Knockout iPSC Cell line

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Many serious diseases cannot be cured by medicines, such as heart failure, Late Stage Diabetes, hemophilia, myeloma, End-Stage Cirrhosis, etc. The best method is allogeneic transplantation. However, due to the limited donors and the risk of immune rejection, researchers are dedicated to finding more efficient and safer treatment besides allogeneic transplantation. Induced pluripotent stem cells (iPSCs) can be derived from the body cells of the patients themselves, which eliminates the risk of immune rejection, and has the potential of differentiation into different cells. Transplantation of cells derived from iPSC, such as cardiomyocytes, hepatocytes, neurocytes, T cells, hematopoietic stem cells (HSCs) and pancreatic cells, is possible to solve many medical problems.
Hepatocyte
The differentiation of liver cells induced by iPSC can alleviate the shortage of sources in liver transplantation and hepatocyte transplantation, which is more conducive to basic and clinical research. In addition, the induced hepatocyte could be used as a tool to simulate and study liver diseases and screen the hepatotoxicity of drugs in the future.
Neural stem cell and neuron
Neural stem cells differentiated from iPSC can be used to generate cell models of nervous system diseases. This approach avoids ethical problems and immune rejection, and is an ideal way to obtain NSC in vitro.
iPSC can differentiate into neuron under appropriate conditions. For example, differentiation into motor neurons (MN) provides the possibility for the treatment and research of MN injury diseases such as Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA).
T cell
iPSC can differentiate into T cell. The CAR-T cell therapy developed on the basis of iPSC has a safer and more effective pharmacological activity. iPSCs based CAR-T cells can be used in T cell immunotherapy without the limitation of Allograft rejection.
Hematopoietic stem cell
The limited number of hematopoietic stem cells (HSC), the difficulty of expansion and culture in vitro and graft versus host disease (GVHD) limit the HSC transplantation. iPSC can proliferate and differentiate into transplantable HSCs in vitro, which brings a bright future for the treatment of malignant blood diseases.
Cardiomyocyte
iPSC derived cardiomyocytes provide a new way for the study of disease-specific and individual-specific pathogenesis of cardiovascular diseases, which has become an effective tool in the field of cardiovascular research and also brings new hope for clinical treatment.
Pancreatic cell
iPSC can differentiate into pancreatic β-cells in vitro, which can be used in the research of disease mechanism, drug development and cell therapy for diabetes. Using this source of pancreatic β-cells for transplantation in the treatment of diabetes can better solve the ethical, limited source problems faced by the previous islet transplantation.
Ubigene’s iPSC platform:
Reprogramming services
By transferring transcription factors, such as Oct3/4、Sox2、c-Myc and KlF4, somatic cells could be reprogrammed into iPSC with the potential of proliferation and differentiation.
Steps of iPSC reprogramming:
· Vectors carrying transcription factors will be transferred into somatic cells to reprogram into iPSC;
· iPSC validation: genotyping and phenotyping.
Gene editing service
The success rate of gene editing in human iPSC is lower because, unlike tumor cell lines, iPSC does not have the characteristics of chromosomal abnormality and strong ability of DNA repair. CRISPR/Cas9 has the advantages of high efficiency, easy to construct and low toxicity in human cells, so it is the most common method in iPSC genome editing. CRISPR-U™ optimizes the targeting efficiency, greatly improve the efficiency of DSB and homologous recombination in iPSCs.
Knockout
CRISPR-U™ gene knockout iPSC cell line: gRNA and Cas9 are transferred into iPSCs by nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.
Type Strategy Application
Short fragment removal Guide RNAs target introns at both sides of exon 2 and the number of bases in exon 2 is not a multiple of 3, which can cause frame-shift mutation. Study of gene function through gene defect
Frame-shift mutation Guide RNA targets the exon, and the base number of deletion is not a multiple of 3. After knockout, frame-shift mutation would cause gene knockout.
Large fragment removal Complete removal of the coding sequence to achieve gene knockout.
Case Study:
The limited T cells and the difficulty of proliferation is the main obstacle of T-cell immunotherapy, which can be overcome by using pluripotent stem cells with proliferation and differentiation ability to generate T-iPSC with antigen specificity. Strict antigen specificity is essential for safe and effective T-cell immunotherapy. However, in the process of double-positive CD4/CD8 differentiation, the rearrangement of the T-cell receptor (TCR) α chain will lose antigen specificity. This TCR rearrangement was prevented by removing the recombinant enzyme gene (RAG2) in T-iPSCs with CRISPR/Cas9. Xenotransplantation of CD8αβ-T cells with stable TCR can effectively inhibit tumor growth in disease models. This contributes to a safe and effective T-cell immunotherapy.
gRNA sequence and RAG2ockout sequence。The positive clones have frameshift mutations in the designated RAG2.
Comparison of the binding ability of WT and RAG2 knockout T-iPSCs to dextramer. RAG2-/- T-iPSCs differentiated into CD8αβ cells expressing stable TCR, while 40% of RAG2wt/wt-iPSC derived CD8αβ cells lost antigen specificity.
