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RL1-00647-1: Establishing Noonan and Leopard syndrome patient-specific iPS cell lines

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

As a biomedical researcher, I firmly believe that many diseases that are incurable today could be treated effectively using human embryonic stem (ES) cell technology, the so-called cell replacement therapy or regenerative medicine. However, one ethical hurdle is the need to use human embryos in the classical way of establishing human ES cell lines. Also, patients may experience tissue rejection upon receiving transplantation of donor stem cell-derived functional cells. In 2007, scientists in Japan and the United States demonstrated that human ES cell-like stem cells can be created by reprogramming adult somatic cells with defined transcription factors, proteins that control gene expression in cells. This exciting result of establishing human inducible pluripotent stem (iPS) cells from somatic cells offers an alternative way to establish human stem cells without the need to use (destroy) human embryos. More importantly, this new technology raises the possibility for establishment of patient-specific human ES-like stem cells for research and therapeutic purposes. We will use this newly developed technology to create novel human iPS cell lines using dermal fibroblast cells derived from Noonan syndrome (NS) and LEOPARD syndrome (LS) patients. The objective of this project is to provide proof of principle in establishment of patient-specific iPS cell lines for dissection of the pathogenesis for complex human diseases and also for drug screening. NS and LS are developmental diseases with many common features. NS is an autosomal dominant disorder affecting 1 in 2500 live births, characterized by facial dysmorphia, typically ocularhypertelorism, cardiac defects, most commonly pulmonary valve stenosis, and proportionate short stature. LS (LEOPARD syndrome) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardio-gramabnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Both NS and LS patients exhibit increased risk of malignancy including myelogenous leukemia and neuroblastoma. We will use the established patient-specific iPS cell lines to investigate the molecular and cellular mechanisms underlying the developmental defects of the patients. We will identify and test small molecules that correct the cellular disorders associated with the diseases. We will also improve and optimize the technology to minimize the risky factors in use of iPS cell-derived functional cells for cell therapy on patients. Therefore, success of this project will provide fundamental insights into the pathogenesis and suggest novel therapeutic strategies for Noonan and LEOPARD syndrome patients. This project will set up an example of how the stem cell research can be beneficial to many otherwise hopeless human patients. However, this type of research is currently not fundable by NIH, and thus we are applying for CIRM award.

Statement of Benefit to California (provided by applicant)

The goal of this project is to establish new human pluripotent stem cell lines by reprogramming human somatic cells with defined transcription factors. As a biomedical researcher, I firmly believe that many diseases that are incurable today could be treated effectively using functional cells differentiated from human ES cells, the so-called cell replacement therapy or regenerative medicine. However, one ethical hurdle to the stem cell biology research field is the need to use human embryos in the classical way of establishing human ES cell lines. Also, a practical problem in clinical treatment is that patients may experience tissue rejection upon receiving transplantation of donor stem cell-derived functional cells. The recent success of establishing human iPS cells from somatic cells offers the feasibility for establishment of patient-specific human ES-like stem cells for diagnostic and therapeutic purpose. We will create novel human iPS cell lines using dermal fibroblast cells derived from Noonan syndrome (NS) and LEOPARD syndrome (LS) patients. Success of this project will benefit California citizens in the following ways. A) Success of this project will put California in the leading position in regenerative medicine. The objective of this project is to provide proof of principle for establishment of patient-specific iPS cell lines for dissection of pathogenesis for complex human diseases and also for drug screening. Therefore, the technology and the model system established will be applicable for stem cell therapy of many diseases, such as spinal cord injury, alzheimer’s disease, diabetes and leukemia. B) This project will directly benefit the NS and LS patients in California and other states in the country. NS is an autosomal dominant disorder affecting 1 in 2500 live births, characterized by facial dysmorphia, typically ocularhypertelorism, cardiac defects, most commonly pulmonary valvestenosis, and proportionate shortstature. LS (LEOPARD syndrome) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardio-gramabnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Both NS and LS patients exhibit increased risk of malignancy including myelogenous leukemia and neuroblastoma. Therefore, success of this project will provide fundamental insights into the pathogenesis and offer novel therapeutic strategies for Noonan and LEOPARD syndrome patients who are otherwise hopeless in their life. During the course of this research project, we will improve and optimize the technology to minimize the risky factors in use of iPS cell-derived functional cells for cell therapy on patients. In essence, success of this project will put us on the driver’s position in the competitive stem cell research field, which will benefit tremendously California’s biomedical research and industry.

