IP Library › Granted Patent US 12,286,646
Granted Patent B2
US 12,286,646 · App. 16/739,182 · Granted Apr 29, 2025

Methods for in vitro evolution of constructs derived from viruses

Inventors: Darrell J. Irvine (Arlington, MA); Ron Weiss (Newton, MA); Yingzhong Li (Quincy, MA)
Assignee: Massachusetts Institute of Technology
C12N7/00A61K47/6929A61K49/00C12N9/0069C12N15/1003C12N15/1086C12N15/1096C12N15/86C12N2770/36121C12N2770/36143C12N2770/36171
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Quick Facts
Patent No.
US 12,286,646
App. No.
16/739,182
Granted
Apr 29, 2025
Kind
B2
Abstract

The disclosure provides methods for an in vitro evolution technique to identify and characterize mutations in the non-structural genes of an alphavirus replicon that increase the strength and persistence of expression of the replicon genome. Also provided herein are in vivo methods for administering to an animal model a mutated alphavirus replicon that codes for a gene of experimental or therapeutic interest in the subgenome of the alphavirus replicon. The mutations identified herein improve the therapeutic potential of self-replicating RNA, which may have implications for cancer immunotherapy and beyond, e.g., for vaccination or gene therapy.

Claims (20)

1. A nucleic acid molecule comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1-18, or any combination of two or more of SEQ ID NOs: 1-18.

2. A nucleic acid molecule comprising a portion of a nucleotide sequence as shown in any one of SEQ ID NOs: 1-18, or any combination of two or more portions of SEQ ID NOs: 1-18, wherein the portion of the nucleotide sequence comprises a combination of two or more mutations relative to the corresponding wild-type Venezuelan equine encephalitis (VEE) virus ribonucleic acid (RNA) selected from the group consisting of A1979G, G3936C, A4311G, A4758G, G4796T, and G4944A, wherein the position of the mutation(s) is relative to SEQ ID NO: 1.

3. An alphavirus replicon comprising the nucleic acid molecule of claim 2 .

4. The replicon of claim 3 , wherein the replicon encodes non-structural proteins for replication, but does not encode structural proteins for viral formation.

5. The replicon of claim 3 , wherein the replicon comprises the untranslated regions, non-structural proteins, and subgenomic promoter of the alphavirus.

6. The replicon of claim 3 , wherein the structural proteins of the replicon are replaced by one or more gene(s) of experimental or therapeutic interest.

7. The replicon of claim 6 , wherein the one or more gene(s) of experimental or therapeutic interest encodes Interleukin-2 (IL-2) or luciferase.

8. The replicon of claim 3 , wherein the replicon is engineered to express a detectable molecule in the subgenomic region of the replicon.

9. The replicon of claim 8 , wherein the detectable molecule is a fluorescent protein.

10. The nucleic acid molecule of claim 2 , wherein the portion of the nucleotide sequence is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length.

11. The replicon of claim 6 , wherein the one or more gene(s) encode cytokines, chemokines, or growth factors.

12. The replicon of claim 8 , wherein the detectable molecule is a nucleic acid or polypeptide.

13. The replicon of claim 9 , wherein the fluorescent protein is mCherry, mKate, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, mVenus, green fluorescent protein, mRaspberry, or mStrawberry.

14. An alphavirus replicon comprising the nucleic acid molecule of claim 1 .

15. The replicon of claim 14 , wherein the replicon encodes non-structural proteins for replication, but does not encode structural proteins for viral formation.

16. The replicon of claim 15 , wherein the structural proteins of the alphavirus replicon are replaced by one or more gene(s) of experimental or therapeutic interest.

17. The replicon of claim 16 , wherein the one or more gene(s) of experimental or therapeutic interest encodes Interleukin-2 (IL-2) or luciferase.

18. The replicon of claim 15 , wherein the alphavirus replicon is engineered to express a detectable molecule in the subgenomic region of the replicon.