Reference:
Minagawa, Atsutaka, et al. "Enhancing T cell receptor stability in rejuvenated iPSC-derived T cells improves their use in cancer immunotherapy." Cell Stem Cell 23.6 (2018): 850-858.
Gene modeling or repair
Point Mutation
iPSC would be co-transfected with gRNA, Cas9 and donor oligo by electroporation. After the DNA DSB caused by the complex of gRNA and Cas9, iPSCs use donor oligo carrying wild-type sequence as a template for homologous recombination repair (HDR) and replace the target sequence with point mutation.
Case Study:
Disease model generation
ssODN carrying point mutation which replaces the WT sequence by HDR.
Disease model rescuing
ssODN carrying WT sequence which replaces the mutated site by HDR.
The limited T cells and the difficulty of proliferation is the main obstacle of T-cell immunotherapy, which can be overcome by using pluripotent stem cells with proliferation and differentiation ability to generate T-iPSC with antigen specificity. Strict antigen specificity is essential for safe and effective T-cell immunotherapy. However, in the process of double-positive CD4/CD8 differentiation, the rearrangement of the T-cell receptor (TCR) α chain will lose antigen specificity. This TCR rearrangement was prevented by removing the recombinant enzyme gene (RAG2) in T-iPSCs with CRISPR/Cas9. Xenotransplantation of CD8αβ-T cells with stable TCR can effectively inhibit tumor growth in disease models. This contributes to a safe and effective T-cell immunotherapy.
CRISPR/Cas9 and ssODN used to repair the point mutation in A79V-hiPSC. A) Genomic sequence surrounding the mutation site: mutated nucleotide (T, red); sgRNA recognition site containing 20 bp (yellow); CRISPR cutting site between the 17th and 18th bp (bold); forward and reverse primers (pink). B) ssODN with 120 bp, 60 bp upstream and 60 bp downstream the mutation site containing the WT nucleotide (C, green).
Sequencing of exon 4 of the PSEN1 gene in hiPSCs.
A) Heterozygous c.236C>T substitution in the mother line previously published.
B) Successful correction of the point mutation (T>C).
Reference:
Pires, C., Schmid, B., Petræus, C., Poon, A., Nimsanor, N., Nielsen, T. T., ... & Freude, K. K. (2016). Generation of a gene-corrected isogenic control cell line from an Alzheimer's disease patient iPSC line carrying a A79V mutation in PSEN1. Stem cell research, 17(2), 285-288.
Gene Knock in
Knock in
CRISPR-U™ Gene Knockin iPSC:iPSC would be co-transfected with gRNA, Cas9 and donor vector by electroporation. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.
Knockin Strategies :
Disease model generation
pic-9
Guide RNA and Cas9 complex cause a double-strand break (DSB) on the target site of DNA. The donor vector carrying knockin sequence is the template for homologous recombination repair (HDR), and it recombines to the target site.
Safe harbor knockin:
pic-10
Gene knockin at Safe harbors such as hROSA26 and AAVS1 not only avoids random insertion in genome, but also achieves overexpression of target gene.
Case Study:
The most common method to treat hemophilia is substitution therapy, but this method has the risk of virus infection, and it is a method that needs lifelong continuous treatment. Gene therapy seems like the only way can cure hemophilia. CRISPR/Cas9 technology can be used for gene therapy of hemophilia. The mutations of coagulation factors, F8 and F9, are the main causes of hemophilia. Previous studies have shown that F9 is a more effective gene therapy target. AAVS1-Cas9-sgRNA plasmid and AAVS1-EF1α-F9 cDNA puromycin donor plasmid were constructed and transferred into iPSC. Human factor IX (hFIX) antigen activity was detected in the culture supernatant. Finally, liver cells differentiated from iPSC were transplanted into NOD/SCID mice by spleen injection, to cure hemophilia B.
After 48 hours of transfection, puromycin was used for drug screening. Most iPSCs died after drug screening, but a few survived. After about 7 days, each surviving iPSC clone was expended to be further testing of insertion (Fig. a, b). Six clones were selected. As shown in Figure C, 1.3kb fragments can be detected in all iPSC clones with primers; 468bp and 4.9kb fragments can be detected in iPSC clones 1, 2, 3, 4 and 6 with another pair of primers, indicating F9 cDNA heterozygous insertion; only 4.9kb fragments can be detected in iPSC clone 5, indicating F9 cDNA homozygous insertion.
Reference:
Lyu, Cuicui, et al. "Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system." Stem cell research & therapy 9.1 (2018): 92.
iPSC differentiation
The study of human embryonic stem cells (hESCs) derived from early embryos has been controversial in ethics, and the rejection of differentiated cells derived from hESCs in transplantation has limited its clinical application. Hepatocytes, nerve cells, T cells, cardiomyocytes, hematopoietic stem cells and islet cells can be differentiated from patients' somatic cells (such as fibroblasts) or existing iPSCs.
Differentiation Process