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.