Abstracts for the 24th Annual Meeting of the American Society for Neural Therapy and Repair

2017 Cell Transplantation  
In a relay race, team members take turns participating in a portion of a race. Success depends on each person performing to the best of his or her abilities. Translational research is like a relay race, in which scientists build upon other scientists' results, ultimately benefitting patients by creating and selecting better treatments. Every scientist, like every racer, is responsible for the quality of his or her contribution, in this case, to the scientific enterprise. In this context, it is
more » ... umbling to realize the high rate of attrition during the clinical translation of neurological treatments. During the 2017 Presidential Symposium, we will have an opportunity to analyze factors affecting the translational process of candidate neurological therapies and discuss strategies to improve the outcomes. The vast majority of neurological drugs put into clinical development fail to demonstrate safety and efficacy-failure rates are especially high for neurodegenerative disorders like Parkinson's disease. These failures exact heavy burdens on patients, research systems, and society in general. Does the high rate of attrition in neurological drug development reflect inevitable uncertainties in the process of clinical development, or are these failures due to correctable factors in the way we conduct preclinical and clinical research? In this presentation, I argue that both explanations are at play. I also suggest that both explanations also harbor important lessons for how we might reorganize and refine the way preclinical and clinical research in neurology is organized and conducted. I close by outlining a set of recommendations for limiting the burdens of failure in neurological drug development, maximizing prospects of success, and capitalizing on failed drug development trajectories. Stroke is the primary cause of severe long-term disabilities in the adult population in the US. Stroke alters functional and structural neuroplasticity, which contributes to neuronal death and cognitive dysfunction. High levels of cognitive ability at an early age have been implicated in neuroprotection in adulthood, characterized by a decreased incidence of cognitive impairments and neurodegenerative diseases. A similar neuroplasticity-based neuroprotective mechanism may influence the neuropathology of stroke after stem cell treatment; therefore, we investigated whether transplantation with human bone marrow stem cells (hBMSCs) in adult smart rats (fast learners) exposed to stroke could produce a robust therapeutic effect and stem cell mobilization compared to slow learners and/or untrained rats. Briefly, to test this hypothesis, we analyzed the cognitive function of adult Sprague-Dawley rats 704 ABSTRACTS prior to stroke using radial arm water maze. After training, rats were divided into fast learners and slow learners as a measure of high cognitive ability and poor cognitive ability, respectively, and then animals were subjected to ischemic stroke. Animals were transplanted intravenously with 2 ´ 10 6 hBMSCs or vehicle (saline) at 3 h after ischemic stroke. Motor, cognitive (radial arm water maze), and neurological tests were evaluated at day 1, day 3, and day 7 after stroke. Results from immunofluorescence revealed a significant increased mobilization of human nuclei-positive (HUNU + ) cells in the hilus and dentate gyrus of stroke-hBMSC fast learners compared to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. Meanwhile, relative fluorescence intensity showed significant increments in nestin and N-methyl-d-aspartate receptor subtype 2B (NMDAR2B) (neuroplasticity markers) in the striatum, subventricular zone (SVZ), and dentate gyrus of stroke-hBMSC fast learners compared to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. Moreover, quantification of the relative expression of NMDAR modulators revealed a significantly increased expression of vascular endothelial growth factor (VEGF + ) and von Willebrand factor (WF + ) cells in the striatum, dentate gyrus, hilus, and CA1 area of stroke-hBMSC fast learners relative to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. These findings indicate that higher cognitive function may positively influence the neuroplasticity and stem cell mobilization after stroke. Support: C.V.B. is supported by NIH NINDS Advances in the gene-editing arena, specifically with clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPRassociated protein-9 nuclease (Cas9), have pushed the demand for efficiently delivering payloads even further. Of the tools available, developments in the field of lipid nanoparticles (LNPs) have allowed for the reliable and efficient delivery of CRISPR components, both in research and clinical settings. Here we bridge that gap by describing the development of an LNP delivery system for CRISPR components, robustly manufactured with clinical-grade materials using microfluidic technology at scales for screening applications, in vitro experiments, and research in animals. We describe the use of LNPs for highly efficient encapsulation and delivery of payloads, such as small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid. In this proof of concept, we show that representative small RNAs, mRNAs, and plasmids can be successfully delivered to primary neurons. LNPs manufactured to encapsulate various nucleic acids can do so with high efficiency, encapsulating more than 95% of the payload, minimizing payload loss. Transfection efficiency of the LNPs is >95%, quantified using a fluorescent dye. The biological endpoint assays used to determine the accessibility of the payloads delivered varies for siRNA, mRNA, and plasmid. Using doses of 1 mg/ml of media, we achieved >90% knockdown with siRNA delivery, >90% of the primary neurons are green fluorescent protein positive (GFP + ) with GFP mRNA delivery, and >60% of the primary neurons are GFP + with GFP plasmid delivery. The LNPs are well tolerated, such that 5´ the required doses have no observable cytotoxicity. We show that the LNPs can also be used to deliver payloads into various regions of the animal brain. The localized injections into the cortex and the striatum are well tolerated and have extensive distribution. These validation studies provide suitable insights in establishing strategies for efficiently delivering CRISPR components into primary cultures and into the animal. The use of LNPs can be extrapolated to CRISPR components with a simple change in payload. We have editing efficiencies associated with delivering guide RNAs (gRNAs) to Cas9expressing cells, as well as simultaneously delivering Cas9 mRNA and gRNAs to cells. Endoplasmic reticulum (ER) stress is observed in a wide range of neurodegenerative diseases and is linked to the intracellular accumulation of misfolded proteins. Many chaperones important for proper protein folding carry a C-terminal Lys-Asp-Glu-Leu (KDEL) or KDELlike sequence, which mediates ER retention by interaction with transmembrane Golgi-localized KDEL receptors. The binding of KDEL ligand to receptor in the Golgi network induces a signaling cascade that
doi:10.3727/096368917x695146 pmid:28494821 pmcid:PMC5657807 fatcat:wpxmgozq2fgyxp6vvb7bk6i5au