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US-20260125686-A1 - COMPOSITIONS AND METHODS FOR MODULATION OF CFTR

US20260125686A1US 20260125686 A1US20260125686 A1US 20260125686A1US-20260125686-A1

Abstract

In some aspects, provided herein are compositions, methods, and kits relating to an agent modulates expression of a CFTR protein. An agent provide herein can modify the CFTR gene or modulate process of the CFTR pre-mRNA. In some embodiments, the compositions, methods, and kits provided herein are applicable for treatment of cystic fibrosis.

Inventors

  • Normand ALLAIRE
  • Matthew Armstrong
  • Jae Seok Yoon

Assignees

  • CYSTIC FIBROSIS FOUNDATION

Dates

Publication Date
20260507
Application Date
20231002

Claims (20)

  1. 1 . A method of modulating expression of a CFTR gene in a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the cell comprises a pre-mRNA, wherein the pre-mRNA is transcribed from the CFTR gene and comprises a first intron that comprises an alternative polyadenylation site, and wherein the agent modifies the CFTR gene or modulates processing of the pre-mRNA, thereby increasing a level of a processed mRNA that is processed from the pre-mRNA and that lacks nucleic acid sequence of the pre-mRNA downstream of the first intron.
  2. 2 . The method of claim 1 , wherein the cell is a human cell, and the CFTR gene is a human gene.
  3. 3 . The method of claim 2 , wherein the first intron is Intron 22.
  4. 4 . The method of claim 2 , wherein the first intron is located at a region from GRCh38.p14/hg38: chr7: 117,627,771 to GRCh38.p14/hg38: chr7: 117,642,437.
  5. 5 . The method of any one of claims 1-4 , wherein the agent removes from genome of the cell nucleic acid sequence of the CFTR gene that is downstream of the first intron.
  6. 6 . The method of claim 5 , wherein the nucleic acid sequence that is removed from the genome is located from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665.
  7. 7 . The method of claim 5 or 6 , wherein the agent comprises gene editing agents based on CRISPR/Cas9, TALEN, Zinc Finger, or any combination thereof.
  8. 8 . The method of claim 5 or 6 , wherein the agent comprises a pair of guide RNAs, and wherein the pair of guide RNAs comprise the sequences of SEQ ID NOs: 94 and 95, respectively.
  9. 9 . The method of any one of claims 1-8 , wherein the agent removes from the pre-mRNA the nucleic acid sequence of the pre-mRNA downstream of the first intron.
  10. 10 . The method of claim 9 , wherein the nucleic acid sequence that is removed from the pre-mRNA is located from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665.
  11. 11 . The method of any one of claims 1-10 , wherein the agent suppresses splicing out of the first intron from the pre-mRNA.
  12. 12 . A method of modulating expression of a CFTR gene in a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the cell comprises a pre-mRNA that is transcribed from the CFTR gene and comprises a first intron that comprises an alternative polyadenylation site, and wherein the agent suppresses splicing out of the first intron from the pre-mRNA during splicing of the pre-mRNA in the cell.
  13. 13 . The method of claim 12 , wherein the cell is a human cell, and the CFTR gene is a human gene.
  14. 14 . The method of claim 13 , wherein the first intron is Intron 22.
  15. 15 . The method of claim 13 , wherein the first intron is located at a region from GRCh38.p14/hg38: chr7: 117,627,771 to GRCh38.p14/hg38: chr7: 117,642,437.
  16. 16 . The method of any one of claims 12-15 , wherein the agent increases a level of a processed mRNA that is processed from the pre-mRNA and that lacks nucleic acid sequence of the pre-mRNA downstream of the first intron.
  17. 17 . The method of claim 16 , wherein the nucleic acid sequence of the pre-mRNA downstream of the first intron is located at a region from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665.
  18. 18 . The method of any one of claim 1-11, 16, or 17 , wherein the level of the processed mRNA is increased in the cell by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20 times as compared to a corresponding cell that is not contacted with the agent or the vector encoding the agent.
  19. 19 . The method of any one of claim 1-11, 16, or 17 , wherein the level of the processed mRNA is increased in the cell by at least about 10 times as compared to a corresponding cell that is not contacted with the agent or the vector encoding the agent.
  20. 20 . The method of any one of claim 1-11 or 16-19 , wherein the processed mRNA comprises, in a 5′ to 3′ order, 22 exons, an intronic sequence encoding nine amino acids, a stop codon, and an alternative 3′ untranslated region.

