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Modeling and correction of genetic skeletal dysplasias in iPSCs

Funding Type: 
Basic Biology III
Grant Number: 
Funds requested: 
$1 743 882
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Achondroplasia is the most common form of skeletal dysplasia, a disease of the bone and cartilage that is characterized by short-limbed dwarfism. Achondroplasia is known to be a heritable genetic disorder, and the gene believed to be responsible is FGFR3. Genetic alterations in FGFR3 are found in most cases of achondroplasia, but how they influence disease progression remains poorly understood. While mouse models of achondroplasia exist, due to the inherent differences in physiology between mice and humans, these models cannot inform scientists of the detailed processes controlling human disease progression. Indeed, this gap in our knowledge has prevented us from completely understanding achondroplasia and inhibited the development of new treatments for this disease. Our laboratory has developed techniques that allow for precise modification of cellular genomes. These techniques are based on zinc finger nuclease (ZFN) proteins that can be designed to modify DNA at a targeted location in the human genome. We are proposing to use ZFNs to recreate the specific gene mutations found in a majority of achondroplasia cases in laboratory based human cell lines. These cell lines will then be used to create induced pluripotent stem cell (iPSC)-based disease models of achondroplasia. iPSCs are cells that have the potential to develop into almost any cell type or tissue. By creating iPSCs that have the mutations associated with achondroplasia, we will be able to monitor the progression of the disease throughout the developmental process by monitoring the formation of bone tissue from stem cells. This strategy will provide a very accurate model of achondroplasia and will allow us to reveal mechanisms controlling disease progression and bone formation. In addition, the generation of iPSC-based models of the disease will permit the development of novel therapies that can be initially analyzed with these cells. Finally, we will use ZFNs to genetically correct the FGFR3 gene in cell lines obtained from patients with achondroplasia. By comparing these corrected cell lines to the disease models we create, we will be able to confirm that the assumed causative FGFR3 mutations are responsible for achondroplasia.
Statement of Benefit to California: 
Stem cell technologies hold the potential to revolutionize medicine, healthcare, and our understanding of human biology. Induced pluripotent stem cells (iPSC) are cells that can be generated from adult tissues and used to give rise to several different cell types. Stem cells hold vast potential for the treatment of disease and injury because they are pluripotent: these cells have the ability to develop into any of the more than 200 cell types in the human body. Our research goal is to use iPSC technology to generate stem cell-based models of the disease achondroplasia. Achondroplasia is the most common cause of skeletal dysplasia, a congenital abnormality of the bone and cartilage that is characterized by short-limbed dwarfism. While some of the genetic factors underlying the disease are known, much about the development and pathology of the disease is unknown. These gaps in our understanding are due to the fact that human-based laboratory models of the disease do not exist. While mouse models are useful for developing insight into human disease, they often cannot reveal the mechanisms of a human disorder. However, with iPSCs we will be able to generate a wide range of disease in the dish models of achondroplasia. Accurate models of human disease will allow for precise study of the underlying mechanisms and the development of new therapies. Scientists believe that stem cells can eventually be used to create therapies to treat previously untreatable injuries and diseases. Devastating and currently incurable conditions such as AIDS, Alzheimer’s, liver disease, diabetes, Parkinson's disease, muscular dystrophies, spinal cord injuries, inborn errors of metabolism, and many other diseases can be studied by creating disease in the dish models with iPSCs. However, in order to realize the full potential of this technology, researchers need to develop new tools that will permit the safe introduction or correction of genes in iPSCs. To achieve this significant goal, we will capitalize on our extensive experience in developing new and effective gene targeting technologies to derive and characterize unique stem cell-based models of achondroplasia. This new technology and the new therapies that may result from this research may result in the reduction of the long-term healthcare costs to the State of California by providing cures and alternative treatments to disease and injury that are chronic and/or untreatable. Support for the development of this new technology will ensure that California is a leader in stem cell technologies, making California better equipped not only to treat its own citizens but also to compete for the multi-billion dollar market that is expected to develop with advances in stem cell technologies. Spurring industry, research, and product development in the biotechnology and healthcare fields will further benefit California by attracting highly skilled and well-educated individuals and tax revenues to the state.
