CIRM Quick Links
Right Column
RL1-00678-1: New Cell Lines for Huntington's Disease
Recommendation: Not recommended for funding. Recommended to be available for funding only if the ICOC makes a programmatic determination to fund this application
Scientific Score: 69
First Year Funds Requested: $455,400
Total Funds Requested: $1,369,800
Public Abstract (provided by applicant)
Huntington’s disease (HD) is a devastating neurodegenerative disease with a 1/10,000 disease risk that always leads to death. These numbers do not fully reflect the large societal and familial cost of HD, which requires extensive caregiving and has a 50% chance of passing the mutation to the next generation. Current treatments treat some symptoms but do not change the course of disease. Symptoms of the disease include movement abnormalities, inability to perform daily tasks and and psychiatric problems. A loss os specific regions of the brain are observed. The mutation for HD is an expansion of a region of repeated DNA in the HD gene and the longer the repeat, in general the earlier the onset of disease. While the length of this polyglutamine repeat largely determines the age-of-onset, there is variance in onset age that is not accounted for by repeat length but is determined by genetic and environmental factors. In addition, the symptoms can vary significantly among patients in a non-repeat dependent manner. To assist in preventing onset of HD, there is a great need to identify genes that are involved in why one individual with 45 repeats will manifest symptoms at age 40 while another manifests symptoms at age 70. Further, there is a lack of early readouts to determine when to begin HD treatments. Because the disease mutation is known, preimplantation genetic diagnosis (PGD) is possible and mutant Htt embryos are available. Stem cell lines can be derived from PGD embryos with varying repeat lengths and genetic backgrounds to provide new methods to identify genetic modifiers and readouts of disease progression. The development of pluripotent stem cells, termed induced pluripotent stem cells (iPS) cells, derived directly from HD patient fibroblasts, would also provide new methods for these analyses. Chemical compound screens to identify drugs that protect against the effect of mutant Htt protein expression in patient derived hESCs cells would allow evaluation of drug responses in on cells having different genetic backgrounds Ultimately, the iPS cells can provide a way to transplant neurons or neuronal support cells from affected individuals or from unaffected family members having a normal range repeat. Such cells would help reduce immune rejection effects likely to occur with transplantation, however, while patient-derived cells overcome the problems of immune rejection, the mutant protein is still expressed. To overcome this problem we will genetically modify these stem cells to reduce the mutant protein and produce a normal gene. Beyond the immediate application to HD, the development of these models is applicable to a range of neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases.
Statement of Benefit to California (provided by applicant)
The disability and loss of earning power and personal freedom resulting from Huntington's disease (HD) is devastating and creates a financial burden for California. Individuals are struck in the prime of life, at a point when they are their most productive and have their highest earning potential. Further, as the disease progressives, individuals require institutional care facilities at great financial cost. Therapies using human embryonic stem cells (hESCs) have the potential to change the lives of hundreds of individuals and their families, which brings the human cost into the thousands. Further, hESCs from HD patients will help us understand the factors that dictate the course of the disease and provide a resource for drug development. For the potential of hESCs in HD to be realized, a very forward approach such as that proposed will allow experienced investigators in HD and stem cell research and clinical trials to come together and create cell lines to more fully mimic the diseases neurons and allow for future treatment options. The federal constraints on hESCs create a critical need for the development of treatments using hESCs supported and staffed with non-federal funds. We have proposed goals and strategies for generating new stem cells derived from patient preimplantation diagnosis embryos and patient fibroblasts. We have put in place critical milestones to be met We will build on existing regional stem cell resources . Anticipated benefits to the citizens of California include: 1) development of new stem cell lines that will allow us to more closely model the disease for mechanistic studies and drug screening, 2) improved methods for following the course of the disease in order to treat HD as early as possible before symptoms are manifest; 3) development of new cell-based treatments for Huntington's disease with application to other neurodegenerative diseases such as Alzheimer's and Parkinson's diseases that affect thousands of individuals in California; 4) development of intellectual property that could form the basis of new biotech startup companies; and 5) improved methods for drug development that could directly benefit citizens of the state.
