The inner workings of the nervous system which regulate normal body movements, thought processes, feelings and senses are highly complex. How the nervous system relays and receives this variety of information is little understood, although significant inroads are being made to deduce underlying causes of many forms of neurological disorders. Many forms of retardation are caused directly by a failure of the cells within the nervous system to survive or work properly. One of the biggest limitations in this research is the inability to study the disease in the laboratory over the course of the disease. This is because neuronal or brain cells cannot be examined experimentally and ethically until postmortem tissues are obtained. Human embryonic stem cells (hESCs) can differentiate in the laboratory into many of the neural tissues within the human brain and spinal cord. Thus, they are a key to exploring human neurogenesis in vitro. In this proposal, we explore yet another vital use of hESCs, which is to study how neurogenesis is effected in vitro with cells carrying mutations that cause different forms of neurological dysfunction. To date, most of the hESC field has been devoted to deriving purified adult tissues for eventual transplantation therapies. Many hurdles remain, including methods to purify tissues, adequate means to avoid tumorigenesis, immunorejection, and transplantation. While using hESCs for regenerative purposes is the great hope of stem cell biology, the creation of disease specific hESCs is a more tangible and immediate means to both understanding disease and developing drugs to treat disease without having the tremendous cost and hurdles that remain for transplantation therapies. Thus, we argue that a vast and immediate effort should be placed upon the development of hESC lines with specific disease mutations that can be tested in vitro. In this grant we will develop more than 15 new hESC lines that carry mutations that cause neurological disease in children. Our goal is to develop, characterize and distribute hESCs as models for Hurlers Syndrome, Fragile X, Tay Sachs, and Canavan Syndrome so that researchers can study these diseases in the laboratory and test drugs to alleviate them.
Our goal is to develop human embryonic stem cells that carry mutations that cause neurological diseases in children, including Canavans, Hurler, Tay Sachs, Fragile X and Adrenoleukodystrophy. hESCs can differentiate into neural cell types which means that these lines would provide a valuable tool to study the mechanisms underlying these debilitating diseases and would provide a critical method for drug testing to alleviate the disease symptoms. There is no doubt the development of these hESCs as a tool to understand neurological diseases would benefit the citizens of California, both emotionally and financially.
The goal of this proposal is to derive, characterize and distribute more than 15 new human embryonic stem cell (hESC) lines containing mutations in disease loci that affect neurogenesis, with an emphasis on specific mutations that cause mental retardation in children. Four reasons are given for the derivation of these new lines, and these highlight the importance of the proposal 1) hESCs carrying these mutations would allow mechanistic examination of the affected neuronal tissues in a way not previously possible (need); 2) hESCs differentiate into neurons at a high frequency (feasibility); 3) diseases targeted by this proposal are commonly screened in pre-implantation genetic diagnosis (PGD) (coordination with clinicians); 4) the phenotypes of these diseases are highly penetrant, thus the phenotypes studied in different hESC lines carrying the same mutations are expected to be relatively consistent (feasibility). A collaboration with a PGD laboratory is cited, as well as a derivation success of four new lines with a success rate of 1 in 10. The new lines will be fully characterized and the proposal contains a detailed sharing plan to make the lines available to researchers.
It is clearly important to obtain defined hESC lines harboring genetic causes of different diseases, and those that cause mental retardation would be a very valuable resource given the protocols for neural differentiation of hESC. The use of PGD embryos is the logical way forward. Though a few of these lines exist, they are not readily available. Importantly, the principal investigator (PI) has developed a relationship with a large commercial laboratory that is a leader in PGD and thereby has access to a large number of in vitro fertilization (IVF) clinics. This innovative and impressive collaboration represents an unprecedented opportunity to identify a relatively large number of embryos affected by genetic causes of mental retardation and use them to derive hESCs, all within the guidelines of the National Academy of Sciences. Despite the strength of the collaboration, one reviewer pointed out that insufficient evidence was presented to convince him/her that the number of PGD embryos needed for 15 new lines would be available. The diseases targeted are very rare, and no data on the frequency of PGD is provided either in the application or collaboration letter. Furthermore, concern was raised about the fact that the diseases cited in the letter from the PGD collaborator do not match those targeted in this application, suggesting insufficient communication between the investigators. Overall, though, the significance of this proposal was deemed high, and the work will likely result in expanding the availability of hESC lines with genetic mutations causing mental retardation. This goal was considered a high priority and unique opportunity in the field.
To achieve the proposed aims, the most critical steps will be obtaining consent for using affected embryos and then transporting the embryos from the various IVF facilities to the institution’s hESC facility. The investigators provide a plan to establish a strong and collegial relationship with the IVF clinics, which will be of utmost importance to allow this process to occur in such a short time frame (~2-3 days).
The PI and collaborators are highly qualified experts, with outstanding track records in the field, and the PI’s institution is a perfect setting for these studies. The proposed research is likely to generate hESC lines that have the ability to differentiate into all three germ layers. The plan to share these newly derived lines with researchers through a website and publicity through disease advocacy groups is adequate.
