Although our grant application was not funded, we received sufficiently encouraging reviews that we are now planning on revising and re-submitting the application for the March, 2018 review cycle. We believe that our proposed work is complementary to developments that are underway by commercial companies.
We have completed and submitted a grant application to the National Institute for Neurological Diseases and Stroke for continued pre-clinical studies of ways to improve delivery of DNA to the regions of the brain most affected in Huntington’s disease. The Scientific Review of the grant application by an NINDS study section will occur this coming fall.
Recently, we successfully completed a legal agreement giving us access to intellectual property, and we are now back to the task of determining how to best help facilitate the development of potential gene therapies for relatively rare central nervous system diseases, still focusing first on Huntington’s disease. We are working with academic collaborators to develop and seek NIH funding for pre-clinical studies to improve the delivery of DNA to the regions of the brain most affected in Huntington’s disease, including not only the striatum (caudate and putamen) but also the cortex. More news will follow in future months as these studies and grant applications are developed.
We have refunded $19,015 to the Hereditary Disease Foundation. This is because we determined that continuing with the project they funded us to do, involving the application of shRNA-encoding plasmids to patient-derived cells, would be unlikely to yield any useful information (see below).
We are disappointed with this outcome, but not irrevocably discouraged. The CGTA Research Group will continue to plan and conduct studies towards the goal of bringing a huntingtin-lowering gene therapy for Huntington’s disease to the people who need it.
Strategic planning is underway for our next projects.
With our collaborators, we decided that proceeding to conduct the intended main study with our plasmids would be unlikely to provide any useful information one way or the other regarding whether the huntingtin-targeting shRNA has beneficial or harmful effects in donor-derived cells as a consequence of suppressing both the mutant and normal-length copy (allele) of huntingtin. Also, the budgeted work plan would not accommodate switching to the use of viral vectors as an alternative.
Consequently, the Board of Directors of the CGTA Research Group met and voted unanimously to refund all the remaining grant monies to the Hereditary Disease Foundation.
Spring, 2015: Update on our research project funded by the Hereditary Disease Foundation
Unfortunately, the second pilot experiment (mentioned in our December, 2014 update) to identify a suitable concentration for applying the shRNA-encoding plasmids onto neuron-like cells derived from the Huntington disease patient donor’s stem cells did not identify a concentration low enough not to have toxic effects on the cells, while still high enough to allow identification and tracking of the cells taking up the plasmids.
Not only the huntingtin-targeting plasmid but also the control plasmid (encoding an shRNA that does not target huntingtin mRNA, nor any other known human gene) was found to be toxic to the cells in culture, even though neither of these shRNA were toxic in monkeys’ brains in our prior work in which the shRNA was delivered using a viral vector.
A possible explanation for this finding in cell culture, despite the lack of toxicity in the live monkeys in our prior study, is the possibility that the cells produce too much shRNA from the plasmids they’ve taken up, even when treated with the lowest plasmid concentration feasible for the study. The technical term for what may be happening to the cells in culture as a result is “mechanistic toxicity,” which is described in more detail here.
We have submitted our first progress report for our research projected funded by the Hereditary Disease Foundation, which began in the summer of 2014. The objective of the project is to determine whether non-allele-specific suppression of huntingtin by an shRNA previously shown to be safe and bioactive in rhesus monkey striatum can enhance the survival and other characteristics of neuron-like cells derived from HD patients.
Work began with the production of batches of two plasmids, pAAV-HD5 and pAAV-CTRL5, from bacterial clones sent by the CGTA Research Group to Aldevron, Inc., (Fargo, ND). The plasmid stocks produced were tested for sterility and endotoxin levels, then shipped to the CGTA Research Group and stored in donated freezer space at the University of Minnesota. There, we thawed and aliquoted the plasmids in a tissue-culture hood under aseptic conditions into 100 μl aliquots. Blind-coded tubes of HD5 and CTRL5 plasmids were packaged into two containers (one for each replication of the planned cell culture experiment) and shipped to our collaborators in San Francisco, along with two additional labeled tubes (one each of HD5 and CTRL5). The latter two unblinded tubes were for the purposes of pilot phase of the project. The main study will be conducted under the blinded conditions.
Accomplishments so far include:
- optimization of the protocol being used for differentiation of HD patient-derived adult stem cells (induced pluripotent stem cells) into exclusively neuronal cell types,
- establishment of the culturing and plasmid transfection methods that produce cell cultures in which patient-derived neurons can be individually identified and tracked over time by imaging, and
- pilot experiments using the unblinded pAAV-HD5 and pAAV-CTRL5 to identify the concentration of plasmids to be used in the main experiment.
So far, results indicate that a high concentration of plasmids is not necessary to yield usable levels of neuron transfection for the experiment’s purpose, and instead can cause some toxicity. Therefore, a second pilot experiment at lower plasmid concentrations than used so far will be conducted in late December and January, before proceeding to the main study.
Our study of the efficacy of our gene therapy (shRNA) in adult stem cells, funded by the Hereditary Disease Foundation, is underway with our collaborator. This study is using skin cells donated by Huntington’s disease individuals. The cells have been engineered to become pluripotent stem cells, then differentiated into neurons to which the shRNA is being applied. If all goes well, results will be available next spring.
One of us (Bill Kaemmerer) just got back from attending the Hereditary Disease Foundation investigators’ conference entitled “HD2014: The Milton Wexler Celebration of Life.” The Hereditary Disease Foundation is funding our collaboration with a laboratory that is part of the HD iPS Cell Consortium, to test our anti-huntingtin gene therapy in cells derived from adult stem cells from persons affected with Huntington’s disease. The abstract for this project may be found here, and the poster we presented at the conference may be found here.
The CGTA Research Group received a response to our application to the U.S. Internal Revenue Service for charitable status. We are recognized as a 501(c)3 public charity, which means in the future, if we seek to raise funds from individuals to “crowd-source” our science, the donations will be tax deductible on the donor’s federal income tax return.
We have completed the step of acquiring (from a supplier) a sufficient quantity of the custom-made DNA needed for our study of the efficacy of our anti-huntingtin gene therapy. This DNA has been shipped to our collaborators who will conduct the cell culture study.
The CGTA Research Group has received word that a grant proposal we submitted with an academic collaborator for a study of the efficacy of HD5 shRNA in cell culture using cells donated by persons affected with Huntington’s Disease has been accepted for funding by the Hereditary Disease Foundation. More news later as this study gets underway.
The CGTA Research Group has reached an agreement with the current assignee of the intellectual property for the DNA construct described in the Grondin et al., 2012, Brain article. Our next step is to plan and then seek funding for a study to obtain evidence regarding the efficacy of this construct in a transgenic animal model of Huntington’s disease.
Grondin et al., 2012: http://brain.oxfordjournals.org/content/135/4/1197.full