Sustained siRNA production from human MSC to treat Huntingtons Disease and other neurodegenerative disorders

During the first year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders. The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease. This year we have shown that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs through direct cell-to-cell transfer of siRNA, and we have filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. A manuscript documenting the results of these studies is in preparation. We continue to explore the precise methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-protein interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project and a collaborative manuscript is in preparation. During year two of the proposed studies we will continue to document levels of reduction of the toxic htt protein in different types of neurons, including medium spiny neurons (MSN) derived from HD patient induced pluripotent stem cells (iPSC). We have made significant advances in developing the tools for these studies, including HD iPSC line generation and MSN maturation from human pluripotent cells in culture. A manuscript on improved techniques for generating MSN from pluripotent cells is in preparation. We have also worked closely with our colleagues at the UC Davis MIND Institute to achieve improved maturation and electrical activity in neurons derived from human pluripotent stem cells in vitro, and we are examining the impact of human MSC on enhancing survival of damaged human neurons. In the second year of funding we will test efficacy of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of our newly developed strain of immune deficient Huntington's disease mice. This strain was developed by our teams at UC Davis to allow testing of human cells in the mice, since the current strains of HD mice will reject human stem cells. A manuscript describing generation of this novel HD mouse strain is in preparation, in collaboration with our nationally prominent Center for Mouse Biology. Behavioral studies will be conducted in this strain with and without the MSC/siRNA-mediated knockdown of the mutant protein, through years 2-3, in collaboration with our well established mouse neurobehavioral core at the UC Davis Center for Neurosciences. We have documented the safety of intrastriatal injection of human MSC in immune deficient mice and will next test the efficacy of human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo. As added leverage for this grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures. From the funded studies 4 manuscripts are now in preparation, a chapter is in press and a review paper on MSC to treat neurodegenerative diseases is in press.

During the second year of funding we have made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders. The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. During the second year we have more fully characterized our development candidate; MSC/anti-htt. We have documented that normal human donor MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neurons in vitro to achieve significant reduction in levels of the htt protein. We reported this work at the Annual meeting of the American Academy of Neurology (G Mitchell, S Olson, K Pollock, A Kambal, W Cary, K Pepper, S Kalomoiris, and J Nolta. Mesenchymal Stem Cells as a Delivery Vehicle for Intercellular Delivery of RNAi to Treat Huntington's disease. AAN IN10-1.010, 2011) and have recently completed and submitted a manuscript describing these results (S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Submitted, In Review, July 2011). We have explored the molecular methods by which the cell-to-cell transfer of small RNA molecules occurs, working in close collaboration with the national Center for Biophotonics Science and Technology at UC Davis. This Center is located across the street from our CIRM-funded Institute for Regenerative Cures (IRC) where our laboratory is located, and has equipment that allows visualization of protein-siRNA interactions in high clarity and detail. The proximity of our HD team researchers in the IRC to the Center for Biophotonics has been an important asset to our project. This work was also presented at AAN 2011, and a collaborative manuscript is in preparation for submission (S Olson, G McNerny, K Pollock, F Chuang, T Huser and J Nolta, Visualization of siRNA Complexed to RISC Machinery: Demonstrating Intercellular siRNA Transfer by Imaging Activity. MS in preparation, Presented at AAN 2011: IN4-1.014). In the second year of funding we developed the models for in vivo efficacy testing of the siRNA-mediated knockdown of the mutant human htt RNA and protein in the brains of established and new strains of Huntington's disease mice. Behavioral studies were conducted in two strains, the R6/2 immune competent mice and our new immune deficient strain, the NSG/HD, in comparison to normal littermate controls that are not affected by HD. We established the batteries of behavioral tests that are now needed to test efficacy of our development candidate in the brain, in year three. Established tests include rotarod, treadscan, pawgrip, spontaneous activity, nesting, locomotor activity, and the characteristic HD mouse hindlimb clasping phenotype. In addition we monitor the status of weight and tremor, grooming, eyes, hair, body position, and tail position, which all change over time in HD mice. These tests are conducted at 48 hour intervals by two highly trained technicians who are blinded to the treatment that the mouse had received. These behavioral and phenotypic tests have been established at the level of Good laboratory practices in our new Institute for Regenerative Cures shower-in barrier facility vivarium. We have documented the biosafety of intrastriatal injection of human MSC in immune deficient mice and are now examining the in vivo efficacy of the development candidate: human MSC engineered to continually produce the siRNA to knock down the mutant htt protein in vivo, which will be completed in year three. As added leverage for this funded grant program, and supported entirely by philanthropic donations from the community committed to curing HD, we have performed IND-enabling studies in support of an initial planned clinical trial that will use normal donor MSC (non-engineered) to validate their significant neurotrophic effects in the brain. These trophic effects have been documented in animal models. The planned study will be a phase 1 safety trial. We have completed the clinical protocol design and have received feedback from the Food and Drug Administration. We will be conducting additional studies in response to their queries, over the next 6-10 months, through a pilot grant obtained from our Clinical Translational Science Center (CTSC), which is located in the same building as our Institute. Upon completion of these additional studies we will submit the updated IND application to the FDA. MSCs for this project have been expanded and banked using standard operating procedures in place in the Good Manufacturing Practice Facility in the CIRM/UC Davis Institute for Regenerative Cures.

