In vivo and ex vivo correction of pathogenic gene mutations using CRISPR-based genome editing

Abstract

Over the last few years, CRISPR (clustered regularly interspaced short palindromic repeats) system derived from an adaptive immune system of bacteria and archaea has been widely used for genome editing. Also, many CRISPR-associated editors have been developed and become promising tools to rescue genetic diseases. In this dissertation, I applied the CRISPR system to correct pathogenic mutations in various disease models. First, we tried to rescue adrenoleukodystrophy (ALD) using in vivo gene therapy. We performed in vivo homology-independent targeted integration (HITI)-mediated gene editing using AAV9 vectors delivered via intravenous administration in the ALD model mice. We found that the ABCD1 mRNA level was significantly increased in HITI-treated mice, and the plasma levels of C24:0-LysoPC (lysophosphatidylcholine) and C26:0-LysoPC, sensitive diagnostic markers for ALD, were significantly reduced. Next, we targeted hereditary tyrosinemia type 1 (HT1) and recessive dystrophic epidermolysis bullosa (RDEB) using ex vivo gene therapy. To rescue HT1, we reprogrammed hepatocytes from a mouse model of HT1 into expandable chemically derived hepatic progenitors (CdHs) and corrected the disease-causing mutation using both adenine base editors (ABEs) and prime editors (PEs). ABE- and PE-corrected CdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased survival of mutant HT1 mice. To rescue RDEB, we applied ABEs to correct the pathogenic mutation or to bypass a premature stop codon in patient-derived fibroblasts. Furthermore, we utilized PEs to expand the targeting scope. Ultimately, we found that the transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice led to C7 deposition within the dermal-epidermal junction. These results show the versatility of CRISPR-mediated gene editing and demonstrate the feasibility of the CRISPR system as a promising therapeutic strategy.