Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE29774 | GEO | 2011/07/26
SECONDARY ACCESSION(S): PRJNA141045
REPOSITORIES: GEO
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Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the ?-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.
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Project description:This SuperSeries is composed of the following subset Series: GSE29772: CNV analysis for Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations GSE29773: Gene Expression Data for Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations Refer to individual Series
2011-07-25 | E-GEOD-29774 | biostudies-arrayexpress
Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this “disease in a dish” approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinson’s disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the α-synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells.
2011-07-26 | GSE29772 | GEO
Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this “disease in a dish” approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinson’s disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the ?-synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells. ZFN-mediated genome edited human iPS cells or ES cells were assayed for genomic variation
2011-07-25 | E-GEOD-29772 | biostudies-arrayexpress
Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this “disease in a dish” approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinson’s disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the α-synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells.
2011-07-26 | GSE29773 | GEO
Project description:Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease in disease relevant cells, as well as a promising source for cell replacement therapies for degenerative diseases. However one of the crucial limitations before realizing the full promise of this “disease in a dish” approach has been the inability to do controlled experiments under genetically defined conditions. This is particularly relevant for disorders with long latency periods, such as Parkinson’s disease (PD), where in vitro phenotypes of patient-derived iPSCs are predicted to be subtle and susceptible to significant epistatic effects of genetic background variations. By combining zinc-finger nuclease (ZFN)-mediated genome editing and iPSC technology we provide a generally applicable solution to this key problem by generating isogenic pairs of disease and control human embryonic stem cells (hESCs) and hiPSCs lines that differ exclusively at a susceptibility variant for PD by modifying a single point mutation (A53T) in the α-synuclein gene. The robust capability to genetically correct disease causing point mutations in patient-derived hiPSCs represents not only a significant progress for basic biomedical research but also a major advancement towards hiPSC-based cell replacement therapies using autologous cells. ZFN-mediated genome edited human iPS cells or ES cells were assayed for gene expression
2011-07-25 | E-GEOD-29773 | biostudies-arrayexpress
Project description:Curative bone marrow transplantation (BMT) therapies for sickle cell disease (SCD) can cause infertility. The Fertility Preservation Program (FPP) in Pittsburgh cryopreserves testicular tissues for SCD patients prior to BMT in anticipation that those tissues can be thawed in the future and matured to produce sperm. Here, we generated and validated two isogenic patient-derived induced pluripotent stem cell (iPSC) lines from testicular biopsy fibroblasts of a 12-year-old SCD patient.
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Project description:Human pluripotent stem cells (hPSCs) are a powerful platform for disease modeling and drug discovery. However, the introduction of known pathogenic mutations into hPSCs is a time-consuming and labor-intensive process. Base editing is a newly developed technology that enables facile introduction of point mutations into specific loci within the genome of living cells. Here, we design an all-in-one episomal vector that expresses a single guide RNA (sgRNA) with an adenine base editor (ABE) or a cytosine base editor (CBE). Both ABE and CBE can efficiently introduce mutations into cells, A-to-G and C-to-T, respectively. We introduce disease-specific mutations of long QT syndrome into hPSCs to model LQT1, LQT2, and LQT3. Electrophysiological analysis of hPSC-derived cardiomyocytes (hPSC-CMs) using multi-electrode arrays (MEAs) reveals that edited hPSC-CMs display significant increases in duration of the action potential. Finally, we introduce the novel Brugada syndrome-associated mutation into hPSCs, demonstrating that this mutation can cause abnormal electrophysiology. Our study demonstrates that episomal encoded base editors (epi-BEs) can efficiently generate mutation-specific disease hPSC models.
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Project description:The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
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Project description:We developed an efficient CRISPR prime editing protocol and generated isogenic-induced pluripotent stem cell (iPSC) lines carrying heterozygous or homozygous alleles for putatively causal single nucleotide variants at six type 2 diabetes loci (ABCC8, MTNR1B, TCF7L2, HNF4A, CAMK1D, and GCK). Our two-step sequence-based approach to first identify transfected cell pools with the highest fraction of edited cells significantly reduced the downstream efforts to isolate single clones of edited cells. We found that prime editing can make targeted genetic changes in iPSC and optimization of system components and guide RNA designs that were critical to achieve acceptable efficiency. Systems utilizing PEmax, epegRNA modifications, and MLH1dn provided significant benefit, producing editing efficiencies of 36-73%. Editing success and pegRNA design optimization required for each variant differed depending on the sequence at the target site. With attention to design, prime editing is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.
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