For many serious diseases, such as heart failure, advanced diabetes, hemophilia, myeloma, end-stage liver cirrhosis, etc., drugs that can completely cure these diseases have not been developed. The most effective method is allogeneic transplantation. However, due to the limited number of donors and the risk of autoimmune rejection, more efficient and safer treatment methods are the current research focus. Induced pluripotent stem cells (iPSCs) are derived from the somatic cells of the recipient itself, eliminating the risk of immune rejection, and have the potential for continuous self-renewal and multidirectional differentiation. The transplantation of cardiomyocytes, hepatocytes, nerve cells, T cells, hematopoietic stem cells, and islet cells derived from iPSC can effectively solve the above medical problems. Using the CRISPR/Cas9 method, introducing mutations that mimic diseases into iPSC, or repairing mutations in iPSC disease models, and then differentiated to obtain the required cells for research or treatment, is the main direction of current iPSC research.
Since iPSC does not have the characteristics of chromosomal abnormalities and abnormally strong DNA damage response like ordinary tumor cell lines, gene editing has a lower success rate in human iPSCs. However, due to its high efficiency, easy construction, and low toxicity in human cells, CRISPR/Cas9 is popular in iPSC gene modification.
The gRNA and Cas9 were efficiently transferred into iPSC cells by nuclear transfection, and the single clones were selected for culture after drug screening. Different clones were selected for target site amplification and sequencing verification, and positive clones with gene knockout were screened out. For example, CRISPR/Cas9 knocks out the RAG2 gene in iPSC, effectively generating CD8αβ-T cells with stable antigen specificity.
The number and proliferation of T cells are the main obstacles to T cell immunotherapy. This obstacle can be overcome by using pluripotent stem cells with "regeneration" ability to induce differentiation into antigen-specific T cells (T-iPSCs). Strict antigen specificity is essential for safe and effective T cell immunotherapy. However, human T-iPSC-differentiated CD8αβ cells during the CD4/CD8 double-positive differentiation process through the T cell receptor (TCR) α chain Rearrangement will lose antigen specificity. The use of CRISPR/Cas9 to knock out the recombinase gene (RAG2) in T-iPSCs prevented this additional TCR rearrangement. CD8αβ-T cells containing stable TCR can effectively inhibit tumor growth in xenograft tumor models. These methods contribute to safe and effective T cell immunotherapy.
Fig.1 iPSC-derived T cells in cancer immunotherapy. (Minagawa, 2018)
The gRNA, Cas9, and Donor were co-transfected into iPSC cells by the nuclear transfection method, and the drug screening was performed. After the drug screening was completed, a single clone was selected for culture. Different clones were selected for target site amplification and sequencing, and positive clones homozygous for knock-in were screened out. Knock-in fluorescent reporter genes can also be initially screened by observing fluorescence.
For example, the AAVS1 safe site of iPSC knocks into coagulation factor 9, and the differentiated hepatocytes can be used to treat hemophilia B. At present, the most common method of treating hemophilia is an alternative therapy, but this method has the risk of viral infection and is a method that requires continuous treatment for life. Only gene therapy can cure hemophilia fundamentally, and the CRISPR/Cas9 system can be used for gene therapy of hemophilia. Mutations in coagulation factors 8 and 9 (F8 and F9) in cells are the main cause of hemophilia. Previous studies have shown that F9 is a more effective target for gene therapy. The AAVS1-Cas9-sgRNA plasmid and the AAVS1-EF1α-F9 cDNA puromycin donor vector targeting the AAVS1 site were constructed and introduced into the iPSC. The iPSCs with F9 cDNA inserted into the AAVS1 site were differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were successfully detected in the culture supernatant. Finally, hepatocytes were transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice by spleen injection to treat hemophilia B.
Fig.2 Insertion of F9 cDNA into AAVS1 locus of HEK293T cells by CRISPR-Cas9. (Lyu, 2018)
The gRNA vector (including Cas9) and Donor are co-transformed into the cell by nuclear transfection. After the gRNA and Cas9 complex cause a DNA double-strand break at the target site, iPSC cells carry the target point mutation Donor as a template for homology. Recombination repair (HDR), recombination of target point mutations to genomic target sites will occur. The gRNA carrier carries resistance and can be screened for drugs. After the drug screening is completed, single clones are selected for culture. Different clones were selected for target site amplification and sequencing, and positive clones homozygous for the point mutation were screened out.
For example, Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease that can lead to nerve cell degeneration and brain atrophy. It is considered the most common form of dementia. The A79V mutation in the PSEN1 gene can cause Alzheimer's disease. By studying the effect of this mutation on the cell phenotype, it can help researchers to study the pathology of this disease in-depth and develop more effective treatments. The researchers induced the somatic cells of Alzheimer's patients into pluripotent stem cells (iPSC), and then corrected the mutated gene by replacing the point mutation sequence with the wild-type sequence. By studying the patient's iPSC and the revised iPSC, we can know the effect of the mutation on the cell phenotype, to further study the pathological effect of the mutation. The A79V mutation of the PSEN1 gene can cause Alzheimer's disease. Studies have shown that the somatic cells of Alzheimer's patients are induced into pluripotent stem cells (iPSC), and then the mutant gene is corrected by replacing the point mutation sequence with the wild-type sequence. By studying the patient's iPSC and the revised iPSC, we can know the effect of the mutation on the cell phenotype, to further study the pathological effect of the mutation.
Fig.3 CRISPR/Cas9 and ssODN in the reparation of the point mutation in A79V-hiPSC. (Pires, 2016)
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References
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