Development of Safe in Utero Gene Editing Technology in Mice

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Abstract In the nearly 20 years since the groundbreaking sequencing of the human genome, the potential for precision genetic medicine has still not been realized. Advances in sequencing have vastly increased our ability to screen patients (including parents and fetuses) for genetic abnormalities, dramatically expanding identification of hereditary diseases in the prenatal period. This opens the door for fetal molecular therapies to address critical genetic conditions in utero, preventing preterm pregnancy termination, newborn death, or irreversible tissue damage resulting in life-long disabilities. The discovery of CRISPR systems and their remarkable ability to perform precision gene editing quickly showed possibilities for clinical applications to treat genetic disorders. However, clinical application of CRISPR is limited due to the introduction of mutations and DNA restructuring at unintended off-target sites within the genome, which can lead to toxicity and cancer. Nowhere is this deficiency more acute than in the application of CRISPR for in utero gene editing, where uncontrolled editing side effects could affect the patient for the entirety of their lives. Controlling CRISPR gene editing is critical to ensure the safety and efficacy of in utero gene therapies. Acrigen Biosciences is commercializing technology to bring safe in vivo CRISPR-based gene editing therapies to the clinic. Acrigen utilizes anti-CRISPR (Acr) proteins as robust inhibitors of Cas nuclease, providing an off-switch for gene editing and preventing off-target effects. The MacKenzie lab at UCSF specializes in fetal surgery and applying molecular therapies to correct neonatal genetic diseases. The combination of controlled CRISPR gene editing with precise in utero delivery will allow us to address previously untreatable genetic disorders, including hematopoietic disorders like alpha thalassemia and neurologic diseases such as neuropathic Gaucher disease. We propose to develop a safe and effective in utero CRISPR gene editing delivery system targeting hematopoietic stem cells and neurons in a fetal mouse model. This Phase I STTR project has three aims. Aim 1: Design SaCas9 guides targeting a mouse reporter gene. Milestone 1: Select guides showing >70% editing in reporter mouse cell line. Aim 2: Construct a single vector (Cas9-sgRNA-Acr) delivery system for controlled in utero editing. Milestone 2: Demonstrate maintained on-target activity and >90% reduction of off-target activity with an AAV compatible single vector system under Acr control. Aim 3: Demonstrate safe and effective editing of a reporter mouse model in utero. Milestone 3: Show >50% on- target editing and <5% off-target editing with AAV6 targeting hematopoietic stem cells and AAV9 targeting neurons.