The Hemophilia work as been updated in adult mice and the percentage of correction is up to 30%! This is an amazing result and a big improvement from the neonatal mice. Hopefully we are getting close to the IND.
 Correction of Hemophilia B Phenotype Following ZFN Mediated Genome Editing in Adult Mice
Rajiv Sharma, Xavier M. Anguela, Yannick Doyon, Sunnie Y. Wong, Hojun Li, Virginia Haurigot, Anand Bhagwat, Robert J. Davidson, Shangzhen Zhou, Philip D. Gregory, Michael C. Holmes, Katherine A. High. Hematology, Children's Hospital of Philadelphia, Philadelphia, PA; Sangamo Biosciences Inc., Richmond, CA; Howard Hughes Medical Institute, The Children's Hospital of Philadelphia, Philadelphia, PA
Genome editing can direct stable long-term expression of a transgene product while preserving endogenous regulation. We have recently reported that persistent and clinically meaningful levels of genome editing can be achieved through AAV-mediated delivery of zinc finger nucleases (ZFNs) and a donor template in neonatal mice (Li et al., Nature 2011). To expand the potential therapeutic applicability of this approach, we sought to investigate whether treatment of adult mice results in targeted genome modification. Tail vein injection of the AAV-ZFN and AAV-Donor, containing a promoterless wild type human factor IX (hF.IX) insert flanked by arms of homology to the target site, into adult (8 week old) mice resulted in stable (>30wk) mean circulating hF.IX levels of ∼30% of normal, a substantial increase over the levels we observed in neonatal mice (3-7% of normal), whereas control mice receiving ZFN alone or AAV-Luciferase + AAV-Donor exhibited only background levels of expression. Importantly, mice lacking the defective human factor IX gene averaged less than 100 ng/mL after receiving AAV-ZFN and AAV-Donor, consistent with hF.IX expression being derived from on-target genome editing. Liver regeneration following 2/3 partial hepatectomy, which is known to substantially reduce expression from non-integrated AAV genomes, had no significant impact on hF.IX expression compared to non-hepatectomized mice. Together, these data suggest that the hF.IX expression in ZFN + Donor treated mice is predominantly a result of stable correction at the intended genomic target site. PCR analysis of genomic DNA from livers of treated mice revealed evidence of targeted gene addition of the corrected donor template by both homology directed repair and non-homologous end-joining at the ZFN target locus. Finally, to assess whether these levels of genome editing are sufficient to normalize the prolonged clotting times characteristic of hemophilic mice, we treated HB mice containing a copy of the defective human F.IX locus. Mice receiving both the ZFN and Donor vectors expressed levels of hF.IX in the range of 30% of normal. Activated partial thromboplastin time, a measurement of clot formation, was corrected to wild-type levels, from a mean of 63 seconds before treatment to 39 seconds at 2 weeks post delivery. In summary, our data suggest that highly efficient genome editing by both homology dependent and independent mechanisms is achieved following AAV mediated ZFN and donor delivery in adult mice. Further, this ZFN-driven genome editing results in substantial levels of circulating hF.IX and a normalization of clotting times in a mouse model of Hemophilia B.
Keywords: Gene Correction/Modification; Zinc-finger nucleases; Hemophilia
Date: Thursday, May 17, 2012
Session Info: Simultaneous Oral Abstract Sessions: DNA Vector Development and Gene Targeting (1:15 PM-3:15 PM)
Presentation Time: 1:15 pm
Room: 201 C