Funding opportunities

Establishing Noonan and Leopard syndrome patient-specific iPS cell lines

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00647
Funds requested: 
$1 588 224
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
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: 
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 Summary: 
Executive Summary 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. Reviewer Synopsis Noonan syndrome and Leopard syndrome are autosomal dominant, multisystem diseases caused by different missense mutations in the protein tyrosine phosphatase SHP2, a component of several intracellular signal transduction pathways involved in embryonic development that modulate cell division, differentiation, and migration. Both syndromes represent a complex of features, mostly involving the skin, skeletal and cardiovascular systems and there is an increased risk of malignancy. Despite the similar disease phenotypes, mutations in SHP2 in NS patients typically result in a dominantly active form of the enzyme while those in LS patients result in a catalytically inactive form of SHP2. A knockin mouse model of NS displays only a portion of the features of the human disease highlighting the current lack of understanding of the molecular pathogenesis of NS and LS. The aim of this project is to establish pluripotent cell lines from dermal fibroblasts derived from NS and LS patients using iPS Reviewer One Comments Significance: 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. While epidemiological data is not available for Leopard syndrome due to its rarity, Noonan syndrome 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. Both syndromes are complex, affecting multiple organs including the cardiovascular system and at this time although both are associated with missense mutations in a single gene, SHP2, precise understanding of the molecular pathologies responsible for either syndrome is lacking. The research proposed in this application if successful will provide a tremendously useful resource in the form of pluripotent cell lines derived from reprogrammed NS and LS patient somatic cells that would represent valuable tools for the research community and pharmaceutical industry. Feasibility: The research plan is well designed for the most part and is supported by a significant body of preliminary data. The research proposed is likely to yield valuable new patient specific iPS lines and their use as a platform for examining the molecular defects associated with NS and LS will represent an important proof of principle of the use of iPS technology to provide insights into the nature and molecular pathology of complex human diseases. The research is divided into three specific aims: In Aim 1, the applicant proposes to establish and characterize iPS lines by reprogramming fibroblasts from NS and LS patients. Given the participation of Dr Huang in providing the patient samples and the contribution of Dr Yu from the Thomson lab in the lentiviral mediated reprogramming, there is little doubt as to the feasibility of this aim. • The proposed alternative approaches to reprogramming (adenoviral delivery; use of recombinant transcription factors tagged with PTDs; cocktails of inhibitors to attempt to prevent ES differentiation) are high risk and their feasibility is questionable. • The various experimental approaches proposed to establish pluripotency of the iPS lines (microarray assay of global gene expression; epigenetic status of OCT4, NANOG and SOX2; in vitro differentiation assays; teratoma formation in SCID mice) are necessary and appropriate. • While the applicant has successfully demonstrated differentiation of murine ESC into cardiomyocytes, a concern in Aim 2 is whether the applicant has successfully established the methodology for cardiomyocyte differentiation from hES cells reported by Laflamme and colleagues (Nat Biotech, 2007). This is key to the success of the studies proposed under Aim 2. • Under Aim 3, the applicant proposes to uses small molecule inhibitors/activators of Shp2 function as a ‘pharmaceutical intervention’ to rescue the developmental defects resulting from defective Shp2 catalytic activity. Evidence of a small molecule inhibitor of Shp2 is presented in the preliminary data which certainly enhances the feasibility of this aim. Conceptually Aim 3 represents a very reasonable and appropriate approach but there are some concerns as to its feasibility and the experimental design. Will the applicant have suitable small molecule agonists of Shp2 function within the time-span of funding? While the proposed studies analyzing the effects of the small molecule inhibitors on the proliferation and self-renewal of the NS and LS derived iPS cells are appropriate and may yield useful data, insufficient emphasis is placed on experiments which utilize the pluripotent iPS cells to probe the developmental defects associated with NS and LS. The PI Dr Feng, a Professor at the Burnham Institute for Medical research is ideally positioned to conduct the proposed studies. A well-established investigator with a very impressive track record in both publications, his earlier work contributed to the discovery of Shp2 which is of central relevance to the present application. The applicant has considerable experience in dissecting molecular signaling events in ES and hematopoietic