Materials and methods for modulating expression

Abstract

The present disclosure relates to methods of modulating transcription of target genes using transcription modulators termed RNA-based transcription modulators or "RPMs". The RPMs attract transcription regulators to the genes.

Description

This application relates to a method for regulating the transcription of target genes using transcriptional regulators known as RNA-based transcriptional regulators, or "RPMs." The RPMs recruit transcriptional regulatory factors to the genes. Abstract

Claims

ClaimsWe claim:

1. An RNA-based promoter modulator (RPM) comprising:(a) a regulatory element tail,(b) a binding sequence, and(c) an Sm OPT consensus sequence and hairpin.

2. The RPM of claim 1 wherein the orientation of (a), (b) and (c) in the RPM is 5' to’ 3' and wherein the (b) binding sequence binds the sense strand of a target gene promoter.

3. An SMN2 RPM comprising:(a) Regulatory Element A Tail encoded by ACCCTTCTCCGGCCGCTGAC (SEQ ID NO: 1 ) or Regulatory Element B Tail encoded by TCCGCTCTGGGGCGCGCACACCCTCGCCCGCACTTCTCCCCTCGTCCGGCACGGACT GACCGCACACGAATAACACATCCGACCAGAGCT (SEQ ID NO: 2), and(b) Binding Sequence 1 encoded by AGACGGGGTTTCGGCATGTT (SEQ ID NO: 3), Binding Sequence 2 encoded by ATTGTGTAGGCTGGTCTGA (SEQ ID NO: 4), or Binding Sequence 3 encoded by GGGGCAGGAAGGAAGGCAGA (SEQ ID NO: 5).

4. A TCF4 RPM comprising:(a) Regulatory Element A Tail encoded by ACCCTTCTCCGGCCGCTGAC (SEQ ID NO: 1 ) or Regulatory Element B Tail encoded by TCCGCTCTGGGGCGCGCACACCCTCGCCCGCACTTCTCCCCTCGTCCGGCACGGACT GACCGCACACGAATAACACATCCGACCAGAGCT (SEQ ID NO: 2), and(b) Binding Sequence 1 encoded by GTGGTAAACAGAGCGCCTAG (SEQ ID NO: 6).

5. A nucleic acid encoding an RPM, the nucleic acid comprising(a) a DNA encoding(i) an RPM of claim 1 or 2,(ii) an SMN2 RPM of claim 3, or(iii) a TCF4 RPM of claim 4;(b) a DNA construct of Figure 7A or 7B; or(c) a DNA at least 90% identical to the DNA of (a) or DNA construct of (b).

6. The nucleic acid of claim 5(a) further comprising a promoter driving expression of the RPM.

7. The nucleic acid of claim 6, wherein the promoter is any of a 117 promoter, 116 promoter, a tRNA promoter, a H1 promoter, a minimal CMV promoter, a T7 promoter, an EF1 -alpha promoter, a Minimal EF1 -alpha promoter.

8. A recombinant adeno-associated virus (rAAV) comprising the nucleic acid of any one of claims 5-7.

9. The rAAV of claim 8, wherein the AAV is a single-stranded AAV (ssAAV) or a self- complementary AAV (scAAV).

10. The rAAV of any one of claims 8-9, wherein the rAAV is rAAV1 , rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVI O, rAAV11 , rAAV12, rAAV13, rAAV- anc80, rAAV rh.74, rAAV rh.8, rAAVrh.10, AAV-B1 , MyoAAV, MYOAAV1A, SLB101 , M1 and M3, or their derivatives.11 . The rAAV of any one of claims 8-10, wherein the rAAV is rAAV9.

12. A composition comprising(a) the nucleic acid of any one of claims 5-7, or(b) the rAAV of any one of claims 8-11 , and a pharmaceutically acceptable carrier.

13. A method of modulating expression of a target gene in a cell comprising delivering an RPM of any of claims 1 -4 to the cell.

14. A method of modulating expression of a target gene in a cell comprising contacting the cell with:(a) the nucleic acid of any one of claims 5-7,(b) the rAAV of any one of claims 8-11 , or(c) the composition of claim 12.

15. The method of claim 14 wherein the expression of the target gene increases.

16. The(a) nucleic acid of any one of claims 5-7,(b) adeno-associated virus (AAV) of any one of claims 8-11 , or(c) composition of claim 12, wherein the nucleic acid, AAV or composition is formulated for intramuscular injection, intrathecal injection, transdermal transport or injection into the blood stream.

17. A method of treating spinal muscular atrophy in a subject comprising delivering a SMN2 RPM of claim 3 to the subject.

18. A method of treating Pitt Hopkins Syndrome in a subject comprising delivering a TCF4 RPM of claim 4 to the subject.

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