Site-specific integration of transgenes into human cells

• US 9,932,607 B2
• Filed: 11/12/2014
• Issued: 04/03/2018
• Est. Priority Date: 11/15/2013
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Calos, Michele Pamela, Tsai, Ruby Yanru, Zhu, Fangfang, Gamboa, Matthew, Farruggio, Alfonso P., Hippenmeyer, Simon, Tasic, Bosiljka, Schle, Birgitt
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/539,909
• Publication Number:
  US 20150140665A1
• Time in Patent Office: 1,238 Days
• Field of Search: None
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Methods for inserting a polynucleotide sequence into the genome of a human cell are provided. The present methods result in insertion of a polynucleotide sequence of interest into the H11 locus in the genome of a human cell. Also provided are nucleic acids that include sequences for integrating a polynucleotide sequence of interest into the H11 locus in the genome of a human cell. A transgenic human cell including site specific recombination sites at the H11 locus is also disclosed.

Claims

1. A method of inserting a polynucleotide sequence into a genome of a human pluripotent stem cell, comprising:

simultaneously introducing into the human pluripotent stem cell;

a circular nucleic acid comprising the polynucleotide sequence flanked by a phiC31 first recombination site and a Bxb1 first recombination site;

a phiC31 integrase; and

a Bxb1 integrase,

wherein the human pluripotent stem cell comprises a phiC31 second recombination site and a Bxb1 second recombination site at H11 locus in chromosome 22;

maintaining the human pluripotent stem cell under conditions that facilitate simultaneous recombination between phiC31 first recombination and phiC31 second recombination sites and between the Bxb1 first recombination and Bxb1 second recombination sites,

wherein the introducing and maintaining results in insertion of the polynucleotide sequence into the genome of the human pluripotent stem cell at the H11 locus an expression of a product of the polynucleotide sequence.

2. The method of claim 1, wherein the method comprises:

inserting the phiC31 second recombination site and the Bxb1 second recombination site at the H11 locus prior to introducing the circular nucleic acid, and the phiC31 and Bxb1 integrases, wherein the inserting comprises;

introducing into the human cell, a circular nucleic acid comprising the phiC31 second recombination site and the Bxb1 second recombination site flanked by a first sequence homologous to a first region of the H11 locus and a second sequence homologous to a second region of the H11 locus; and

maintaining the human cell under conditions that facilitate recombination between the first sequence and the first region and between the second sequence and the second region,

wherein the introducing and maintaining results in insertion of the phiC31 second recombination site and the Bxb1 second recombination site at the H11 locus.

3. The method of claim 1, wherein the phiC31 first recombination site is attB and the phiC31 second recombination site is attP.

4. The method of claim 1, wherein the phiC31 first recombination site is attP and the phiC31 second recombination site is attB.

5. The method of claim 1, wherein the Bxb1 first recombination site is attB and the Bxb1second recombination site is attP.

6. The method of claim 1, wherein the Bxb1 first recombination site is attP and the Bxb1second recombination site is attB.

7. The method of claim 1, wherein said phiC31 and Bxb1 integrases are introduced into the human cell by introducing a nucleic acid encoding the phiC31 and Bxb1 integrases into the cell.

8. The method of claim 1, wherein said phiC31 and Bxb1 integrases are introduced into the human cell by introducing a first nucleic acid encoding the phiC31 integrase and a second nucleic acid encoding the Bxb1 integrase.

9. The method of claim 7, wherein the nucleic acid is an mRNA.

10. The method of claim 7, wherein said nucleic acid is a circular DNA.

11. The method of claim 8, wherein the first and second nucleic acids are mRNA.

12. The method of claim 8, wherein the first and second nucleic acids are circular DNA.

13. The method of claim 1, wherein the polynucleotide encodes a polypeptide.

14. The method of claim 13, wherein the polypeptide is a transcription factor.

15. The method of claim 1, wherein the PS cell is an embryonic stem (ES) cell.

16. The method of claim 1, wherein the PS cell is an induced pluripotent stem (iPS) cell.

17. The method of claim 1, wherein the H11 locus is 4500-bp downstream of DRG1 and 683-bp downstream of EIF4ENIF1.