Massachusetts Chapter of the American College of Surgeons

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Fetal Liver-directed Gene Editing Results in Supratherapeutic Enzyme Levels in a Lysosomal Storage Disease
Sourav K Bose1,2, Apeksha Dave1, Rohan Palanki1, Brandon M White1, Pallavi Menon1, William H Peranteau1.
1Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, PA; 2Department of Surgery, Brigham and Women's Hospital, Boston, MA

In Mucopolysaccharidosis I, neuroinflammation results in hydrocephalus, vision loss, and developmental delay. Systemic gene therapeutic strategies are limited by the blood brain barrier (BBB). We hypothesize that 1. fetal therapy may facilitate efficient homology-directed repair (HDR) and 2. targeting the hepatic albumin locus may result in supratherapeutic enzyme production that could correct both systemic and neuropathology.

We delivered Cas9/gRNA targeting the albumin locus, and homology template encoding GFP in embryonic day 13.5 fetuses or postnatal day 0 (P0) neonates. Constructs were delivered either via adeno-associated virus serotype 8 (AAV8) or nanoparticle. Expression was assessed via flow cytometry at 2 weeks. In a preliminary experiment, Cas9/gRNA and homology template encoding human IDUA was delivered to IDUA W392X neonates with AAV8 and IDUA expression and genome modification were assessed at 2 weeks.

Neonates and fetuses showed similar rates of insertions and deletions (indels) ~5% but for similar modification rates, fetuses demonstrated significantly higher GFP expression (~3% vs 1%, p = 0.0044). Nanoparticle-mediated editing in fetuses was less efficient than AAV (~0.2% indels). In neonates injected with the IDUA HDR system, indel rate was ~5%, liver IDUA enzyme activity was ~240% normal, and brain activity was ~6.8% of normal.

Fetal Cas9-mediated homology-directed repair is more efficient than postnatal repair. Targeting the albumin locus led to supratherapeutic liver enzyme expression and above-threshold brain enzyme expression. This proof-of concept is promising with respect to the potential of a systemic therapeutic approach for multiorgan disease correction.

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