The goal of this proposal is to derive, characterize and distribute more than 15 new human embryonic stem cell lines containing mutations in disease loci affecting neurogenesis, with an emphasis on those causing Canavan’s, Hurler, Tay-Sachs, Fragile X and Adrenoleukodystrophy in children. Four reasons are given for the derivation of these new lines: 1) hESCs carrying these mutations would allow mechanistic examination of the affected neuronal tissues in a way not previously possible; 2) hESCs differentiate into neurons at a high frequency; 3) diseases targeted by this proposal are commonly screened in PGD; 4) the diseases the application is focused on are highly penetrant to ensure less variation in phenotype effect between hESCs carrying the same mutation. A collaboration with a PGD lab is cited, as well as a derivation success of four new lines with a success rate of 1 in 10.
Reviewer One Comments
The investigators aim to derive hESC with genetic mutations that cause mental retardation (MR). hES cells harboring genetic causes of MR would be useful to researchers. Though a few of these lines exist, they are not readily available. The investigators have developed a relationship with Genesis Genetics, a large PGD laboratory, which identifies 30-70 embryos over a 3 year period for disorders such as Fragile X, Canavan’s, ALD, Hurler, Tay-Sachs. This collaboration is an innovative approach to identify a relatively large number of embryos affected by genetic causes of MR and use them to derive hESCs. The lines will be fully characterized and then be made available to researchers.
To achieve the proposed aims, the most critical steps will be obtaining consent for using affected embryos and then transporting the embryos from the various IVF facilities to the Stanford hESC facility.
During the first year of the project, the IVF coordinator will play a key role in ensuring these relationships are developed and maintained. The IVF coordinator will travel to each new IVF clinic to have a meeting to discuss the efforts and consenting plan with the local IVF coordinator/nurse and team. A small financial incentive will be offered to the participating IVF clinics to compensate for any time devoted to consenting and shipping embryos and will work toward fostering good relationships with all participants. Though Genesis Genetics has worked with over 200 IVF clinics, the majority of their work comes from approximately 15-20 of these clinics. The majority of networking efforts will be focused on this subset of the clinics.
After an embryo has been diagnosed, the IVF coordinator of the study (or local IVF staff) will obtain consent to use the embryo for research. After consent has been obtained, the embryos will be shipped to the derivation facility at Stanford and used immediately. The investigators point out that this strong and collegial relationship with the IVF clinics will be critical to allow this process to occur in such a short time frame -- a 2-3 day window approximately.
The hES cell derivation experience of the investigators and the facilities at their disposal appear more than adequate. The relationship with Genesis Genetics will allow access to many embryos harboring disease. Networking with the IVF facilities and having a means to effectively communicate with patients to enroll them in a timely fashion after the embryos are diagnosed will be of utmost importance. The investigators may want to consider obtaining consent while the couple is undergoing IVF and prior to the actual testing, to avoid the narrow time window between genetic diagnosis and using the embryo for hES cell derivation.
Responsiveness to RFA:
The proposed research will likely generate truly pluripotent human stem cell lines. The applicant provides an adequate plan to share or commercialize the new cell lines to make them available to other researchers.
Reviewer Two Comments
In an innovative approach to identifying targeted embryos through a collaboration with Genesis Genetics, the significance of this proposal is high, and will likely result in expanding the availability of hESC lines with genetic mutations causing mental retardation. This represents a high priority in the field.
This proposal includes three specific aims. The first is to derive hESC with genetic mutations that cause neurological disease in children. The second aim is the expansion and characterization of derived hESCs. The third aim is distribution of the new lines to researchers and clinicians.
The PI and collaborators are outstanding researchers, with significant track records in the field. Stanford University is a perfect setting for these studies.
Preliminary results include two key areas. The first is the successful derivation in a collaborative effort (between the Baker and Behr Labs) of four hESCs in standard and xeno-free conditions, with a success rate of just over 9% (4 lines generated from 43 embryos). The second area of preliminary results is the PGD analysis of neurological disease conducted at Genesis Genetics. (Letter of collaboration is included in the application).
The success of this proposal depends upon obtaining consent from the IVF facilities, and it seems that the investigators have structured their collaboration to make this hurdle surmountable.
Responsiveness to RFA:
This proposal is likely to generate hESC lines that have the ability to differentiate into all three germ layers.
The plan to share these new derived lines with researchers through a website and publicity through disease advocacy groups is adequate.
Reviewer Three Comments
It is clearly important to obtain defined hES cell lines from different diseases, and those that cause mental retardation would be a very valuable resource, given the protocols for neural differentiation of hES cells. The use of PGD embryos is the logical way forward.
The strengths are the links with a large PGD company, the expertise of the applicants in hES cell work and the straightforward research plan – derivation of new lines.
However there is not enough evidence presented to be confident that they will get the number of PGD embryos they need for 15 new lines. The diseases targeted are very rare, and no data on the frequency of PGD is provided either in the application or cover letter. They “estimate” 30-70 embryos/yr – where is the proof I would expect for a $1.5M award? It is telling that the collaboration letter from the PGD company talks about availability of embryos for common AD diseases, and not the diseases that form the target of this application!