Review
In this application, the applicant proposes to use stem cell technology to develop an in vitro model of Huntington’s disease (HD), and to produce cells that could be used to replace dead and dying cells in HD patients. New cell lines will be derived from two sources: 1) from embryos from HD patients undergoing pre-implantation genetic diagnosis (PGD) in Aim 1, or 2) using induced pluripotent stem cell (iPS) technology using fibroblasts from patients. Cells will be differentiated into neurons, and specifically into neuronal sub-types involved in HD. In Aim 3, the applicant proposes to reduce expression of the mutant Huntingtin protein (Htt) using shRNA technology in human embryonic stem cells (hESCs) or iPS cells from HD patients, and then express the wild type protein in these cells. These cell lines can then be used to search for biomarkers of the disease in culture or for modifier genes that may be involved in development of the disease, as well as to provide cells that could be used for transplantation.
This is a clear, well-written proposal from an expert in HD. The proposal outlines experiments geared toward taking the first steps in developing a hES cell-based therapy for HD. The significance of this proposal is high, although reduced due to the fact that there are HD lines already in existence. Reviewers’ enthusiasm for this proposal was lessened by the final Aim, as the feasibility of the gene therapy approach was debated. There were also questions regarding the ability to generate striatal neurons from stem cells.
Most of the criticism of this grant came over Aim 3, in which the applicant proposes to “cure” HD in patient-derived iPS cells. Reviewers commented that this is a great idea and if feasible might be a step towards developing a therapy for this disease. However, there are a number of potential problems with this aim. An shRNA-resistant gene needs to be placed in the iPS cells since the shRNA will knockdown both the endogenous wild type (wt) and mutant copies (as pointed out by the applicant). Due to the large size of the Htt gene (~10kb), the applicant proposes to use technology derived at a company, and lists the company web page address as a reference. On the stated web page there is not enough information to convince a reviewer that the Htt cDNA can be produced as proposed.
Reviewers commented on the lack of preliminary data from the lab for generating iPS cells and, more importantly, on the lack of evidence to support the claim that striatal neurons can be selected for in the population of differentiated neurons derived from the human ES cells, a criticism that weakened the entire grant.
A minor concern expressed by reviewers was that the use of shRNA to knockdown gene function has the potential to target other genes. The groups’ data using the mouse model clearly shows that shRNA can knockdown Htt expression. In patient-derived iPS cells, it will be essential to demonstrate that the shRNA used to target Htt is specific for this locus and does not result in off-target effects.
There are a number of strengths in this application. Deriving pluripotent stem cells both from embryos and by reprogramming of somatic cells should ensure that HD cell lines are obtained, although as mentioned above HD lines are already in existence. The plans to take samples from patients and unaffected siblings (who have presumably been genotyped in a predictive testing program) will provide an interesting resource for studies of the effect of the CAG repeat expansion, the molecular cause of HD, on genomic and proteomic characteristics of the cell. Reviewers were interested in the possibility of comparing samples from other family members with similar repeat sizes but where the age of onset was rather different, so as to get a handle on what determines the variability in age of onset for a given repeat size. However this experiment was not suggested by the applicant. Finally, reviewers were intrigued by the antisense knockdown of endogenous Huntingtin with simultaneous expression of another shRNA resistant form, which would provide proof of concept for genetic engineering to treat other autosomal dominant diseases. However, some reviewers were skeptical that cells generated through iPS and manipulated genetically would be acceptable therapeutic tools to the regulatory authorities.
Aims 1 and 2, deriving HD hESC lines and iPS lines, should be feasible. These reagents would be very useful for investigating HD. The direct comparison of iPS lines derived from presymptomatic and symptomatic HD patients could be used for a number of interesting experiments.
Other strengths were that the applicant is a senior investigator and expert in Huntington’s disease who is very well funded and has a very good publication record. S/he is clearly a leader in the field and, in collaboration with the two named colleagues, has the experience to carry out the proposed work. Additional listed collaborations will be helpful with the shRNA work. The applicant has access to all the required equipment and facilities.
In summary, doubts about the collaborating company’s technology and the potential problems of off-target effects in Aim3 detract from the proposal. There were also questions regarding the source of embryos, as exclusion testing would not yield acceptable material. Finally, reviewers were concerned that it would be difficult to generate striatal neurons from stem cells. These concerns weakened the application considerably.
Programmatic Discussion
During programmatic review, reviewers discussed this application due to its focus on HD. Reviewers were supportive of the investigator's strong HD background, and the importance of comparing pluripotent cells using PGD and iPS techniques. However, the significance of this approach was questioned as there are already a number of freely-available HD lines (panelists could think of 8). Reviewers felt that programmatic reasons were not strong enough to override their previous vote, given that the experiments proposed are technically challenging and that there were questions about the ability to produce striatal neurons. Therefore, the review panel did not make a motion to move this application into the funding category.
- None