Polypeptide variants of sleeping beauty transposase

Inventors: Zsuzsanna Izsvak (Berlin, DE); Zoltan Ivics (Berlin, DE); Lajos Mates (Berlin, DE); Namitha Manoj (Louisville, CO); Carmen-Anisia Judis (Berlin, DE); Andrea Katzer (Wandlitz, DE)

Assignee: Max-Delbruck-Centrum Fur Molekulare Medizin

C12N9/22 C12N15/90 A61K48/00 C12N2800/90

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Quick Facts

Patent No.: US 9,228,180
App. No.: 12/667,527
Granted: Jan 5, 2016
Kind: B2

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Abstract

The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and/or RSDs). Furthermore, applications of these transposase variants, the transpsoson, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

Claims (124)

1. An isolated sleeping beauty 10 (SB10) polypeptide variant of SEQ ID NO: 1, wherein said variant has transpositional activity at least twice the transpositional activity of SEQ ID NO: 1 wherein the amino acid sequence of said SB10 polypeptide variant differs from SEQ ID NO:1 by 1 to 20 amino acids and wherein one mutation is K14R.

2. The isolated SB10 polypeptide of claim 1 , wherein the variant comprises 2 to 20 mutations.

3. The isolated SB10 polypeptide of claim 1 , wherein the SB10 polypeptide variant is a combination of mutations selected from the group consisting of:

Variant 1: K14R//R214D/K215A/E216V/N217Q;

Variant 3: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;

Variant 7: K14R/T83A/M243Q;

Variant 8: K14R/T83A/II00L/M243Q;

Variant 9: K14R/T83A/R143L/M243Q;

Variant 10: K14R/T83A/R147E/M243Q;

Variant 11: K14R/T83A/M243Q/E267D;

Variant 12: K14R/T83A/M243Q/T314N;

Variant 13: K14R/K30R/I100L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;

Variant 14: K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;

Variant 15: K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H;

Variant 16: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;

Variant 17: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;

Variant 18: K14R/K30R//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;

Variant 19: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H;

Variant 20: K14R/K30R/R147E//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;

Variant 21: K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/E267D;

Variant 22: K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/T314N;

Variant 23: K14R/K30R/R143L//A205K/H207V/K208R/D210E//R214D/K215A/E216V/N217Q//M243H/G317E;

Variant 24: K14R/K33A/R115H/R143L//R214D/K215A/E216V/N217Q//M243H;

Variant 25: K14R/K33A/R115H/R147E//R214D/K215A/E216V/N217Q//M243H;

Variant 26: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/E267D;

Variant 27: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/T314N;

Variant 28: K14R/K33A/R115H//R214D/K215A/E216V/N217Q//M243H/G317E; and

Variant 29: K14R/T83A/M243Q/G317E.

4. The isolated SB10 polypeptide of claim 1 , wherein the variant further comprises mutations

R214D/K215A/E216V/N217Q.

5. The isolated SB10 polypeptide of claim 4 , wherein the variant is a combination of mutations selected from the group consisting of: