Genome editing finds extensive application especially in stem cell research for developing therapeutics. The genome editing tools can be used to disrupt any disease-causing genes via insertion, deletion, or point mutations. Stem cells are potential candidates for therapeutic development using genome editing since they have remarkable self-renewal capacity. Stem cells are also capable of expression endogenous proteins such as coagulation factor IX, VEGF, sRAGE and others. Genome editing technologies that rely on the engineered nuclease-based platform such as CRISPR/Cas utilise the endogenous DNA repair mechanism to introduce the desired gene editing at almost any location within the target genome.
Stem cell genome editing for cellular therapy broadly employs two strategies. The first strategy involves ex vivo genome editing in stem cells. In this approach in vitro transduction or correction of therapeutic genes is performed followed by their transplantation into the host. The second strategy involves in vivo genome editing in stem cells in which transduction or correction of therapeutic genes is performed upon delivery of the genome editing components into the host cells. The ex vivo approach is preferred over the in vivo approach since the former is relatively safe, simple and efficient.
To date, CRISPR/Cas systems have been used to probe and treat genetic disorders, cancers, infectious diseases, and immunological diseases. A large number of preclinical and clinical trials have been conducted to evaluate the potential of CRISPR/Cas therapy in treating inheritable genetic disorders.
Haemoglobinopathies such as β-thalassemia and sickle cell disease (SCD) arise due to mutations in the β-globin subunit of haemoglobin. The therapeutic strategy involves re-expression of paralogous γ-globin genes and has been achieved using the CRISPR/Cas9 system. Inherited eye disease such as Leber congenital amaurosis (LCA) is a genetic disease manifesting at birth or at infancy and is caused by mutations in the CEP290 gene. Similarly, autosomal dominant cone-rod dystrophy (CORD6) is caused by a mutation in the GUCY2D gene. The CRISPR/Cas technique has demonstrated its potential to treat such genetic retinal diseases.
Other genetic diseases include genetic deafness, muscular genetic disease, genetic lung and liver diseases. CRISPR/Cas9-mediated in vivo disruption of the deafness-associated allele in the humanised transmembrane channel-like 1 (Tmc1) in the mouse model can ameliorate the deafness. Similarly, in the case of cystic fibrosis, researchers have used CRISPR technology to accurately repair homozygous F508del mutation in the CFTRgene in the induced pluripotent stem cells (iPSC) isolated from patients. The method displayed a correction efficiency of 90% and recovery of CFTR function in the epithelial cells or lung organoids derived from the genetically repaired iPSCs. The sgRNA directed CRISPR/Cas9 system has also been reported to rectify the C676T mutation in the CYBB gene in human hematopoietic stem and progenitor cells (HSPCs) derived from patients suffering from X-linked chronic granulomatous disease.
Both in vivo and ex vivo approaches based on CRISPR/Cas technique has shown immense potential in rectifying monogenic mutations involved in genetic disorders. Additionally, the CRISPR-based generation of disease models has accelerated the pace of drug discovery and development. The therapeutic domain of CRISPR and stem cells is likely to expand further to treat genetic diseases in humans.