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RS1-00213-1: Generation of Inherited Disease Human Embryonic Stem Cell Lines

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

The development of human embryonic stem (hES) cell lines that carry a disease causing mutation can provide insight into the mechanisms underlying disease progression as well as into the development of therapies that can ameliorate that pathology. The primary goal of this proposal will be the development of novel hES cell lines from embryos that will manifest a given genetic disease upon further development. This will be achieved following two distinct approaches. The first will be through the identification of embryos that are homozygous for a given mutation and the generation of novel cell lines from these embryos. These embryos will be identified by preimplantation genetic diagnosis (PDG). The afflicted embryos will then be grown according to established protocols known to generate human ES cell lines. The second approach will involve the generation of disease associated homozygote cell lines through a technique that will specifically modify a gene sequence and introduce a disease associated mutation. This technique, small fragment homologous replacement (SFHR), has been shown to be effective at modifying DNA sequences in human cells. SFHR-mediated changes are caused by small DNA fragments (SDFs) that are introduced into the cells. The SDFs are effectively the same as the gene target sequences except for the changes to be introduced. Initial studies will target genes on the X-chromosome of normal male hES cells that carry only one X-chromosome. The genetic diseases that are anticipated to be served by this proposal include, but are not limited to, cystic fibrosis, sickle cell disease, ?- and ?-thalassemia, Duchenne’s and Becker muscular dystrophy, X chromosome-linked severe combined immune deficiency (SCID-X1), spinal muscular atrophy (SMA), Guacher’s disease, Fanconi anemia, and Lesch-Nyhan syndrome.

Statement of Benefit to California (provided by applicant)

This proposal will provide benefit to the citizens of California by increase our knowledge of the basis of genetic diseases and by providing a means to develop new and more effective therapies for these diseases. This project is focused on improving the health and well being of the citizens of California and could have far reaching positive implications for health and economic factors influencing the quality of life for the citizens of this state.

Review

SYNOPSIS: The principal goal of the proposal is to develop disease-speciific hESC that could be used to unravel mechanisms of disease and then lead to new therapies. Two methods are proposed. First, hESC lines from preimplantation genetic diagnosis embryos will be derived. Second, genes will be mutated (specifically two on the X-chromosome) by a specific replacement method. In the first instance, preimplantation genetic diagnosis embryos will be identified by collaborators in fertility. The availability of disease-specific hESC lines is a worthwhile goal.

SIGNIFICANCE AND INNOVATION: The generation of disease-specific hESC is an important step in considering the mechanisms of disease and new therapeutic strategies. However, the idea is neither novel nor innovative. The approaches to this goal include use of preimplantation genetic diagnosis embryos, somatic cell nuclear transfer, and gene targeting. This proposal deals with the first and last approach.

There has been little progress in this field to date so the studies proposed are potentially significant. Derivation of novel hESC lines from preimplantation genetic diagnosis embryos have been done by other groups but there is difficulty obtaining these cell lines, and additional disease models are very much needed and will be valuable to many investigators.

STRENGTHS: The Principal Investigator (PI) has made appropriate collaborative agreements for obtaining preimplantation genetic diagnosis embryos for hESC derivation. In addition, he/she has experience with the specific replacement method technique for gene modification.

Again, this is a very innovative and highly important area of investigation. The use of small DNA fragments is also novel, though the robustness of this approach is a little unclear compared to standard methods used to knock out genes. However, there are advantages for the approach used here and the PI has used this method with other stem cells. The ability to transfer this methodology into human ES cells now will be of interest not just for the disease models studied here but also for many other genetic disease models.

WEAKNESSES: There are several weaknesses. With regard to generation of hESC lines from preimplantation genetic diagnosis embryos, there is no information provided as to the numbers of cases and the types of diseases that are handled by the PI’s collaborators. Access to preimplantation genetic diagnosis embryos can be difficult in that there is no guarantee that these embryos will be available for the studies proposed. However, this should not be a very difficult hurdle. Also, the early studies are a nice compliment if the preimplantation genetic diagnosis embryos are in short supply. More information on the number and types of embryos that are actually available through the collaborative preimplantation genetic diagnosis clinic should be provided.

The major focus of the proposal appears to be on the generation of mutations in the X-chromosome genes. It is claimed that the proposed replacement method is more efficient than standard homologous recombination and also better in that no selection markers are used. While the PI has published on this method, there is little in the independent literature to substantiate the efficiency and advantage of this method. There is some skepticism on the part of one reviewer as to whether it works at all. Also, if the method is so efficient, it is unclear to the reviewer why the PI selected the genes he did for study. The primary rationale provided by the PI is their X-chromosome localization. But with an efficient method one might seek to generate double hits in other disease genes or alternatively single hits in dominant disorders for which the pathogenesis is less well understood. There is relatively little justification for why the approach is significantly better than standard approaches. More information on the benefits of the approach would be useful. Preliminary data showing that the replacement method actually is as efficient as claimed is important to include.

There is no description of what will be done with these genetically modified hESC lines after they are either made or derived. Specifically, there is no description of studies for differentiation of these cells either in vitro or in vivo such as teratoma formation. There is also no defined method to characterize the hESC including methods such as karyotype analyses, surface antigen expression, and typical gene expression.

The investigators do not have hands-on experience themselves in deriving hESC lines but should be able to work with collaborators to obtain this experience, they will need some time and effort to get this system up and running.

Additional information about what will be done with the new hESC lines is essential to make a more complete and comprehensive proposal. Also, some additional discussion on the relative strengths and weaknesses of the replacement approach in comparison to standard approaches would be helpful.

DISCUSSION: The reviewers noted that since Lesch-Nyhan (a disease focus of this application), is fairly well understood, the proposed new lines may not be that useful. In addition, a major weakness of the proposal was the apparent focus on the specific replacement method for generating mutations. Some skepticism was expressed that this method works since no other group has confirmed the technology yet. There was enthusiasm and support for deriving new lines, particularly disease lines.

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