During the three years of funding we made significant progress toward the goals of the funded CIRM grant TR1-01257: Sustained siRNA production from human MSC to treat Huntington’s disease and other neurodegenerative disorders. The overall goal of the grant is to use human mesenchymal stem cells (MSC) as safe delivery vehicles to knock down levels of the mutant Huntingtin (htt) RNA and protein in the brain. There is mounting evidence in trinucleotide repeat disorders that the RNA, as well as the protein, is toxic and thus we will need to significantly reduce levels of both in order to have a durable impact on this devastating disease. We initially demonstrated that human MSC engineered to produce anti-htt siRNA can directly transfer enough RNA interfering molecules into neuronal cells in vitro to achieve significant reduction in levels of the htt protein. This is a significant achievement and a primary goal of our proposed studies, and demonstrates that the hypothesis for our proposed studies is valid. The transfer occurs either through direct cell-to-cell transfer of siRNA or through exosome transfer, and we filed an international patent for this process, working closely with our Innovation Access Program at UC Davis. This patent has IP sharing with CIRM. An NIH transformative grant was awarded to Dr. Nolta to further explore these exciting findings. This provides funding for five years to further define and optimize the siRNA transfer mechanism. A manuscript documenting the results of these studies was published: S Olson, A Kambal, K Pollock, G Mitchell, H Stewart, S Kalomoiris, W Cary, C Nacey, K Pepper, J Nolta. Examination of mesenchymal stem cell-mediated RNAi transfer to Huntington's disease affected neuronal cells for reduction of huntingtin. Molecular and Cellular Neuroscience; 49(3):271-81, 2012. Also a review was published with our collaborator Dr. Gary Dunbar: S Olson, K Pollock, A Kambal, W Cary, G Mitchell, J Tempkin, H Stewart, J McGee, G Bauer, T Tempkin, V Wheelock, G Annett, G Dunbar and J Nolta, Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s disease. Molecular Neurobiology; 45(1):87-98, 2012. We examined the potential efficacy of injecting relatively small numbers of MSCs engineered to produce ant-htt siRNA into the striata of the HD mouse strain R6/2, in three series of experiments. Results of these experiments did not reach significance for the test agent as compared to controls. The slope of the decline in rotarod performance was less with the test agent, and development of clasping behavior was slightly delayed after injection of MSC/aHtt, but this caught up to the controls and was not significant after day 60. Our conclusions are that the R6/2 strain is too rapidly progressing to see efficacy with the test agent, and also that improved methods of siRNA transfer from cell to cell are needed. We are currently working on this problem through the NIH transformative award, and will use the YAC 128 strain, which has a more slowly progressing phenotype, for all future studies. These mice are now bred and in use in our vivarium, for the MSC/BDNF studies funded through our disease team grant. Through this translational grant funding we have also developed in vitro potency assays, using human embryonic stem cell-derived neurons and medium spiny neurons, as we have described in prior reports. The differentiation techniques (funded through other grants to our group) have now been published:1-3 1. Liu J, Githinji J, McLaughlin B, Wilczek K, Nolta J. Role of miRNAs in Neuronal Differentiation from Human Embryonic Stem Cell-Derived Neural Stem Cells. Stem Cell Rev;8(4):1129-37, 2012. 2. Jun-feng Feng, Jing Liu, Xiu-zhen Zhang, Lei Zhang, Ji-yao Jiang, Nolta J, Min Zhao. Guided Migration of Neural Stem Cells Derived from Human Embryonic Stem Cells by an Electric Field. Stem Cells. Feb; 30(2):349-55, 2012. 3. Liu J, Koscielska KA, Cao Z, Hulsizer S, Grace N, Mitchell G, Nacey C, Githinji J, McGee J, Garcia-Arocena D, Hagerman RJ, Nolta J, Pessah I, Hagerman PJ. Signaling defects in iPSC-derived fragile X premutation neurons. Hum Mol Genet. 21(17):3795-805. 2012.