cells and has established a solid track record in this area. Dr Feng has assembled a very competent group of investigators to conduct the research outlined in the current proposal. The collaboration with Dr Junying Yu from the Thomson lab at the University of Wisconsin-Madison is key to the success of the studies devoted to generating iPS cells from the NS and LS patient fibroblasts. Dr Huang, also at the Burnham Institute, will provide fibroblast samples from the NS and LS patients. Several postdocs (Drs. Raquil and Wang) and a postdoctoral research assistant are also contributing to the project. In terms of the experience and track record of the investigators involved in this project coupled with the access to the excellent facilities and research support infrastructure available at the Burnham Institute, there is little doubt as to the feasibility of the majority of the stated aims of this application. Responsiveness to RFA: There is little doubt that this proposal will yield pluripotent stem cell lines. The applicant has stated his intention is to make the iPS lines widely available to the scientific community once they have been shown to be stable and are well characterized. Reviewer Two Comments Significance: The investigators propose to derive induced pluripotent stem cell lines from Noonan syndrome and Leopard syndrome patient cells, not clear why these were chosen over other genetic diseases but could be significant. Feasibility: • reprogramming feasible, it is likely that they will be successful in generating these cells. • collaboration: Yu will give the plasmids; no expertise in human ES cell culture. I am not sure that this is really a collaboration. Yu states that plasmids will be shared but there is no plan on how a collaboration will work. • differentiation into cardiac myocytes is feasible, however it is not clear how the experiments will be conducted. • The tests for shp2 inhibitors appear feasible and interesting. • It is not clear why iPS cells are really necessary since other cell lines are already available • Phenotype is complex and it is not clear how this will be studied using simple cell lines. Reviewer Three Comments Significance: This is a project to develop iPSC lines from patients with Noonan Syndrome (NS) or Leopard Syndrome (LS). Many of these patients have mutations in the PTPN11 locus, encoding the Shp2 phosphatase. These patients suffer from a variety of issues, including cardiomyopathy and susceptibility to leukemia. The syndromes are related in that they represent as autosomal dominant via dominant activated (NS) or dominant negative (LS) Shp2 variants. The goal is to develop iPS to understand the pathogenesis of these diseases and to provide assays to screen for drugs to ameliorate disease. It is however a bit unclear why iPS are needed for this purpose, or that these are stem cell based disorders. Strong mouse models can (and have been) generated, and cells can be obtained from patients. Feasibility: Aim 1 is to generate the iPS cells. The expertise of the PI is in signaling, for which he is a recognized Shp2 expert. He will be assisted by Dr. Yu, who was the first author on the Thomson human iPS paper. Dr. Feng is an expert at cell signaling and starting to work in the area of ES cells at the Burnham. Dr. Yu is in Wisconsin, and will send the viruses (Oct3/4, Sox2, Nanog, lin28) and provide consult. It would be useful to have local expertise involved in the project. The goal is to derive 5 NS and 5 LS lines from patients, presumably (although this is not indicated) from those who carry Shp2 mutations. The lines will be extensively characterized by markers, ChIP assays, EBs, and teratoma assays. The PI will also plan to generate mouse KIs with specific Shp2 mutations, although this is not relevant to the Aim. In the second Aim, the Laflamme protocol will be used to generate cardiomyocytes from control and patient iPS lines. Previous data from the PI indicated a suppression of cardiac differentiation from Shp2 KO mES, although this might represent a general developmental delay. There are several concerns. It is unclear that the mES KO data is relevant to NS or NS isoforms, or that the Laflamme protocol will replicate in the iPS cells. Most importantly, it is unclear that embryonic cardiac differentiation is an assay that represents the NS or LS phenotype. The major cardiac problem with patients is pulmonary valvular stenosis (50%) with a lower (10%) incidence of septal defects. Is there data that cardiac specification or differentiation markers are altered in patients? Experiments in the third Aim will attempt to rescue the cardiac (or other ESC related) phenotypes with small molecule inhibitors, derived from an ongoing screen. Molecules will be tested for effects on self-renewal, proliferation, cardiac differentiation etc. Of course, this will depend on if there is an in vitro phenotype, but is otherwise feasible. Responsiveness to RFA: The proposal is likely to lead to the derivation of new human iPS lines, derived from patient fibroblasts. The rationale for doing this is justified, but is not extremely compelling, since strong mouse models have already been developed (Robbins, Neel, et al.), and the ability to phenocopy elements of these disorders, in particular valvular disease, might be quite limited.
Conflicts: 

© 2013 California Institute for Regenerative Medicine