Review Summary: 
Project Synopsis: The overall objective of this proposal is to generate human stem cell-based models of achondroplasia for examination of the intracellular mechanisms involved in chondrocyte differentiation and proliferation, cartilage development, skeletal morphogenesis and skeletal dysplasia. The central hypothesis is that induced pluripotent stem cell (iPSC)-based models of achondroplasia can provide new insights into bone development and disease pathophysiology by permitting advanced analysis in a human cell-based system. The applicant proposes to use zinc finger nucleases (ZFN) in human fibroblasts to recreate mutations in FGFR3 that have been implicated in achondroplasia. The fibroblasts will be reprogrammed to iPSC and subsequently differentiated into chondrocytes for in vitro analysis of affected pathways and elucidation of the relationship between FGFR3 mutation and disease phenotype. The applicant will also attempt to use ZFN to repair the FGFR3 locus in fibroblasts taken from a patient with achondroplasia, thereby demonstrating proof of principle for the utility of this technology. Significance and Innovation: - It is not clear how the proposed studies in human cells would provide better insights into signaling defects in achondroplasia than could be obtained using targeted mutations in mice. A clear correlation between clinical phenotypes and in vitro mechanisms has not been provided. - Reviewer opinion on the significance of this work was mixed. Some felt the application describes a novel approach for developing a disease in the dish model for achondroplasia that could be used to explore various aspects of chondrocyte biology and could potentially be developed as a model for screening therapeutic molecules. Others noted that achondroplasia is a relatively rare disorder and were uncertain that this work would have measurable impact. - The proposed ZFN genetic manipulation technology represents a novel, sophisticated, and imaginative means to generate mutations. The applicant’s experience and skills would bring new technical tools to manipulate the genome to the human pluripotent stem cell field. Feasibility and Experimental Design: - While the applicant is quite capable of generating the ZFNs needed for this work, the major flaw with this proposal is his/her lack of experience working with or generating human induced pluripotent stem cells (hiPSCs) and relevant derivatives. - The methods for generating iPS cells from mutant fibroblast lines were briefly described, yet it is critical that these iPS cell lines be carefully validated if they are to be used to study the disease phenotypes. - Reviewers presumed that control iPS cell lines would be generated, although this was not specifically stated in the application. Further, not all lines will differentiate as easily as others so natural variation between cell lines must be considered when analyzing results - a fact not discussed in the application. - Given that the applicant has no experience in studying chondrogenesis or in understanding the clinical phenotypes of achondroplasia, it is unclear how these studies will actually help understand the human syndrome. Inclusion of collaborators with expertise in achondroplasia and cartilage/bone development would have lessened this concern. - The experiments proposed in Aim 3 are purely proof of principle experiments describing a technical advance, and there is no biological justification for the repair of the FGFR3 gene. It is not clear how this aim contributes to the overall goal of the proposal of understanding the mechanisms by which mutations in FGFR3 affect bone and cartilage formation. - The proposal lacks acknowledgement of potential difficulties and alternative approaches. - Although not specifically stated by the applicant, reviewers presumed the rationale for employing genetically engineered cell lines for mechanistic studies, rather than starting with iPSC derived directly from human subjects with achondroplasia, was to allow for the mutations to be made on the same genetic background, thereby minimizing the variability that could be introduced from different patients. Principal Investigator (PI) and Research Team: - While the principal investigator (PI) has a stellar track record and considerable experience and skills in developing ZFNs and in other molecular biology technologies, the PI and the team lack experience in the biological and diseases areas proposed in the application. - Reviewers suggested that the team should include a clinician or someone who understands what the major unknowns are in achondroplasia that would aid in its treatment and should include investigators with expertise in making and analyzing the cell types (chondrocytes) involved in the disease. Responsiveness to RFA: - The concept of studying disease processes in iPS cells in vitro is responsive to the RFA. However, the strongest part of this proposal is the technical aspect of gene manipulation rather than any innovation or deep mechanistic studies on the biology of achondroplasia.
Programmatic review: 
  • This application scored below the initial scientific merit funding line, no programmatic reason to fund the application was proposed, and the GWG voted to place the application in Tier 3, Not Recommended for Funding.

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