Description

CROSS-REFERENCE This application claims the benefit of U.S. Provisional Application No. 63/412,771, filed Oct. 3, 2022, which application is incorporated herein by reference. BACKGROUND Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disease among populations of Northern European descent, with a frequency of 1 in 2000 to 3000 live births. Despite progress in the treatment of CF, there is no cure. Cystic fibrosis can be caused by pathogenic mutations in the CFTR gene, which comprises 250 kilobases of genomic sequence that encodes an epithelial cell protein that is composed of 1480 amino acids in its mature state. Premature Termination Codons (PTC) mutations in the CFTR gene represent the largest class of cystic fibrosis (CF) causing mutations for which there is no therapy. PTC mutations can result in a poorly functional or non-functional protein product and can trigger a dramatic reduction of CFTR mRNA template via Nonsense Mediated mRNA Decay (NMD). Certain C-terminal truncated CFTR protein can retain some chloride channel function in the cells. Therefore, without wishing to be bound by a certain theory, inhibition of NMD to increase truncated protein expression is an attractive approach for treating diseases or conditions caused by PTC mutations near the 3′ end of CFTR. However, given the critical role of NMD as a global quality control mechanism of the cell, a CFTR-specific mechanism to evade NMD is desirable. SUMMARY In some aspects, the present disclosure provides a method of modulating expression of a CFTR gene in a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the cell comprises a pre-mRNA, wherein the pre-mRNA is transcribed from the CFTR gene and comprises a first intron that comprises an alternative polyadenylation site, and wherein the agent modifies the CFTR gene or modulates processing of the pre-mRNA, thereby increasing a level of a processed mRNA that is processed from the pre-mRNA and that lacks nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the cell is a human cell, and the CFTR gene is a human gene. In some embodiments, the first intron is Intron 22. In some embodiments, first intron is located at a region from GRCh38.p14/hg38: chr7: 117,627,771 to GRCh38.p14/hg38: chr7: 117,642,437. In some embodiments, the agent removes from genome of the cell nucleic acid sequence of the CFTR gene that is downstream of the first intron. In some embodiments, the nucleic acid sequence that is removed from the genome is located from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665. In some embodiments, the agent comprises gene editing agents based on CRISPR/Cas9, TALEN, Zinc Finger, or any combination thereof. In some embodiments, the agent comprises a pair of guide RNAs, and wherein the pair of guide RNAs comprise the sequences of SEQ ID NOs: 94 and 95, respectively. In some embodiments, the agent removes from the pre-mRNA the nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the nucleic acid sequence that is removed from the pre-mRNA is located from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665. In some embodiments, the agent suppresses splicing out of the first intron from the pre-mRNA. In some aspects, the present disclosure provides a method of modulating expression of a CFTR gene in a cell, comprising contacting an agent or a vector encoding the agent to the cell, wherein the cell comprises a pre-mRNA that is transcribed from the CFTR gene and comprises a first intron that comprises an alternative polyadenylation site, and wherein the agent suppresses splicing out of the first intron from the pre-mRNA during splicing of the pre-mRNA in the cell. In some embodiments, the cell is a human cell, and the CFTR gene is a human gene. In some embodiments, first intron is Intron 22. In some embodiments, the first intron is located at a region from GRCh38.p14/hg38: chr7: 117,627,771 to GRCh38.p14/hg38: chr7: 117,642,437. In some embodiments, the agent increases a level of a processed mRNA that is processed from the pre-mRNA and that lacks nucleic acid sequence of the pre-mRNA downstream of the first intron. In some embodiments, the nucleic acid sequence of the pre-mRNA downstream of the first intron is located at a region from GRCh38.p14/hg38: chr7: 117,642,438 to GRCh38.p14/hg38: chr7: 117,668,665. In some embodiments, the level of the processed mRNA is increased in the cell by at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 20 times as compared to a corresponding cell that is not contacted with the agent or the vector encoding the agent. In some embodiments, the l