Granted Patent B2

US 12,467,043 · App. 17/626,561 · Granted Nov 11, 2025

Inventors: Qianben Wang (Durham, NC); Fuwen Yuan (Durham, NC); Wei Li (Irvine, CA)

Assignees: Duke University; Baylor College of Medicine

C12N9/22C12N15/11C12N15/1135C12N2310/14C12N2310/20

Abstract

Claims (27)

1 . A system for modifying the length of a 3′ untranslated region (UTR) of an mRNA transcript, the system comprising one or more nucleic acid molecules comprising:

(i) a nucleic acid sequence encoding a direct repeat RNA sequence that can bind a catalytically dead Cas13;

(ii) a nucleic acid sequence encoding a guide RNA (gRNA) sequence that can bind to a target site that is proximal and/or distal to a polyadenylation site (PAS) of the 3′ UTR of the mRNA transcript; and

(iii) a nucleic acid sequence encoding a catalytically dead Cas13, wherein the system causes modification of the length of the 3′ untranslated region (UTR) of an mRNA transcript,

wherein the one or more nucleic acids molecules are contained in one or more vectors and wherein the vector comprises the nucleic acid sequence set forth in SEQ ID NO: 02.

2 . The system of claim 1 , wherein components (i), (ii), and (iii) are located on the same nucleic acid molecule or components (i) and (ii) are located on the same nucleic acid molecule and component (iii) is located on a separate nucleic acid molecule.

3 . The system of claim 1 , wherein the catalytically dead Cas13 is Cas13d-NLS from Ruminococcus fiavefaciens XPD3002 (dCasRx).

4 . The system of claim 1 , wherein the catalytically dead Cas13 is dCas13a, dCas13b, dCas13c, or dCas13d.

5 . The system of claim 1 , wherein the target site is proximal or distal to the PAS of the 3′ UTR of the mRNA transcript.

6 . The system of claim 1 , wherein the mRNA transcript is from a CDC26, INPPLI, DLLI, HEATR3, SLC25A40, SPSBI, MRH, MED18, GABPA, TTC82 DUSP4, TUBB2B, ELAVL1, CHGA, MAP3K4, and/or FHIT gene.

7 . The system of claim 1 , wherein the vector comprises a dCas13d gRNA all-in-one vector or a lenti-gRNA-RFP-PuroR and a lenti-dCas13Rx-EGFP vector, wherein the dCas13 gRNA all-in-one vector comprises nucleic acid sequences encoding one or two nuclear localization peptides (NLS), a dCas13, an epitope tag, a linker peptide, a 2A self-cleaving peptide, and a fluorescent protein.

8 . The system of claim 1 , wherein the gRNA sequence targets an INPPLI mRNA transcript or a SPSBI mRNA transcript.

9 . The system of claim 8 , wherein the gRNA sequence is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 73, and SEQ ID NO: 77.

10 . The system of claim 1 , wherein the gRNA sequence targets a DUSP4 mRNA transcript.

11 . The system of claim 10 , wherein the gRNA sequence is selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84.

12 . A method of modifying the length of a 3′ UTR of an mRNA transcript, the method comprising:

introducing into a cancer cell the system of claim 1 , wherein the system causes modification of the length of the 3′ UTR of the mRNA relative to a cancer cell that has not been transformed with the system of claim 1 .

13 . The method of claim 12 , wherein the modification of the 3′ UTR causes the 3′ UTR to increase, decrease, or stay about the same length.

14 . The method of claim 12 , wherein the modification of the 3′ UTR prevents the progression of a prostate cancer cell to an advanced androgen-dependent prostate cancer (ADPC) cell, castration-resistant prostate cancer (CRPC) cell, or a neuroendocrine prostate cancer (NEPC) cell.

15 . The method of claim 12 , wherein the system prevents PAS cleavage or polyadenylation.

16 . The method of claim 12 , wherein the cancer cell is a primary prostate cancer cell, an ADPC cell, a CRPC cell, or a NEPC cell.

17 . A method of treating or preventing the progression of prostate cancer, the method comprising administering to a subject a therapeutically effective amount of the system of claim 1 .

18 . The method of claim 17 , wherein the prostate cancer is CRPC or NEPC.

19 . The method of claim 17 , wherein the method further comprises improving sensitivity to an androgen receptor (AR) inhibitor.

20 . The method of claim 19 , wherein the AR inhibitor is enzalutamide, bicalutamide, abiraterone acetate, darolutamide or apalutamide.

21 . The method of claim 17 , wherein the system of claim 1 is contained in a lipid nanoparticle.

22 . The method of claim 17 , wherein the lipid nanoparticle consists of DLin-KC2-DMA, DOPE, Cholesterol, and PEG-DMG.

Assignments (4)

Continuity (2)

References Cited (89)