Alternative export pathways for vector expressed rna interference

Patent metadata

Field	Value
Publication number	US-2016348106-A1
Application number	US-201615165898-A
Country	US
Kind code	A1
Filing date	May 26, 2016
Priority date	Nov 29, 2006
Publication date	Dec 1, 2016
Grant date	—

Abstract

The present invention is directed to nucleic acid molecules containing a loop sequence designed to circumvent exportin-5 mediated export, and methods using these novel molecules.

First claim

What is claimed is:

1 . An isolated nucleic acid molecule comprising
a first portion, wherein the first portion is no more than 30 nucleotides in length; a second portion, wherein the second portion has a sequence that is complementary to the first portion; and a loop portion comprising a sequence designed to circumvent exportin-5 mediated export; wherein the first portion and the second portion are operably linked by means of the loop portion to form a hairpin structure comprising a duplex structure and a loop structure.

2 . The nucleic acid molecule of claim 1 , wherein the loop portion is about 12 to 50 nucleotides long.

3 . The nucleic acid molecule of claim 1 , wherein the loop portion is about 20 to 40 nucleotides long.

4 . The nucleic acid molecule of claim 1 , wherein the loop portion is about 25 to 35 nucleotides long.

5 . The nucleic acid molecule of claim 1 , wherein the loop portion is a 31 or 32 nucleotide L1 motif.

6 . The nucleic acid molecule of claim 1 , wherein the loop portion comprises between 12 and 32 nucleotides of SEQ ID NO:1.

7 . The nucleic acid molecule of claim 1 , wherein the loop portion comprises between 12 and 32 contiguous nucleotides of SEQ ID NO:1.

8 . The nucleic acid molecule of claim 1 , wherein the loop portion consists of SEQ ID NO:5.

9 . The nucleic acid molecule claim 1 , wherein the duplex is less than 30 nucleotides in length.

10 . The nucleic acid molecule claim 1 , wherein the duplex contains from 19 to 25 nucleotides.

11 . The nucleic acid molecule claim 1 , wherein the molecule is a short hairpin RNA (shRNA).

12 . The nucleic acid molecule claim 1 , wherein the molecule is a microRNA (miRNA).

13 . An expression cassette comprising a sequence encoding a nucleic acid molecule comprising:
a promoter; a first portion, wherein the first portion is no more than 30 nucleotides in length; a second portion, wherein the second portion has a sequence that is complementary to the first portion; and a loop portion comprising a sequence designed to circumvent exportin-5 mediated export; wherein the first portion and the second portion are operably linked by means of the loop portion to form a hairpin structure comprising a duplex structure and a loop structure.

14 . A vector comprising the expression cassette of claim 13 .

15 . A cell comprising the nucleic acid of claim 1 .

16 . A cell comprising the expression cassette of claim 13 .

17 . A cell comprising the vector of claim 14 .

18 . A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the nucleic acid of claim 1 .

19 . A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the expression cassette of claim 13 .

20 . A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the vector of claim 14 .

Assignees

• Univ Iowa Res Found

Inventors

• Harper Scott
• Davidson Beverly L

Classifications

• C12N2320/50

  Methods for regulating/modulating their activity · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title