Induced pluripotent stem cells (iPSCs) developed from patients have enabled the investigation of disease mechanisms in the lab without dependence on animal models, which do not always mimic human disease conditions. As the cells differentiated from patient iPSCs also show the disease phenotype, they are very valuable in drug screening and testing. However, obtaining patient samples has issues of ethical concerns and accessibility. Further, the mutation from a specific patient may not represent the most prevalent disease variant.
Using genome editing, it is now possible to disrupt a gene function by introducing the mutation of interest and to correct a disease associated mutation. Therefore, to study a specific genetic disorder, the mutation of interest can be introduced in the target gene in a pluripotent stem cell derived from a normal donor. Once generated, the cell line can be differentiated to lineages to study disease pathogenesis or drug screening. Using the gene editing-based approach, we have successfully generated iPSC-based disease models for Pompe and ACADVL. In addition, using patient samples, iPSCs have been generated for the skeletal disorder Osteogenesis Imperfecta (OI). Currently, drug screening is being carried out using OI-iPSC-derived osteogenic cells.
Team
Vasanth Thamodaran
Evolutionary conservation and ease of handling have made small mammalian models like mice and rats into valuable tools for investigating human diseases and in drug discovery. However, about 20% of human genes do not have orthologues in mice. Further, some disease phenotypes do not mimic the human condition. In such cases, human cell-based in vitro models are used. Conventionally, either the primary cells derived from a donor with the disorder under investigation or an immortalised cell line is used. However, primary cells cannot be maintained indefinitely and immortalised cell lines carrying genomic abnormalities may not faithfully display the disease phenotype.
The drawbacks associated with primary and immortalised cells can be overcome by using human pluripotent stem cells (hPSCs). hPSCs have the potential to differentiate into any cell type in the body and can be maintained in vitro indefinitely. Thus, a hPSC generated from an individual with a specific genetic disorder will enable in vitro derivation of cell types affected in that disorder. The cells so derived can be used in studying disease pathogenesis and drug screening. hPSCs can either be derived from an early-stage embryo or by reprogramming somatic cells to pluripotent state by expressing specific transcription factors. Somatic cell derived induced pluripotent stem cells (iPSCs) also obviate ethical concerns associated with stem cell generation from embryos.
iPSCs can be routinely derived from patient subjects and used in disease modelling studies. iPSCs present an invaluable therapeutic platform when combined with CRISPR-Cas based gene editing approaches. When obtaining patient samples is not possible, the mutation in the gene of interest can be introduced by gene editing. The gene edited lines subsequently generated can be used in vitro to study the disease mechanisms.
The lack of stem cell models for a majority of these RGDs has hindered the generation of insights on them. Thus, establishment of pluripotent stem cell lines carrying the mutation for these disorders will enable detailed study of RGD pathogeneses. With this goal in mind, we have initiated the following steps to develop models for a variety of disorders:
- Establishing mutant iPSC lines
- Disease modelling
- Drug testing and screening
Over the last few months, we have had some success in working on models for multiple disorders while others have just been initiated, at TIGS and in partnership with other institutes, as described below
A. Pompe Disease
» Generation of Pompe stem cell disease model: Using the identified optimal nucleofection condition, the GAA gene which is defective in Pompe patients was targeted in hESC. The gene edited cell line when differentiated to cardiac and skeletal muscle cells they successfully recapitulated the pompe disease phenotype.
B. Gaucher’s Disease
Gaucher’s Disease is the most prevalent LSD globally and also in India. It is caused by mutations in GBA1 gene, which encodes for glucocerebrosidase that enables breakdown of glucosylceramide to ceramide and glucose. Accumulation of glucocerebroside in GD affects neurons, liver, blood cells and skeletal system. Specific mutations of GBA1 gene are associated with Parkinson’s disease.
Currently we are working towards correction of the GBA1 mutation, prevalent in India by gene editing.
C. Disease Modelling and Drug Discovery for Osteogenesis Imperfecta (OI)
D. Establishing iPSC-based Disease Model for Epidermolysis Bullosa (EB)
E. Skeletal myopathies (in collaboration with DBT – Institute for Stem cell science and Regenerative medicine (inStem)
Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is caused by defect in the gene ACADVL, which codes for the enzyme acyl-CoA dehydrogenase very long chain.
Using gene editing we have established stem cell model for VLCADD and the skeletal muscles derived showed increase in intermediates of fatty acid metabolism.
Investigator: Vasanth Thamodaran