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US-12618053-B2 - Methods and compositions for cell and tissue rejuvenation

US12618053B2US 12618053 B2US12618053 B2US 12618053B2US-12618053-B2

Abstract

The present disclosure provides compositions, methods and kits for the rejuvenation of target cells. In some aspects, the compositions, methods and kits comprise mRNAs the promote the expression of TERT and/or TERC.

Inventors

  • IGOR KOGUT
  • GANNA BILOUSOVA
  • Nicole Frances DIETTE
  • Patrick Sean MCGRATH

Assignees

  • THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE

Dates

Publication Date
20260505
Application Date
20200914

Claims (19)

  1. 1 . A composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a biologically active exogenous human telomerase reverse transcriptase (TERT) having telomere elongation activity; and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the at least a portion of the at least one DNA targeting polypeptide increases transcription of endogenous human telomerase RNA component (TERC) compared to untreated cells wherein none of the DNA-targeting polypeptides target an endogenous TERT gene, wherein the DNA-targeting polypeptide does not target an endogenous TERT gene, and wherein the combination of a) and b) increases telomere length compared to untreated cells.
  2. 2 . The composition of claim 1 , wherein the at least one first polynucleotide molecule comprises: i) an mRNA molecule encoding at least a biologically active human TERT having elongation activity; or ii) a plasmid comprising a nucleic acid sequence encoding at least a biologically active human TERT operably linked to at least one promoter to drive detectable expression of the at least one biologically active human TERT.
  3. 3 . The composition of claim 1 , wherein the at least one second polynucleotide molecule comprises: i) an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide; or ii) a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter to drive detectable expression of the at least one portion of the at least one DNA targeting polypeptide.
  4. 4 . The composition of claim 1 , wherein the DNA targeting polypeptide comprises at least one CRISPR-associated protein 9 (Cas9) molecule, at least one Cas9 variant that remains functionally active, or at least one Cas9 ortholog thereof that remains functionally active, at least one Transcription Activator-Like Effector (TALE) molecule, at least one zinc-finger molecule, at least one meganuclease molecule or any combination thereof.
  5. 5 . The composition of claim 1 , wherein the DNA targeting polypeptide comprises at least one transactivation molecule.
  6. 6 . The composition of claim 1 , wherein when the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant that remains functionally active, or at least one Cas9 ortholog thereof, the composition further comprises at least one guide RNA (gRNA).
  7. 7 . The composition of claim 5 , wherein the transactivation molecule comprises at least one single guide RNA MS2 bacteriophage (sgRNA-MS2) molecule, wherein the at least one sgRNA-MS2 molecule comprises a nucleic acid sequence complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene and at least one MS2 RNA aptamer.
  8. 8 . The composition of claim 1 , further comprising: i) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA in the plurality is complementary to a nucleic acid sequence located upstream, within, or downstream of the endogenous TERC gene; or ii) at least one plasmid comprising at least one nucleic acid sequence encoding at least one gRNA operably linked to at least one promoter to drive detectable expression of the at least one species gRNA.
  9. 9 . The composition of claim 1 , wherein the nucleic acid sequence encoding at least a portion of the at least one DNA targeting polypeptide in b) comprises at least one modified mRNA molecule and wherein the at least one DNA targeting polypeptide comprises dCas9 and a VP64-P65-Rta (VPR) molecule; and further comprising: c) a plurality of guide RNA (gRNA) molecules, wherein at least one gRNA is selected from the group consisting of SEQ ID nos. 1-1276.
  10. 10 . The composition of claim 1 , further comprising at least one polynucleotide encoding at least one rejuvenating factor.
  11. 11 . The composition of claim 1 , wherein the composition is packaged or encoded in at least one viral particle, at least one exosome, at least one microvesicle, at least one liposome, or at least one nanoparticle.
  12. 12 . A method of rejuvenating at least one cell, the method comprising contacting the at least one cell in need of rejuvenation with the composition of claim 1 .
  13. 13 . A method of treating, reducing the risk of onset of, or preventing a health condition in a subject comprising: a) contacting at least one cell in vitro with the composition of claim 1 ; b) expanding the at least one cell in vitro to produce a plurality of rejuvenated cells; and c) administering a therapeutically effective amount of the plurality of rejuvenated cells to the subject in need of cell therapy and treating, reducing onset of, or preventing the health condition in the subject.
  14. 14 . A method for rejuvenating at least one cell in a subject comprising administering to the subject a therapeutically effective amount of the composition of claim 1 .
  15. 15 . The composition of claim 1 , further comprising a pharmaceutically acceptable excipient.
  16. 16 . A kit comprising the composition of claim 1 , and at least one container.
  17. 17 . A method for preparing a composition of claim 1 comprising combining a) and b) into a medium.
  18. 18 . The composition of claim 1 , wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant that remains functionally active, or the at least one Cas9 ortholog molecule thereof that remains functionally active and the at least one Cas9 molecule, the at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof comprises at least one of eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D11 35E), Cas9 (QQR1), SaCas9 (KKH, Nme1Cas9, Nme2Cas9, Nme3Cas9, Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9 , Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae Cas9 or any combination thereof.
  19. 19 . The composition of claim 4 , wherein the DNA targeting polypeptide comprises at least one Cas9 molecule, at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof and the at least one Cas9 molecule, the at least one Cas9 variant, or the at least one Cas9 ortholog molecule thereof comprises at least one of SpCas9, SaCas9, SpyFi Cas9, Cpf1 and xCas9, or variant or ortholog molecule thereof.

