C4 - Targeted Gene Insertion (integrase mediated insertion -targeted or safe harbor)
194: In Vivo Expansion of Gene-Targeted Hepatocytes through Transient Inhibition of an Essential Gene
Type: Oral Abstract Session
Presentation Details
Session Title: Targeted Gene Insertion
Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. For example, HDR requires cell division, generally limiting targeting of adult liver to ~1% of hepatocytes. The goal of this work is to develop a system for selective expansion of gene-targeted hepatocytes using essential genes, which we call “Repair Drive”. Repair Drive consists of: (1) transient conditioning of the liver by knocking down an essential gene - Fumarylacetoacetate hydrolase (Fah), which is important for tyrosine catabolism, and (2) delivery of an untargetable version of the essential gene in cis with the therapeutic transgene. To test this approach, we used AAV vectors to insert FAH in tandem with a fluorescent marker (TdTomato) into a highly expressed locus in the liver (Apoa1). Mice were injected with AAV8 vectors encoding CRISPR/Cas9 (AAV-CRISPR) and a Donor template (AAV-Donor) with FAH and a TdTomato transgene. Monthly injections of N-acetylgalactosamine (GalNac)-modified siRNA targeting murine Fah were used as a conditioning agent to deplete untargeted hepatocytes and drive expansion of correctly repaired cells. Three months later, mice receiving the conditioning siRNA - Repair Drive mice - showed a dramatic increase in the number of TdTomato positive hepatocytes, versus those receiving the viral vectors alone - Unselected mice (~10% vs 0.5%). Selective expansion could be further improved to >25% of hepatocytes by manipulation of tyrosine catabolism with a high protein diet. To evaluate the translatability and long-term safety of this approach, we inserted a human Factor IX (FIX) transgene into the Apoa1 locus. Following genome editing, the mice were injected monthly with the conditioning siRNA for three months, and then followed for 1 year. Repair Drive mice showed sustained FIX expression over 1 year with a ~5-fold increase as compared to Unselected mice. Liver conditioning caused only a transient elevation of ALT at early time points (4 to 8 weeks), which fully resolved over time. Histology revealed no evidence of liver toxicity and full reconstitution of Fah expression. Importantly, the expansion protocol did not increase the risk of tumorigenesis. SMRT-seq analysis at the Apoa1 locus revealed 20- to 50-fold increase of correct HDR-, 5’-HDR/3’-NHEJ- or partial integration of the Donor cassette in Repair Drive mice. NGS data showed a dramatic reduction in the relative frequency of off-target to on-target editing in Repair Drive mice. Overall, these data show a novel strategy for selectively expanding targeted hepatocytes for liver gene therapy.
Marco De Giorgi1, So Hyun Park2, Adam Castoreno3, Mingming Cao2, Ayrea Hurley1, Lavanya Saxena2, Marcel A. Chuecos1, Christopher J. Walkey1, Alexandria M. Doerfler1, Mia N. Furgurson1, M. Cecilia Ljungberg1, Kalyani R. Patel4, Sarah Hyde3, Tyler Chickering3, Stephanie Lefebvre3, Kelly Wassarman3, Patrick Miller3, June Qin3, Mark K. Schlegel3, Ivan Zlatev3, Rich Gang Li1, Jong Kim1, James F. Martin1, Karl-Dimiter Bissig5, Vasant Jadhav3, Gang Bao2, William R. Lagor1
1Baylor College of Medicine, Houston, TX,2Rice University, Houston, TX,3Alnylam Pharmaceuticals Inc, Cambridge, MA,4Texas Children's Hospital, Houston, TX,5Duke University, Durham, NC"
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