Targeting the Apoa1 locus for liver-directed gene therapy

Clinical application of somatic genome editing requires therapeutics

that are generalizable to a broad range of patients. Targeted

insertion of promoterless transgenes can ensure that edits

are permanent and broadly applicable while minimizing risks of

off-target integration. In the liver, the Albumin (Alb) locus is

currently the only well-characterized site for promoterless transgene

insertion. Here, we target the Apoa1 locus with adeno-associated

viral (AAV) delivery of CRISPR-Cas9 and achieve rates of

6% to 16% of targeted hepatocytes, with no evidence of toxicity.

We further show that the endogenous Apoa1 promoter can drive

robust and sustained expression of therapeutic proteins, such as

apolipoprotein E (APOE), dramatically reducing plasma lipids

in a model of hypercholesterolemia. Finally, we demonstrate

that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can

correct and rescue the severe metabolic liver disease hereditary

tyrosinemia type I. In summary, we identify and validate

Apoa1 as a novel integration site that supports durable transgene

expression in the liver for gene therapy applications.