19. The replicon of claim 18 , wherein the detectable molecule is a nucleic acid or polypeptide.

20. The replicon of claim 18 , wherein the detectable molecule is a fluorescent protein.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 14, 2020
From: IRVINE, DARRELL J.; WEISS, RON; LI, YINGZHONG; HOWARD HUGHES MEDICAL INSTITUTE
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 051821/0001 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 14, 2020
From: IRVINE, DARRELL
To: HOWARD HUGHES MEDICAL INSTITUTE
Reel/Frame 051821/0023 →
Continuity (2)
Provisional Application 62790589 · Jan 10, 2019
Related Publication 20200224174A1 · Jul 16, 2020
References Cited (56)
US 4650764A · Temin et al. · 1987 [cited by applicant]
US 7332322B2 · Frolov et al. · 2008 [cited by applicant]
US 20150159143A1 · Dowdy · 2015 [cited by examiner]
WO WO2013177133A2 · 2013 [cited by applicant]
GenBank Accession# DQ322642, VEEV replicon vector YFV-C3opt, complete sequence., Jan. 2006. [cited by examiner]
Li et al., In vitro evolution of enhanced RNA replicons for immunotherapy. Sci Rep. May 6, 2019;9(1):6932. doi: 10.1038/s41598-019-43422-0. PMID: 31061426; PMCID: PMC6502795. [cited by applicant]
Li, Mutations from in vitro evolution affect existence of replicon RNA and transcription of subgenomic. 5 [cited by applicant]
Lundstrom, Replicon RNA Viral Vectors as Vaccines. Vaccines (Basel). Nov. 7, 2016;4(4):39. doi: 10.3390/vaccines4040039. PMID: 27827980; PMCID: PMC5192359. [cited by applicant]
Petrakova et al., Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells. J Virol. Jun. 2005;79(12):7597-608. doi: 10.1128/JVI.79.12.7597-760… [cited by applicant]
Rose et al., In vitro evolution of high-titer, virus-like vesicles containing a single structural protein. Proc Natl Acad Sci U S A. Nov. 25, 2014;111(47):16866-71. doi: 10.1073/pnas.1414991111. Epub Nov. 10, 2014. PMID… [cited by applicant]
Anderson et al., Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucleic Acids Res. Nov. 2011;39(21):9329-38. doi: 10.1093/nar/gkr586. … [cited by applicant]
Aznar et al., Intratumoral Delivery of Immunotherapy—Act Locally, Think Globally. J Immunol. Jan. 1, 2017;198(1):31-39. [cited by applicant]
Beal et al., Model-driven engineering of gene expression from RNA replicons. ACS Synth Biol. Jan. 16, 2015;4(1):48-56. doi: 10.1021/sb500173f. Epub Jun. 6, 2014. [cited by applicant]
Boyman et al., The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol. Feb. 17, 2012;12(3):180-90. [cited by applicant]
Davis et al., In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology. Jul. 1989;171(1):189-204. [cited by applicant]
Drake, Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci U S A. May 1, 1993;90(9):4171-5. [cited by applicant]
Duportet et al., A platform for rapid prototyping of synthetic gene networks in mammalian cells. Nucleic Acids Res. Dec. 1, 2014;42(21):13440-51. doi: 10.1093/nar/gku1082. Epub Nov. 5, 2014. [cited by applicant]
Frolova et al., Roles of nonstructural protein nsP2 and Alpha/Beta interferons in determining the outcome of Sindbis virus infection. J Virol. Nov. 2002;76(22):11254-64. [cited by applicant]
Garmashova et al., Sindbis virus nonstructural protein nsP2 is cytotoxic and inhibits cellular transcription. J Virol. Jun. 2006;80(12):5686-96. [cited by applicant]
Gelderblom, Structure and classification of viruses. Chapter 41. Medical Microbiology. 4 [cited by applicant]
Guan et al., Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Ther. Mar. 2017;24(3):133-143. doi: 10.1038/gt.2017.5. Epub Jan. 17, 2017. [cited by applicant]
Guo et al., Effect of alpha interferon on the hepatitis C virus replicon. J Virol. Sep. 2001;75(18):8516-23. [cited by applicant]
Jensen et al., Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion. J Virol. Mar. 2012;86(6):2900-10. doi: 10.1128/JVI.05738-11. Epub Jan. 18, 2012. [cited by applicant]
Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. Jun. 27, 2017;9(1):60. [cited by applicant]
Kariko et al., Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. Nov. 2008;16(11):1833-40. doi: 10.1038/mt.2008.200.… [cited by applicant]
Kawasaki et al., Recognition of nucleic acids by pattern-recognition receptors and its relevance in autoimmunity. Immunol Rev. Sep. 2011;243(1):61-73. [cited by applicant]
Kinney et al., Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5′-noncoding region and the E2 envelope glycoprotein. J Virol. Mar. 1993;67(3):1269-77. [cited by applicant]
Kuhn et al., Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther. Aug. 2010;17(8):961-71. doi:… [cited by applicant]
