IN VIVO GENOME EDITING WITH ZINC FINGER PROTEINS
Gregory PD
Sangamo BioSciences Inc, Richmond, CA, USA
Editing of the human genome to correct disease-causing mutations provides a novel approach to the
treatment of monogenic disorders. This possibility is now being realized using a class of customizable,
sequence-specific endonucleases, called zinc finger nucleases (ZFNs), that can be designed to introduce
a discrete cleavage event at any user-selected location within the genome. By adjusting the conditions
under which this cleavage event is subsequently repaired, one may efficiently and precisely edit the
targeted locus to disrupt, repair or even site-specifically integrate a large, gene-sized DNA fragment.
This technology has permitted efficient ex vivo targeted gene addition to putative "safe harbor" loci in
primary human cells. To evaluate genome editing in vivo, we have selected hemophilia B as a model
monogenic disease. In this setting, we recently reported that the systemic co-administration of adenoassociated virus (AAV) vectors carrying the ZFNs and an appropriately designed gene targeting cassette
stimulated both homology-directed and homology-independent targeted gene addition at the ZFNspecified locus. Moreover, the levels of gene targeting achieved fully corrected the clotting defect in
these hemophilia B mice even following induced liver regeneration. In addition to correcting a diseased
locus, ZFNs could be used to insert the therapeutic transgene into a genomic “safe harbor.” For liverderived protein replacement, albumin represents an attractive target due to its robust and exclusive
expression from the liver. Indeed, transgene insertion into only a fraction of hepatocytes should yield
therapeutically relevant levels of protein expression. We show that ZFN-driven targeted gene addition
to the albumin locus can achieve expression of a variety of transgenes (including Factor 9, αGalactosidase A, β-Glucosidase, Iduronate-2 Sulfatase, and α-L-Iduronidase for the treatment of
Hemophilia B, Fabry, Gaucher, Hunter, and Hurler’s disease/syndromes, respectively). Thus, in vivo
genome editing via ZFN-mediated transgene insertion into the albumin locus could represent a general
strategy to express a wide variety of transgenes missing or mutated in monogenic disease.