Nanoparticle delivery of CRISPR/Cas9 gene therapy for Wilson's disease

• Online Access: http://hdl.handle.net/2047/D20356168

CRISPR/Cas9 is being investigated by the larger medical and research community to cure genetic disease that place large burden on patients. This new method of Gene Editing has been investigated for addressing unmet medical need in patients with genetic disease. Wilson's Disease (WD) is a genetic disease that has been identified with over 500 mutations in the ATP7B gene; the most prolific has been identified as H1069Q. This point mutation interrupts the regulation and hydrolysis of ATP in hepatocytes who are essential for the export of copper into bile. Mis-sense and non-sense mutations found in WD make it attractive for gene therapy. The ATP7B P1-ATPase plays a crucial role in copper homeostasis and is imperative for the excretion of metabolic copper. WD is known to have a number of mutations found in the ATP7B gene; if untreated, disease progresses with copper accumulation in organs, eventual liver transplantation, psychiatric manifestations and in some cases death. The current standard of care for patients relies on chelation therapy to bind free copper and facilitate it's excretion from circulation. Previous ATP7B gene therapy has included: implantation of Lentivirus (LV)-engineered hepatocytes, in utero gestational LV-gene transfer of ATP7B, and Adeno-associated virus (AAV)-ATP7B gene introduction. Animal studies show improved copper homeostasis with transgene expression declining rapidly, thought to be due to episomal expression rather than stable integration into the genome. Work with CRISPR/Cas9 gene editing is more stable than these traditional gene therapies. We hypothesize that gene correction of a WD-like mutation in Toxic Milk (TxJ) mice with CRISPR/Cas9 could improve copper homeostasis. To test our hypothesis, we identified loci for either therapeutic gene delivery or gene correction in vivo. Our hyaluronic acid-based nanoparticle will contain the programmable nuclease Cas9/sgRNA plasmid as well as a DNA or oligo payload for gene knock-in or gene correction. TxJ mice harbor a WD-like mutation that interrupts copper homeostasis and was used as our predictive model. The biomarker Ceruloplasmin responsible for copper transport in circulation was used to evaluate the efficacy and potential therapeutic outcome from this CRISPR/Cas9 gene therapy.--Author's abstract