Over twenty human genetic diseases are caused by expansion of simple DNA sequences composed of repeats of three nucleotides (such as CAG, CTG, CGG and GAA) within essential genes. These repeats can occur within the region of a gene that encodes the protein, generally resulting in proteins with large stretches of repeats of just one amino acid, such as runs of glutamine. These proteins are toxic, cause the death of specific types of brain cells and result in diseases such as Huntington’s disease (HD) and many of the spinocerebellar ataxias (a type of movement disorder). Other repeats can be in regions of genes that do not code for the protein itself, but are copied into messenger RNA, which is a copy of the gene that serves to generate the protein. These RNAs with expanded repeats are also toxic to cells, and sometimes these RNAs sequester essential cellular proteins. One example of this type of disease is Myotonic Dystrophy type 1, a form of muscular dystrophy. Lastly, there are two examples of repeat disorders where the repeats silence the genes harboring these mutations: these are Friedreich’s ataxia (FRDA) and Fragile X syndrome (FXS). One limitation in the development of drugs to treat these diseases is the lack of appropriate cell models that represent the types of cells that are affected in these human diseases. With the advent of the technology to produce induced pluripotent stem cells from patient skin cells, and our ability to turn iPSCs into any cell type, such as neurons (brain cells) that are affected in these triplet repeat diseases, such cellular models are now becoming available. Our laboratories have generated iPSCs from fibroblasts obtained from patients with HD, FXS and FRDA. By comparing cells before and after reprogramming, we found that triplet repeats were expanded in the FRDA iPSCs, but not in HD iPSCs. This application is aimed at the understanding the molecular basis underlying triplet repeat expansion/instability that we have observed during the establishment and propagation of iPSCs from disease-specific fibroblasts. While artificial systems with reporter gene constructs have reproduced triplet repeat expansion in bacteria, yeast and mammalian cells, no cellular models have previously been reported that recapitulate repeat expansions at the endogenous cellular genes involved in these diseases. Therefore, our observations that repeat expansion is found in FRDA iPSCs provides the first opportunity to dissect the mechanisms involved in expansion at the molecular level for the authentic cellular genes in their natural chromatin environment. Repeat expansion is the central basis for these diseases, no matter what the outcome of the expansion (toxic protein or RNA or gene silencing), and a fuller understanding of how repeats expand may lead to new drugs to treat these diseases.
A major obstacle in the development of new drugs for human diseases is our lack of cell models that represent the tissues or organs that are affected in these diseases. Examples of such diseases are the triplet-repeat neurodegenerative diseases, such as Huntington’s disease, the spinocerebellar ataxias, forms of muscular dystrophy, Fragile X syndrome and Friederich’s ataxia. These diseases, although relatively rare compared to cancer or heart disease, affect thousands of individuals in California. Recent advances in stem cell biology now make it possible to generate cells that reflect the cell types at risk in these diseases (such as brain, heart and muscle cells), starting from patient skin cells. Skin cells can be turned into stem cell-like cells (induced pluripotent stem cells or iPSCs), which can then give rise to just about any cell type in the human body. During the course of our studies, we found that iPSCs derived from Friedreich’s ataxia patient skin cells mimic the behavior of the genetic mutation in this disease. A simple repeat of the DNA sequence GAA is found in the gene encoding an essential protein called frataxin, and this repeat increases in length between generations in human families carrying this mutation. Over a certain threshold, the repeats silence this gene. It is also known that the repeats expand in brain cells in individuals with this disease. With the advent of patient derived iPSCs and neurons, we now have human model systems in which to study the mechanisms responsible for repeat expansion. We have already identified one set of proteins involved in repeat expansion and we now wish to delve more deeply into how the repeats expand. In this way, we may be able to identify new targets for drug development. We will extend our studies to Huntington’s disease and Fragile X syndrome. We have identified two possible therapeutic approaches for Friedreich’s ataxia, and identified molecules that either reactivate the silent gene or block repeat expansion. Our studies in related diseases may provide possible therapeutic strategies for these other disorders as well, which will be of benefit to patients suffering from these diseases, both in California and world-wide.
Trinucleotide (triplet) repeat expansions within the genome can generate toxic gene products or lead to outcomes that negatively impact essential proteins. These triplet repeats are implicated in numerous human diseases including Huntington’s disease (HD), Friedreich’s ataxia (FRDA), and Fragile X syndrome (FXS). For these types of disorders, the difficulty in obtaining affected cell types and the lack of appropriate cellular models of disease has limited efforts towards therapeutic discovery. The applicant has previously generated induced pluripotent stem (iPS) cells from HD, FXS, and FRDA patients and observed that triplet repeats are expanded in iPS from FRDA but not HD. He/she now proposes three specific aims in which these disease models will be exploited to elucidate mechanisms of triplet repeat expansion. In the first Aim, the investigators will use molecular approaches to assess the role of chromatin structure and transcription in expansion of triplet repeats. In Aim 2, they will explore role of various DNA repair, recombination, replication, and gene regulatory factors in mediating triplet repeat expansion. Lastly, they will use disease-specific iPS to investigate the mechanism by which the factors studied in Aim 2 are recruited to triplet repeats and how they contribute to their subsequent expansion.
Significance and Innovation:
- The mechanisms underlying the expansion of triplet repeats are largely unknown yet may be important for understanding age of disease onset in familial disease, the appearance of spontaneous mutations, and mechanisms of pathology. This research could be important for developing new therapeutic approaches.
- Reviewers noted a significant caveat that strongly impacts the significance and impact of the proposal: it cannot be known at this stage whether the mechanisms involved in the changes between the fibroblasts and iPS cells are the same as those involved in generational repeat expansion and in human disease.
- The findings described in the proposal are novel and, consequently, the innovation in this proposal is good.
- The cell lines developed and utilized in this project might also provide a tool to support future therapeutic compound screens.
Feasibility and Experimental Design:
- The applicant provides solid preliminary data supporting both the experimental rationale and the applicant’s ability to perform the described experiments. Many of the necessary tools are already in place to complete the proposed experiments.
- Reviewers noted that the observed triplet repeat expansions in FRDA iPS cells might be a cell culture artifact. While preliminary data alleviated this concern for some, others were concerned that the mutational load from the reprogramming process (iPS generation) may have impacted those data.
- The experimental plan is logical, with achievable milestones and realistic timelines.
Principal Investigator (PI) and Research Team:
- The principal investigator (PI) has a strong track record and has the appropriate expertise and experience to direct this project.
- The PI has assembled an excellent team that is well suited to perform the proposed experiments. The expertise of the co-investigator is complementary to that of the PI.
Responsiveness to RFA:
- The proposed research, which employs human pluripotent stem cell derived models to study the molecular basis of disease, is fully responsive to the RFA.