Kwong et al., Localized immunotherapy via liposome-anchored Anti-CD137 + IL-2 prevents lethal toxicity and elicits local and systemic antitumor immunity. Cancer Res. Mar. 1, 2013;73(5):1547-58. doi: 10.1158/0008-5472.CA… [cited by applicant]
Lächelt Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50 Years (and Beyond). Chem Rev. Oct. 14, 2015;115(19):11043-78. doi: 10.1021/cr5006793. Epub Apr. 15, 2015. [cited by applicant]
Lastarza et al., Genetic analysis of the nsP3 region of Sindbis virus: evidence for roles in minus-strand and subgenomic RNA synthesis. J Virol. Sep. 1994;68(9):5781-91. [cited by applicant]
Li et al., Persistent Antigen and Prolonged AKT-mTORC1 Activation Underlie Memory CD8 T Cell Impairment in the Absence of CD4 T Cells. J Immunol. Aug. 15, 2015;195(4):1591-8. doi: 10.4049/jimmunol.1500451. Epub Jul. 10,… [cited by applicant]
Lundstrom, Replicon RNA Viral Vectors as Vaccines. Vaccines (Basel). Nov. 7, 2016;4(4):39. [cited by applicant]
Macdonald et al., The zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses. J Virol. Dec. 2007;81(24):13509-18. doi: 10.1128/JVI.00402-07. Epub O… [cited by applicant]
Marabelle et al., Intratumoral immunization: a new paradigm for cancer therapy. Clin Cancer Res. Apr. 1, 2014;20(7):1747-56. [cited by applicant]
Mayuri et al., Role for conserved residues of sindbis virus nonstructural protein 2 methyltransferase-like domain in regulation of minus-strand synthesis and development of cytopathic infection. J Virol. Aug. 2008;82(15… [cited by applicant]
Merly et al., Murine RAW 264.7 cell line as an immune target: are we missing something? Immunopharmacol Immunotoxicol. Apr. 2017;39(2):55-58. doi: 10.1080/08923973.2017.1282511. Epub Feb. 2, 2017. [cited by applicant]
Naka et al., Interferon resistance of hepatitis C virus replicon-harbouring cells is caused by functional disruption of type I interferon receptors. J Gen Virol. Oct. 2005;86(Pt 10):2787-2792. [cited by applicant]
Pietschmann et al., Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J Virol. Feb. 2001;75(3):1252-64. [cited by applicant]
Prabakaran et al., Post-translational modification: nature's escape from genetic imprisonment and the basis for dynamic information encoding. Wiley Interdiscip Rev Syst Biol Med. Nov.-Dec. 2012;4(6):565-83. doi: 10.1002… [cited by applicant]
Puri et al., Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit Rev Ther Drug Carrier Syst. 2009;26(6):523-80. [cited by applicant]
Ramaswamy et al., Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc Natl Acad Sci U S A. Mar. 7, 2017;114(10):E1941-E1950. doi: 10.1073/pnas.1619653114. Epub Feb. 15, 2017. [cited by applicant]
Rodriguez-Madoz et al., Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas. Mol Ther. Jul. 2005;12(1):153-63. [cited by applicant]
Sanjuan et al., Viral mutation rates. J Virol. Oct. 2010;84(19):9733-48. doi: 10.1128/JVI.00694-10. Epub Jul. 21, 2010. [cited by applicant]
Schott et al., Viral and Synthetic RNA Vector Technologies and Applications. Mol Ther. Sep. 2016;24(9):1513-27. doi: 10.1038/mt.2016.143. Epub Jul. 5, 2016. [cited by applicant]
Shin et al., Structural and functional insights into alphavirus polyprotein processing and pathogenesis. Proc Natl Acad Sci U S A. Oct. 9, 2012;109(41):16534-9. doi: 10.1073/pnas.1210418109. Epub Sep. 25, 2012. [cited by applicant]
Stein et al., Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells. J Virol. Apr. 2012;86(8):4527-37. doi: 10.1128/JVI.07071-11. Epub Feb. 15, 2012. [cited by applicant]
Strauss et al., The alphaviruses: gene expression, replication, and evolution. Microbiol Rev. Sep. 1994;58(3):491-562. [cited by applicant]
Van den Boorn et al., Turning tumors into vaccines: co-opting the innate immune system. Immunity. Jul. 25, 2013;39(1):27-37. [cited by applicant]
Wroblewska et al., Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Nat Biotechnol. Aug. 2015;33(8):839-41. doi: 10.1038/nbt.3301. Epub Aug. 3, 2015. [cited by applicant]
Ying et al., Cancer therapy using a self-replicating RNA vaccine. Nat Med. Jul. 1999;5(7):823-7. [cited by applicant]
Youn et al., Modified mRNA as an alternative to plasmid DNA (pDNA) for transcript replacement and vaccination therapy. Expert Opin Biol Ther. 2015;15(9):1337-48. doi: 10.1517/14712598.2015.1057563. Epub Jun. 30, 2015. [cited by applicant]
Yoshioka et al., Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell. Aug. 1, 2013;13(2):246-54. [cited by applicant]
Zavala-Cerna et al., The clinical significance of posttranslational modification of autoantigens. Clin Rev Allergy Immunol. Aug. 2014;47(1):73-90. [cited by applicant]
PCT/US2020/013004, Apr. 23, 2020, International Search Report and Written Opinion. [cited by applicant]
PCT/US2020/013004, Jul. 22, 2021, International Preliminary Report on Patentability. [cited by applicant]