Description

RELATED APPLICATIONS This application is a U.S. National Phase application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2020/050665, filed Sep. 14, 2020, which claims priority to, and the benefit of, U.S. Provisional Application No. 62/899,861, filed Sep. 13, 2019, the contents of each of which are incorporated herein by reference in their entireties. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 18, 2022, is named “UNCO-028_N01_SeqList.txt” and is about 241 KB in size. BACKGROUND OF THE INVENTION The shortening of telomeres, repetitive DNA sequences at the ends of linear chromosomes, can lead to cellular senescence, apoptosis, or malignancy. In particular, the shortening of telomeres in cells cultured in vitro is an obstacle to the production of therapeutic cell populations, as shortened telomeres can limit further expansions of the therapeutic cell populations as well as degrade the cells' biological activity, leading to a decrease clinical efficacy. Increasing telomere length in cells can lead to cellular rejuvenation, but can also cause deleterious side-effects such as oncogenic cellular immortalization. Thus, there is a need in the art for compositions, kits and methods directed to effectively and safely increasing the length of telomeres in cells in a controllable way, thereby rejuvenating the cells. SUMMARY OF THE INVENTION The present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one second polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC). The present disclosure provides a composition comprising: a) at least one first polynucleotide molecule comprising a nucleic acid sequence encoding at least a portion of telomerase reverse transcriptase (TERT); and b) at least one DNA targeting polypeptide, wherein the DNA targeting polypeptide increases transcription of telomerase RNA component (TERC). A first polynucleotide molecule can comprise an mRNA molecule encoding at least a portion of TERT. A first polynucleotide molecule can comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of TERT operably linked to at least one promoter sufficient to drive expression of the at least one portion of TERT. A second polynucleotide molecule can comprise an mRNA molecule encoding at least a portion of at least one DNA targeting polypeptide. A second polynucleotide molecule can comprise a plasmid comprising a nucleic acid sequence encoding at least a portion of at least one DNA targeting polypeptide operably linked to at least one promoter sufficient to drive expression of the at least one portion of the at least one DNA targeting polypeptide. A DNA targeting polypeptide can comprise at least one Cas9 molecule, at least one Cas9 variant molecule, at least one Cas9 ortholog molecule or any combination thereof. A Cas9 molecule, a Cas9 variant molecule or a Cas9 ortholog molecule can be nuclease-deficient or nuclease-dead. A Cas9 variant molecule can comprise eSpCas9 (K855A), eSpCas9 (1.0), eSpCas9 (1.1), SpCas9-HF1 (VP12), HypaCas9, xCas9, SpyFi Cas9, iSpy Cas9, iSpyMac, Cas9 (VQR), Cas9 (EQR), Cas9 (VRER), Cas9 (D1135E), Cas9(QQR1), SaCas9 (KKH), Nme1Cas9, Nme2Cas9, Nme3Cas9 or any combination thereof. A Cas9 ortholog molecule can comprise Streptococcus pyogenes Cas9 (spCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus Cas9 (SaCas9), Neisseria meningitidis Cas9 (NmCas9; NmeCas9), Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9), Streptococcus thermophilus CRISPR3-Cas9 (St3Cas9), Campylobacter jejuni Cas9 (CjCas9), Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1), Streptococcus canis Cas9 (ScCas9), Treponema denticola Cas9 (TdCas9), Streptococcus macacae Cas9 (SmacCas9), Casφ (Cas12j), Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, Campylobacter lari CF89-12 Cas9, Mycoplasma gallisepticum str. F Cas9, Nitratifractor salsuginis str DSM 16511 Cas9, Parvibaculum lavamentivorans Cas9, Roseburia intestinalis Cas9, Neisseria cinerea Cas9, Gluconacetobacter diazotrophicus Cas9, Azospirillum B510 Cas9, Sphaerochaeta globus str. Buddy Cas9, Flavobacterium columnare Cas9, Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, Mycoplasma mobile Cas9, Lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphylococcus pseudintermedius Cas9, Filifactor alocis Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, Corynebacter diphtheriae