CRISPR/Cas9 Genome Editing to Repair Receptor‐Mediated Endocytosis in Homozygous Familial Hypercholesterolemia Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSC) derived from patients carrying pathological genetic mutations have become a major focus in prospective cell therapy and regenerative medicine because of their immunological compatibility and limitless supply. iPSC, in sequence with latest genomic editing technology, CRISPR/Cas9, are tools to potentially treat genetic diseases. Using familial hypercholesterolemia (FH) as a model, our goal is to use patient‐specific iPSC (FH‐iPSC) in combination with CRISPR/Cas9 genome editing to permanently restore low‐density lipoprotein receptor (LDLR) mediated LDL endocytosis. Skin fibroblasts from a homozygous FH patient (Coriell Cell Repositories; #GM03040) were reprogrammed into iPSC (3040‐iPSC) using transient, synthetic modified RNA ( Oct4, Sox2, Klf4, Lin28, cMyc ). LDLR genotyping found a homozygous 3‐nucleotide deletion (c.654_656delTGG) in exon 4 of chromosome 19, thereby identifying the mutation as Class II defective transport showing less than 5% normal receptor activity of binding, internalization, and degradation of LDL. With this mutation, immature LDLR (120kDa) is misfolded and not transported out of the endoplasmic reticulum for further processing to the mature protein form (160kDa). The defective protein is degraded by an MG132 proteasome inhibitor dependent pathway. After identifying the homozygous 3bp deletion, we utilized online CRISPR/Cas9 guide design software (e.g. CRISPR Design Tool) to identify potential Cas9 guides (sgRNA). To reduce potential off‐target mutations, we elected to utilize the D10A nickase Cas9 (Cas9n) with offset sgRNA pairs for homology‐directed repair (HDR). We designed a repair template of single‐strand oligodeoxynucleotides (ssODN) that included the missing 3bp. The ssODN also introduced a novel restriction site (XmnI) as well as silent mutations to minimize Cas9n re‐binding and cleavage post‐repair. We transfected 293T/17 cells with CRISPR/Cas9 using lipofectamine to verify the cleavage efficiency of our CRISPR design. FACs analysis determined we had a 52% transfection efficiency. We have also transfected 3040‐iPSCs with CRISPR/Cas9 system via electroporation with the Nepa21. After going through several parameters, we identified that 150 V for 5 msec yielded the highest transfection efficiency of 41%. We are currently selecting positive clones through double antibiotic selection with puromycin (0.2 μg/mL) and hygromycin (10 μg/mL). Clonal colonies will be screened for correction efficiency using restriction fragment length polymorphism (RFLP), mismatch mutation detection (Surveyor Nuclease) and Sanger sequencing analysis to determine heterozygous/homozygous insertion of the missing 3bp and examine for off‐target mutations. After verification of a corrected LDLR gene, we will investigate restoration of normalized LDLR mediated endocytosis in hepatocytes derived from the 3040‐iPSC compared to non‐corrected controls. This work will demonstrate that CRISPR/Cas9 can be utilized to correct the genetic mutation in cells of patients with FH to provide functionally normal iPSCs that could be further differentiated into hepatocytes and delivered as therapeutic cells. Support or Funding Information NIH 1R21EB022185‐01; Jewish Heritage Fund for Excellence