Methods and compositions for increasing transgene activity

• US 10,179,918 B2
• Filed: 04/28/2016
• Issued: 01/15/2019
• Est. Priority Date: 05/07/2015
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Cost, Gregory J.
• Primary Examiner(s): Li, Qian Janice
• Application Number:
  US15/141,333
• Publication Number:
  US 20160326548A1
• Time in Patent Office: 992 Days
• Field of Search: None
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/16045 Special targeting system for viral vectors
• CPC Class Codes: C12N 2830/48 regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Abstract

Methods and compositions for increasing transgene expression and/or activity, including for increasing nuclease-mediated genomic modifications.

Claims

1. A method of increasing nuclease activity in a cell, the method comprising:

introducing an mRNA comprising a 3′

untranslated region (3′

UTR) and encoding at least one component of one or more nucleases into the cell, wherein the mRNA comprises a woodchuck hepatitis virus posttranslational regulatory element (WPRE) as shown in any of SEQ ID NOs;

1-3 in the 3′

UTR.

2. The method of claim 1, wherein the WPRE is a wild-type WPRE.

3. The method of claim 1, wherein the nuclease is a zinc finger nuclease, a TALEN and/or a CRISPR/Cas and/or a Cfp1 CRISPR/Cas nuclease system.

4. The method of claim 1, further comprising subject the cells to cold-shock conditions.

5. An isolated mRNA comprising a WPRE sequence as shown in any of SEQ ID NO:

1-3 in the 3′

UTR and a sequence encoding at least one component of a nuclease or a transcription factor (TF).

6. The mRNA of claim 5, wherein the sequence encodes a nuclease.

7. The mRNA of claim 5, wherein the nuclease is a zinc finger nuclease, a TALEN and/or at least one component of a CRISPR/Cas nuclease system.

8. The mRNA of claim 5, wherein the Cas protein is a Cas and/or Cfp1 protein.

9. The mRNA of claim 5, wherein the sequence encodes a transcription factor.

10. The mRNA of claim 9, wherein the transcription factor is a ZF-TF, a TALE-TF or a CRISPR/Cas TF.

11. The mRNA of claim 5, further comprising a polyA sequence.