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:
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.
SYNOPSIS: The principal goal of the proposal is to develop disease-speciific hES cells that could be used to unravel mechanisms of disease and then lead to new therapies. Two methods are proposed. First, derivation of lines from preimplantation genetic diagnosis embryos will be done. Second, genes will be mutated (specifically two on the X-chromosome, Lesch-Nyhan and IL2RG) by small fragment homologus replacement method (SFHR). In the first instance, PGD embryos will be identified by fertility collaborators (Drs Main and Chenette). The availability of disease-specific hES cell lines is a worthwhile goal.
SIGNIFICANCE AND INNOVATION: The generation of disease-specific hES cells is an important step in considering the mechanisms of disease and new therapeutic strategies. The idea though is not novel or innovative. The approaches to this goal include use of PGD embryos, SCNT, and gene targeting. This proposal deals with the first and last approach.
There has been little is progress in this field to date so the studies proposed are potentially significant. Derivation of novel human ES cell lines from PGD 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 PI has made appropriate collaborative agreements for obtaining PGD embryos for ES cell derivation. In addition, he has experience with the SFHR technique for gene modification.
Again, this is a very innovative and highly important area of investigation. The use of small DNA fragments to achieve homologous recombination is also novel, though the robustness of this approach is a little unclear compared to standard homologous recombination used to knock out genes. However, there are advantages for the SDF approach used here and the PI has used this method with mouse ES cells to create a model of cystic fibrosis. The ability to now transfer this methodology into human ES cells will be of interest not just for the disease models studied here (HPRT and the x-linked SCID mutation of the IL-2 receptor gamma gene) but would subsequently be practical for many other genetic disease models.
WEAKNESSES: There are several weaknesses. With regard to generation of hES cell lines from PGD 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. The major focus of the proposal appears to be on the SFHR-generation of mutations in the X-chromosome genes. It is claimed that SFHR is more efficient that 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 of which this reviewer is aware to substantiate the efficiency and advantage of this method. Frankly, there is some skepticism as to whether it works at all. Also, if the method is so efficient, it is unclear why the PI has selected the genes he has for study. The primary rationale 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 the which the pathogenesis is less well understood than Lesch-Nyhan disease of X-linked SCID.
There is no description of what will be done with these genetically modified human ES cell 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 human ES cells lines including methods such as karyotype analyses, surface antigen expression, and typical gene expression.
The investigators do not have hands-on experience themselves in deriving human ES cell 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.
There is relatively little justification of why the SDF approach is significantly better than standard homologous recombination approaches. More information on the benefits of the SDF approach would be useful.
Access to PGD embryos can be difficult in there is no guarantee that these embryos will be available for these studies proposed in Specific Aim 2. However, this should not be a very difficult hurdle. Also, the studies proposed in Specific Aim 1 are a nice compliment if the PGD embryos are in short supply.
More information on the number and types of embryos that are actually available through the collaborative PGD clinic should be provided. Also, preliminary data showing that the SFHR actually is as efficient as claimed is important to include.
Additional information about what will be done with the new ES cell lines is essential to make a more complete and comprehensive proposal. Also, some additional discussion on the relative strengths and weaknesses on the SDF approach in comparison to standard HR approach would be helpful.
DISCUSSION: The primary reviewer noted that since Lesch-Nyhan (a disease focus of application), is fairly well understood, the proposed new lines may not be that useful. In addition, the reviewer considered a major weakness to be the apparent focus of the proposal on SFHR for generation of mutations, skeptical that this method works since no it was believed that no other group has confirmed the technology. The secondary reviewer was enthusiatic and supportive of deriving new lines - particularly disease lines. The secondary reviewer was also concerned about the use of SFHR instead of 'standard' homologous recombination.
There was concern that the applicant did not discuss what would be done with lines once derived.
A discussant was concerned about the lack of feasibility for recombination technology. There was discussion about how much of a weakness the lack of cvonsideration of available PGD embroys was since the applicant might not have access to actual numbers of extant enbryos.
Some concern also focused on the fact that some of the published work on recombination has not been confirmed by others.