iPSC and ESC gene editing service

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with an incidence rate of about one in 400-1000 people. About half of patients with this disease will develop end-stage renal disease, requiring dialysis or kidney transplantation. ADPKD is associated with two gene defects, of which 85% are caused by mutations in the gene PKD1 (trpp1) located on chromosome 16. Romano et al. generated isogenic iPSC with heterozygous knockout and homozygous knockout in PKD1 gene by using CRISPR/Cas9 technology. And verified that iPSC maintained stem cell like morphology, normal karyotype, pluripotency and differentiation ability in three germ layers. It can be used as a model for studying the pathogenesis of ADPKD and drug screening.

β-Thalassemia is a monogenic disease caused by point mutation or fragment deletion of the HBB gene leading to loss of normal β-globin peptide chain or insufficient synthesis of β-globin. The prevalence of β- thalassemia carriers in southern China is 2.54%, which has been threatening the lives of millions of people for decades. At present, hematopoietic stem cell transplantation is the only available radical treatment for patients with severe β-thalassemia. However, hematopoietic stem cell transplantation is limited by the lack of HLA-matched healthy donors in most patients. With the wide application of gene editing technology, researchers used CRISPR/Cas9 system to perform gene editing on patient-derived iPSCs (genotype: homozygous 41/42 deletion), mainly designed gRNA targeting HBB mutation position, and recombined PCR products containing normal sequences (WT) as donor templates, and then screen cells with mutation repaired genotype. The successfully corrected iPSCs were re-differentiated into hematopoietic stem cells (HSCs), and then the HSCs were transplanted to the irradiated NSI immunodeficient mice. Finally, it was found that the mice transplanted with the corrected iPSCs could have normal hematopoiesis and produce normal HBB protein. This study brings new hope for the treatment of β-thalassemia.

Chemokine receptor 5 (CCR5), as a co-receptor of HIV virus, is essential for the cellular infection of CCR5 tropic virus. Studies have shown that the loss of CCR5 function can prevent HIV infection. Through gene editing of immune cells in patients, it is possible to treat HIV. However, due to the low transfection efficiency of immune cells and the difficulty of cell culture, it is difficult to directly gene edit these cells. Kang et al. performed gene editing on iPSC by using CRISPR/Cas9 technology, designed a single gRNA knockout strategy and a double gRNA knockout strategy targeting CCR5, and successfully screened the CCR5 knockout homozygous clones. iPSCs with homozygous CCR5 mutations still show the typical characteristics of pluripotent stem cells and effectively differentiate into hematopoietic cells and macrophages. In vitro HIV infection experiment showed that the KO cells had unique resistance to the attack of CCR5 tropic virus. This study shows that combining iPSC technology with CRISPR/Cas9 technology has a promising application in the treatment of HIV infection.