Development of Safe in Utero Gene Editing Technology in Mice

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 andgt;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 andgt;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 andgt;50% on-
target editing and andlt;5% off-target editing with AAV6 targeting hematopoietic stem cells and AAV9 targeting
neurons.Narrative
Combining precision fetal surgery with controlled gene editing will enable in utero correction of severe genetic
disorders. Acrigen Biosciences, Inc and the MacKenzie group at UCSF are developing technology to control
CRISPR gene editing and deliver precision genetic medicines directly to the developing fetus, enabling safe
and effective therapies for neonatal genetic diseases.