# US 2014/0194322 A1 · Methods for Creating and Identifying Functional RNA Interference Elements

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- Number: US 2014/0194322 A1
- Document ID: US20140194322A1
- Status: Published application
- Kind: A1
- Application number: 14/102,440
- Filed: 2013-12-10
- Published: 2014-07-10
- Earliest claimed: 2007-12-24
- Assignees: Bergenbio AS
- Applicants: BerGenBio AS
- Inventors: DAVID MICKLEM; JAMES LORENS
- Web page: https://patentgrep.com/patent/US20140194322A1

## CPC

- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12Q 1/6897: involving reporter genes operably linked to promoters
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2320/10: in screening processes
- C12N 2330/30: chemically synthesised
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention relates to the control of gene expression. Specifically, the invention provides compositions and methods for the production and use of recombinant nucleic acid molecules that have the ability to specifically downregulate an expressed target gene in vivo. In some aspects, the invention provides methods for producing a hairpin DNA molecule where part of the molecule is derived from an mRNA that is a target for a small interfering RNA (siRNA) derived from the hairpin. In other aspects, the invention provides synthetic hairpin adapter oligonucleotides that are used in the construction of siRNA-producing cassettes. In other aspects, the invention provides methods for testing for the presence or absence of specific inhibitory activity of an RNAi trigger molecule, and in still other aspects, the invention provides methods for identifying an active RNAi trigger molecule from a library of RNAi trigger molecules. In still other aspects, the invention provides methods for identifying a polynucleotide from a plurality of candidate target polynucleotides that is specifically targeted by an RNAi trigger molecule. In other aspects, the invention provides epi-allelic series of hypomorphic RNAi trigger molecules specific for any gene of interest, where the series of RNAi trigger molecules have a variety of uses including analysis of gene function and drug target development.

## Claims

### Claim at position 1

-33. (canceled)

### Claim 34

A method of testing for the presence or absence of specific inhibitory activity of an RNAi trigger molecule for a polynucleotide target, the method comprising:
(a) providing a cell comprising:
i) a reporter construct comprising a reporter transcription unit, the reporter transcription unit comprising (A) a translatable reporter gene polynucleotide encoding a reporter polypeptide, and (B) a nontranslatable target polynucleotide or a portion thereof, the target polynucleotide positioned 3′ to the reporter gene polynucleotide; and
ii) a vector encoding an RNAi trigger molecule;
(b) culturing said first cell under conditions that permit expression of the reporter transcription unit and the RNAi trigger molecule;
(c) detecting a signal associated with the cell, said signal corresponding to a level of expression of the reporter polypeptide in the cell; and
(d) correlating the detected signal with specific inhibitory activity of the RNAi trigger molecule for the target poynucleotide, thereby testing for the presence or absence the specific inhibitory activity of the RNAi trigger molecule for the polynucleotide target.

### Claim 35 (depends on 34)

The method of claim 34, wherein the target polynucleotide is (i) an mRNA, (ii) a cDNA encoding the mRNA, or (iii) a portion of (i) or (ii).

### Claim 36 (depends on 34)

The method of claim 34, wherein the RNAi trigger molecule is selected from a double-stranded RNA, a short hairpin RNA (shRNA), a micro RNA (miRNA), a shmiRNA and a small interfering RNA (siRNA).

### Claim 37 (depends on 34)

The method of claim 34, wherein the reporter gene polynucleotide encodes a fusion reporter polypeptide.

### Claim 38 (depends on 34)

The method of claim 34, wherein the reporter gene polynucleotide further encodes a PEST motif, and said reporter polypeptide is a fusion polypeptide comprising the PEST motif.

### Claim 39 (depends on 34)

The method of claim 34, wherein detecting comprises detecting with single-cell resolution.

### Claim 40 (depends on 34)

The method of claim 34, wherein the reporter transcription unit is operably linked to a transcription promoter.

### Claim 41 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a fluorescent reporter polypeptide.

### Claim 42 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a fluorescent reporter polypeptide selected from a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein or a blue fluorescent protein.

### Claim 43 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a cell surface marker polypeptide.

### Claim 44 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a Lyt2 cell surface marker polypeptide.

### Claim 45 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a negative selection marker.

### Claim 46 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a negative selection marker selected from thymidine kinase (tk), HRPT and APRT.

### Claim 47 (depends on 34)

The method of claim 34, wherein the reporter gene encodes a reporter polypeptide selected from a luciferase polypeptide, a β-galactosidase, α-lactamase, an alkaline phosphatase and a horse-radish peroxidase.

### Claim 48 (depends on 34)

The method of claim 34, wherein the vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, a eukaryotic expression vector.

### Claim 49 (depends on 34)

The method of claim 34, wherein detecting a signal associated with each cell comprises detecting fluorescence by fluorescence activated cell sorting (FACS).

### Claim 50 (depends on 34)

The method of claim 34, wherein detecting a signal associated with each cell comprises detecting a magnetic tag by magnetic flow cell sorting.

### Claim at position 19

-56. (canceled)