Review

Noonan syndrome (NS) and Leopard syndrome (LS) are autosomal dominant, multisystem diseases caused by different mutations in the gene encoding a signal transduction molecule involved in embryonic development. Both syndromes have complex features, mostly involving the skin, skeletal and cardiovascular systems and there is an increased risk of malignancy. Despite the similar disease phenotypes, mutations in NS patients typically result in a dominantly active form of the affected signal transduction molecule while those in LS patients result in a catalytically inactive form of that molecule. The aims of this project are to establish pluripotent cell lines from dermal fibroblasts derived from NS and LS patients using induced pluripotent stem cells (iPSCs), to investigate the effect of the genetic defect on cardiomyocyte differentiation, and to identify small molecules that can rescue the mutant phenotype.

The significance of the proposal was debated among the reviewers. While LS is very rare, NS is one of the most common genetic syndromes associated with congenital heart disease with an estimated 1 in 1000 to 1 in 2500 children born worldwide with NS. Reviewers recognized that the creation of pluripotent stem cell lines from patients with complex diseases has long been invoked as an important application of stem cell technology towards the end of gaining a better understanding of the molecular pathology of such diseases and as a means to develop drugs to provide badly needed treatments for such diseases. Both aims are met in the current application. However, one reviewer commented that the rationale why iPSCs are needed for this purpose was not compelling, and the ability to recapitulate the disease phenotype, in particular that of valvular disease, might be quite limited in cultured cells.

With respect to the research plan, reviewers felt that it was well designed, but had mixed opinions on the feasibility of each research aim. With respect to the first aim, reviewers felt it likely that the principal investigator (PI) would obtain the desired iPSC lines. The PI has enlisted appropriate collaborators to obtain the required patient samples, and to advise on the reprogramming experiments. Assays for pluripotency are necessary and appropriate. One criticism is that the collaborator who will assist in reprogramming is from out of state, and the letter of collaboration did not detail any on-site assistance. This was felt to be an important gap in the proposal, since the PI does not have direct stem cell experience. While the reprogramming as planned should yield the desired cells, one reviewer felt that the alternative strategies for reprogramming are all high risk and their feasibility questionable.

Reviewers noted previous data from the PI supporting one aspect of feasibility of the second research aim, indicating a suppression of cardiac differentiation of murine embryonic stem cells (ESCs) lacking the function of the murine homolog of the signal transduction molecule mutated in NS and LS patients. Reviewers did raise concerns whether the PI has successfully established the methodology for cardiomyocyte differentiation from human (h)ESCs in the PI’s own laboratory, and another reviewer questioned whether the protocol would replicate in iPSCs. Finally, a reviewer questioned whether embryonic cardiac differentiation is an assay that represents the NS or LS phenotype, since the major cardiac problem with patients is pulmonary valvular stenosis, a structural defect.

Reviewers felt that evidence of a small molecule inhibitor of the signal transduction molecule presented in the preliminary data enhances the feasibility of the third research aim, to find small molecules that influence self-renewal, proliferation, and cardiac differentiation. However, it was not clear to reviewers that the PI would be able to find an agonist within the allotted time for the research proposal. One reviewer noted that insufficient emphasis is placed on experiments which utilize the pluripotent iPSC to probe the developmental defects associated with NS and LS.

The PI was judged to be a well-established investigator in the field of signaling, and is a recognized expert in the signaling pathways central to this proposal. The PI has an impressive track record both in publications and in his/her earlier work which is of central relevance to the present application. The PI has enlisted an appropriate collaborator to consult on reprogramming, though it would be useful to have local expertise involved in the project. The applicant institution was noted to have excellent facilities and research support infrastructure.

Reviewers felt the proposal was responsive to the RFA, and that it would likely lead to the derivation of new human iPSC lines, derived from patient fibroblasts. The applicant stated the intention to make the iPSC lines widely available to the scientific community once the lines have been shown to be stable and are well characterized.

In summary, the panel did not find a compelling case for the use of iPSC lines to study cardiomyocyte differentiation in these syndromes, and was not convinced of the feasibility of finding small molecules that could rescue the disease phenotype.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None