Novel Highly Accurate Single Cell Genotyping Technology for use in Discovering Late Onset Disease-Specific Stem Cell Lines and Monitoring Aneuploidy in Developing Stem Cell Lines
Tools and Technologies I
The creation, propagation, understanding and free distribution of novel human embryonic stem cell (hESC) lines are urgently needed in order to help medical researchers find treatments for many debilitating diseases. We propose to develop and implement a tool that can simultaneously determine the identity of hundreds of genes and also detect for aneuploidy at all 24 chromosomes in a single cell, something not previously possible to do simultaneously, and each with far greater accuracy than current methods. This will have wide ranging application in the field of stem cell research. We will demonstrate our technology on two applications. First, we will use it to try and understand the prevalence and characteristics of spontaneous aneuploidy (a change in the number of chromosomes), in developing stem cell lines, currently a significant problem. Second, we will use it to identify embryos harboring interesting disease genes and develop them into stem cell lines. Aneuploidy plagues a large percentage of normal and disease hESC lines, rendering them unsuitable for clinical applications. Very little is known about aneuploidy in early stage hESC development. Our technology, which can detect aneuploidy with high accuracy from a single cell, will be used to monitor ploidy in developing stem cell lines to provide insight into this problem. Disease stem cells are a very important tool for studying diseases because they respond to treatments more similarly to humans than do other models. Preimplantation genetic diagnosis (PGD) of embryos during in vitro fertilization (IVF) is the most effective method for derivation of disease-specific hESC lines. A major drawback of current PGD methods is their inability to detect both aneuploidy and multiple genes known to cause a disease. Since most PGD screens either for aneuploidy or for diseases that develop during early childhood, there is very little opportunity to serendipitously discover embryos predisposed to diseases that develop during adulthood. Because ours is the only method that simultaneously measures both multiple disease-linked genes and aneuploidy in a single cell isolated from an embryo during PGD, we will be uniquely positioned to routinely screen embryos during IVF for genes causing a predisposition to important diseases such as diabetes, Alzheimer’s disease, type II diabetes, and breast cancer. In collaboration with our key California IVF clinic partners we will apply our technology to screen thousands of embryos per year. With parental consent, embryos that test positive for susceptibility to diseases of interest will be used to derive two novel hESC lines that will be made available for free distribution to the research community worldwide. If funded, our research will result in several critical contributions to the field of stem cell research, ranging from empowering scientists to answer fundamental biological questions to facilitating the development of targeted therapeutics.
Statement of Benefit to California:
Our company is headquartered in [REDACTED]. We provide technical, high-salaried employment for dozens of California residents. With Series A venture backing from Sequoia Capital and Claremont Creek Ventures, and we have outlined an ambitious plan for growth, promising to bring even more jobs to the state. Additionally, we have close strategic partnerships with three California-based IVF clinics: [REDACTED] These IVF clinics, and their patients, will be among the first beneficiaries of our ground-breakingtechnologies. Most importantly, if this grant is funded, our company will be able to develop and validate a new technology for high accuracy genotyping of embryos. This technology will have many applications in the field of stem cell research, two of which we will demonstrate with this grant. First, in collaboration with the [REDACTED] Department of Genetics, we will help bring novel human embryonic stem cell lines to California scientists. These disease-specific lines will be a superb resource for Califonia researchers working to understand, prevent, and cure debilitating genetic diseases. Second, we will do basic research into the prevalence of aneuploidy in developing stem cell lines. These resources and knowledge will help keep California at the forefront of stem cell research worldwide, enriching our local economy and earning further prestige for nonprofit research institutions. Local drug companies will benefit from local resources in nonprofit stem cell research. Finally, California residents couldeventually benefit from advances in medicine that result from this important local biological resource.
This proposal aims to develop and apply a tool that can simultaneously assess the identity of thousands of genes and detect abnormal copy numbers among all 24 chromosomes from a single cell with high accuracy. The Principal Investigator (PI) intend to use this technology to understand the prevalence and characteristics of spontaneous aneuploidy (i.e., a change in the number of chromosomes) in developing stem cell lines and also to identify embryos harboring interesting disease genes for subsequent derivation of disease stem cell lines. The PI and research team feel uniquely positioned to routinely screen embryos during IVF for genes related to a variety of important diseases and hope to apply the technology to screen thousands of embryos per year. The goal of this study is to generate at least two novel human embryonic stem cell (hESC) lines that will be made available for free distribution to the scientific community as a tool for basic research or to aid in the development of targeted therapeutics. Reviewers agreed that this proposal addresses significant issues with respect to the genomic stability of hESCs, and also to the derivation of disease-specific lines from discarded embryos or from stem cells obtained for preimplantation genetic diagnosis (PGD). Technologies to quickly, cheaply, and reproducibly screen hESCs for genetic changes during routine cell growth are required, particularly as hESCs move towards clinical development. Reviewers felt that the technology has obvious benefits for fertility clinics, and potentially also for oncology and other areas in which assessment of the genomes of individual cells would be of great value. The proposed work has the potential to therefore advance both research and clinical translation. Reviewers also felt that the technology was novel and that the research plan was well designed and likely to generate important data. The applicants will screen embryos and hESCs at multiple gene loci using a proprietary technology that eliminates the ‘noise’ created in genotyping data when the starting material is very limited (one cell). Reviewers acknowledged that the potential impact of this analytic approach could be large, including the generation of new cell lines that carry mutations for diseases that are not routinely screened for in PGD. It was also noted that the value of generating new disease stem cell lines from PGD might be dampened by the current availability of induced pluripotent stem cell (iPSC) technology, which can be readily used to generate the proposed disease lines. Generation of iPSC lines may be advantageous in circumstances where one can derive cells from individuals where a specific disease has actually manifested rather than from cells that are genetically predisposed. The PI was noted to be a talented investigator with a mix of academic and entrepreneurial interests. The PI’s expertise is primarily in engineering and s/he has an impressive publication and patent record, and also appears to have been successful in specific applications in genomics. Other team members provide complementary skills and appear well qualified, though their precise roles on the project were insufficiently described, and costs associated with the project were not appropriately justified. For example, a consultant providing 10% effort was budgeted for $100,000. Some concern was also raised over a support letter that was greater than a year old. PROGRAMMATIC REVIEW A motion was made to move this application from Tier 2 (Recommended for Funding if Funds Are Available) to Tier 3 (Not Recommended for Funding). Reviewers recognized the proposal’s technical merit, but they had grave concerns about how and why this technology is being developed and whether this type of research should be funded by CIRM. The technology is being developed as a commercial service to genetically screen embryos on an almost industrial scale. In the proposal, the PI states that, starting this year and rapidly ramping up, s/he anticipates using the technology to screen thousands of embryos per year from IVF clinical partners. The large-scale genome-wide screening of embryos other than for diagnosing the presence or absence of a specific disease-causative genetic mutation, has potentially grave ethical consequences that were not addressed by the PI, though a prominent ethicist contributed a letter of support. The PI provides very little information as to how this technology will be used in clinical practice, the nature of the patient population referred for screening and whether there is any ethical oversight or governance of the IVF partners’ clinical practice and genetic screening. Without assurances of strict ethical oversight, reviewers were concerned that CIRM should not fund the application despite its technical strengths. Additionally, reviewers viewed the benefit of this technology to be clearer for IVF clinics than for any immediate impact on the field of stem cell biology. The motion carried.