mol Med. Nov. 2017;9(11):1558-1573. [cited by applicant]
Nikolic et al., Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nat Commun. Mar. 12, 2018;9(1):1029. doi: 10.1038/s41467-018-03432-4. [cited by applicant]
Ou et al., Specific targeting of human interleukin (IL)-13 receptor α2-positive cells with lentiviral vectors displaying IL-13. Hum Gene Ther Methods. Apr. 2012;23(2):137-47. doi: 10.1089/hgtb.2012.054. Epub May 21, 201… [cited by applicant]
Peach et al., Both extracellular immunoglobin-like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28. J Biol Chem. Sep. 8, 1995;270(36):21181-7. [cited by applicant]
Sela-Culang et al., The structural basis of antibody-antigen recognition. Front Immunol. Oct. 8, 2013;4:302. [cited by applicant]
Sevier et al., Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs. Mol Biol Cell.… [cited by applicant]
Sirin et al., AB-Bind: Antibody binding mutational database for computational affinity predictions. Protein Sci. Feb. 2016;25(2):393-409. doi: 10.1002/pro.2829. Epub Nov. 6, 2015. [cited by applicant]
Taube et al., Lentivirus display: stable expression of human antibodies on the surface of human cells and virus particles. PLoS One. Sep. 11, 2008;3(9):e3181. [cited by applicant]
Urban et al., Retroviral display in gene therapy, protein engineering, and vaccine development. ACS Chem Biol. Jan. 21, 2011;6(1):61-74. doi: 10.1021/cb100285n. Epub Dec. 20, 2010. [cited by applicant]
Urban et al., Selection of functional human antibodies from retroviral display libraries. Nucleic Acids Res. Feb. 24, 2005;33(4):e35. [cited by applicant]
Winkler et al., Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody. J Immunol. Oct. 15, 2000;165(8):4505-14. [cited by applicant]
Yang et al., Cell type-specific targeting with surface-engineered lentiviral vectors co-displaying OKT3 antibody and fusogenic molecule. Pharm Res. Jun. 2009;26(6):1432-45. doi: 10.1007/s11095-009-9853-y. Epub Mar. 4, 2… [cited by applicant]
Yang et al., Targeting lentiviral vectors to specific cell types in vivo. PNAS. Aug. 1, 2006;103(31):11479-84. [cited by applicant]
Yu et al., Engineered cell entry links receptor biology with single-cell genomics. Cell. Dec. 22, 2022;185(26):4904-4920.e22. doi: 10.1016/j.cell.2022.11.016. Epub Dec. 13, 2022. [cited by applicant]
Zhang et al., Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus. Retrovirology. Jan. 25, 2010;7:3. [cited by applicant]
Zhang et al., Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein. J Biol Chem. Oct. 6, 2017;292(40):16527-1653… [cited by applicant]
Cited By (2)
US 12,631,621 US 12,631,622