US 11,873,479 B2
Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption
Andrew M. Scharenberg, Seattle, WA (US); Michael T. Certo, Seattle, WA (US); and Kamila Sabina Gwiazda, Seattle, WA (US)
Assigned to SEATTLE CHILDREN'S RESEARCH INSTITUTE, Seattle, WA (US)
Filed by SEATTLE CHILDREN'S HOSPITAL, Seattle, WA (US)
Filed on Apr. 29, 2021, as Appl. No. 17/244,190.
Application 14/949,744 is a division of application No. 14/173,705, filed on Feb. 5, 2014, abandoned.
Application 14/173,705 is a division of application No. 13/405,183, filed on Feb. 24, 2012, granted, now 8,673,557, issued on Mar. 18, 2014.
Application 17/244,190 is a continuation of application No. 15/215,405, filed on Jul. 20, 2016, granted, now 11,008,565.
Application 15/215,405 is a continuation of application No. 14/949,744, filed on Nov. 23, 2015, granted, now 10,995,332.
Claims priority of provisional application 61/447,672, filed on Feb. 28, 2011.
Prior Publication US 2021/0261946 A1, Aug. 26, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); C12N 9/22 (2006.01); C12N 15/62 (2006.01); A61K 38/45 (2006.01); A61K 38/46 (2006.01); A61K 38/52 (2006.01); C12N 9/12 (2006.01); C12N 9/90 (2006.01); A61K 9/00 (2006.01); A61K 35/28 (2015.01); A61K 35/12 (2015.01)
CPC C12N 15/102 (2013.01) [A61K 9/0019 (2013.01); A61K 35/28 (2013.01); A61K 38/45 (2013.01); A61K 38/465 (2013.01); A61K 38/52 (2013.01); C12N 9/1252 (2013.01); C12N 9/22 (2013.01); C12N 9/90 (2013.01); C12N 15/62 (2013.01); A61K 2035/124 (2013.01); C07K 2319/60 (2013.01); C12N 2800/80 (2013.01); C12N 2840/203 (2013.01)] 16 Claims
 
1. A polynucleotide encoding a fusion polypeptide, wherein said polypeptide comprises a homing endonuclease that (i) binds and cleaves a selected dsDNA target site in a mammalian cell, (ii) is selected from the group consisting of: I-LtrI, I-GpiI, I-GzeI, I-MpeMI, I-PanMI, I-CreI, I-OnuI, and I-HjeMI, and (iii) is linked by a linker domain to Trex2 or a biologically active fragment thereof, wherein said linker comprises from about 4 to 30 amino acids and is flexible such that said homing endonuclease and said Trex2 or biologically active fragment thereof retain their respective biological activities.