Screening for novel drugs is one of the costliest steps in drug development. Modelling rare genetic disorders using Drosophila and cell culture allows for fast, cost-effective screening of novel therapeutic interventions. This also reduces the animal burden in drug development.
We use a cost-effective Drosophila model for screening small molecules, mRNA therapy and anti-sense oligos (ASOs) against less explored diseases and rare genetic disorders. Drosophila shows more than 80% conservation with human genes and pathways. Over the last hundred years, Drosophila researchers have developed excellent genetic tools and behavioural assays useful for testing the effect of drugs and for evaluating their impact on key pathways, behaviours and lifespan.
At TIGS we are developing a model of primary screening in flies, followed by testing of potential candidates in cell culture and animal models. Developing in vitro models using stem cells and induced pluripotent stem cells (iPSCs) provides a ready ‘disease-in-a-dish’ model for studying the effect of interventions on the target cell type. This will make it possible to develop low cost and effective treatments for rare genetic disorders.
Spinal muscular atrophy (SMA) is caused by defects in survival motor neuron 1 (SMN1) gene. Currently, there are small molecular drug screens established in cell culture and animal models worldwide to increase functional SMN protein. However, performing such screening in cell lines or animal models is very costly and time consuming. Additionally, cell line-based screens are limited in assessing systemic effects of the drug while the animal model cannot be used for high throughput screens. Drosophila disease models can successfully bridge this gap. Drug screen in flies can be done on a large scale, help assess systemic effects and are comparatively cheap. This can help reduce the overall price of the drug development process and develop cost-effective treatments for SMA. We have initiated the following goals at TIGS:
1. To establish humanized Drosophila SMA model.
2. To design, synthesize and test small molecular drugs on the SMA fly model.
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:
- Generation of mutant iPSCs
- Characterisation of the mutant iPSCs
- 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. Lysosomal storage disorders (LSDs)
» Optimization of gene editing in iPSCs: The optimal nucleofection condition that provides efficient gene editing in iPSC/ESC was identified using guide RNAs that target the OCLN gene.
» Generation of Pompe-iPSC: Using the identified optimal nucleofection condition, the GAA gene which is defective in Pompe patients was targeted in hESC (hem20). Successful gene editing was validated by Sanger sequencing and the cells were single cell cloned to isolate a cell line that carries homozygous mutation.
» Establishment of differentiation protocols: As cardiac and skeletal muscles are defective in Pompe’s disorder, the differentiation procedures were successfully established to mimic the same
B. 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.
» Gene editing of ACADVL gene: To generate iPSC-based disease model for VLCADD, the exon 12 of ACADVL gene was targeted using CRISPR-Cas9 and one of the clones was identified as carrying a homozygous 21 bp deletion. The clone was karyotypically normal and also confirmed to be pluripotent.
» Investigating VLCADD in cardiac and skeletal muscle: Differentiation of VLCADD iPSC to cardiac and skeletal muscle lineages showed hypertrophy.
Investigator: Vasanth Thamodaran