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.
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.
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.
Reviewer One Comments
This is one of a number of applications using the iPS technology to generate disease specific cells from patients. Here the target is Huntington’s Disease, using both pre-implantation genetic diagnosis (PGD) embryos found to have the abnormal expansion and also fibroblasts from patients. There are a number of strengths in this application.
1) The dual approach should ensure that cell lines are obtained.
2) The plans to take samples from patients and unaffected sibs (who have presumably been genotyped in a predictive testing program) will provide an interesting resource for studies of the effect of the expansion on genomic and proteomic behavior of the cell. Equally interesting though, it would have been to have taken 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.
3) The addition of an application for the cells generated; here the antisense knockdown of endogenous Huntingtin with simultaneous expression of another shRNA resistant form. As the authors point out, this will provide proof of concept for genetic engineering in autosomal dominant diseases, although I doubt that such cells will also be useful for transplantation as the authors claim in the first part of the grant.
Design and feasibility
The use of PGD embryos to create human ES cell lines is clearly feasible, although we are not told how many embryos are available. The section on generating iPS cells from fibroblasts is weakened by:
1) The lack of any preliminary data, although the techniques have been used in a number of labs
2) 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. As with many applications, the effect of multiple random insertions is not considered. Preliminary data is provided to support the shRNA experiments, but this is a very high risk part of the application and not one that is central to the success of the project.
One more general concern I have here is the assumption that the expanded repeat is stable with repeated passage in culture. I would have liked to have seen evidence that this is the case. Patients with myotonic dystrophy, another disease caused by genetic expansion, show significant variability from one tissue to the other in the size of the expansion, so it is possible that changes could occur as the iPS cells are repeatedly passaged and differentiated in culture. If so, the utility of these cells will be limited in that it will be essential to genotype each passage if studies of the effect of repeat size on cell behavior are being analyzed
Responsiveness to RFA:
Responsiveness to call
Pluripotent cells will be generated, although the assays of pluripotency and differentiation here have their weaknesses, as I have discussed above.
Reviewer Two Comments
This proposal outlines experiments geared toward taking the first steps in developing a hES cell-based therapy for HD disease. The significance is high. In addition, the successful completion of the Aims of this grant will potentially serve as a “proof-of-principle” for using hES cells to develop gene therapies for other neurodegenerative diseases.
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.
In Aim 3 the PI proposed to “cure” HD in patient-derived iPS cells. This is a great idea and if it could be done, would be a large step towards developing a therapy for this disease. However, there are a number of potential problems with this aim. First, it is not clear the role the company CODA is playing in this approach. An Htt resistant gene needs to be placed in the iPS cells since the shRNA will knockdown both the wt and mutant copies (as pointed out by the PI). Due to the large size of the Htt gene (~10kb) the PI proposes to use CODA-derived technology and lists the CODA web page address as a reference. On the CODA web page there is very little information about the company and they list only 5 references, none of which appear to address the technique that the PI will use in the proposed study. In addition, the PI states that CODA has made a 4,398 bp reagent for a different project. Since the Htt gene is more than twice this size, it is not clear if the same technique can be used for replacing the Htt gene. The PI states that CODA has made a 9 kb protein but there is no data to support this statement.
The use of shRNA to knockdown gene function has the potential to target other genes either through RNAi or the microRNA pathway. 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.
Doubts about the CODA technology and the potential problems of off-target effects, which are not addressed in Aim3, detract from the proposal.
Responsiveness to RFA:
The research plan should generate pluripotent human stem cell lines since the generation of iPS cells and hESC lines from embryos has been done before. The novel aspect of this proposal is that the PI proposes to accomplish the above task using HD embryos and HD fibroblasts. This approach should be successful unless expression of mutant HD protein in hES cells causes a defect in these cells. I have doubts that Aim 3 can be accomplished based on the data presented.
Reviewer Three Comments
Huntington’s disease is an important therapeutic target, hence the use of stem cell technology to explore its pathophysiology and therapy are highly relevant. This proposal is a classic case of the utility of stem cell technology, that is the development of an in vitro system where the target cells can be studied followed by the correction of the defect in ES or iPS derived neurons prior to their use in transplant-induced repair. The proposal contains approaches that are being used by many to isolate pluripotent cells and differentiate them toward specific neuronal populations. In that sense it is not novel yet still important to do. The novelty comes in the two-pronged genetic correction approach that is high risk yet might provide exciting new information.
This proposal has been clearly thought out and considerable detail has been given to the experimental plan. They will generate pluripotent cells from embryos and from fibroblasts from HD patients and confirmation of their pluripotency is satisfactorily described. Having both populations of cells will allow an ideal comparison between the two and the ability to isolate these cells in combination with PGD is a real plus. In Specific Aim 1 the final goal is to derive purified populations of striatal neurons from the ES cells, yet how they will achieve this is not exactly defined and I am not sure it has been reported by others. They do address, however, some of the other potential pitfalls and solutions. Their preliminary data shows that they have isolated blastocyst-derived ES cells and differentiated those toward a neuronal linage. Here, the collaboration with Drs. Donovan and Keirstead is extremely important.
In the second Aim they will utilize the current technology to create iPS cells. Although no preliminary data is shown here, given their combined experience, this should work. Specific Aim 3 carries a much higher degree of risk however. They have experience in shRNA knockdown as shown in the Preliminary Data and some experience with the expression of a synthetic gene using technology developed by the company CODA. They acknowledge however the size of Huntingtin protein is such that this is the riskiest aspect of the proposal.
The applicant is a senior investigator and expert in Huntington’s disease. She is very well funded and has a very good publication record. She is clearly a leader in the field and with her two named colleagues at UC-Irvine, has the experience to carry out the proposed work. In addition, the collaboration with Dr. B. Davidson of the University of Iowa will be helpful with the shRNA work. They have access to all the required equipment and facilities.
This proposal was well and clearly written and was one of the few to detail timelines and milestones that seemed reasonable.
Responsiveness to RFA:
This proposal clearly follows all the guidelines of the RFA and pluripotential cells should be produced that will be shared appropriately.
Reviewer Four Comments
The goal of this proposal is to create human cell-based models for Huntington’s disease (HD) by deriving human embryonic stem (hES) cell lines from embryos that carry an expanded CAG repeat in the HD gene as well as creating induced pluripotent stem (iPS) cells from HD patient fibroblasts. HD is a devastating and not uncommon disease with no cure. Reliable cell-based models for this disease would provide a means to study the cellular and molecular mechanisms underlying HD.
This proposal is clear, coherent and consistent in its design and objectives. It is also very focused, improving the overall chance for success. As the proposal relies mainly on validated and previously published protocols it is very feasible given the expertise of the group. The major weakness of the proposal is the lack of preliminary data demonstrating that the relevant neuronal cell-types (e.g. medium spiny neurons) can be generated from pluripotent cells.
This proposal is very responsive and is likely to produce new HD hES and hIPS cell lines.