UC San Diego

Spinal cord injury (SCI) affects upwards of one million people in the U.S. alone each year, with high costs to standards of living and healthcare (NSCISC 2015, #8). Present therapy targets increasing motor function and coping with medical complications though extensive rehabilitation. However, recovery remains difficult and restricted in scope for many, revealing the need for more therapeutic SCI development. Current research conceptualizes that a “neuronal relay” can be formed to bridge injuries of the central nervous system in order to communicate motor and sensory signals that may perhaps aid recovery. Recently, Lu et al. 2012 showed that NSCs (Neural Stem Cells) have the potential for implantation, integration and synapse formation with host neurons over long distances when combined with growth factor cocktail and an extracellular matrix (Lu et al. 2012, #34). We theorize that signal cues are essential for guidance and synaptogenesis for growing axons from developing neurons, and Mai et al 2009 demonstrates that bound BDNF gradients in in vitro are involved in axon guidance (Mai et al. 2009, #36).

BDNF (brain-derived neurotrophic factor) is an important cue for neural development (Chen et al. 2007, #9) and synapse formation that has previously been investigated in the spinal cord by Park et al. (Park et al. 2013, #42). We propose two gene therapy techniques to over-express BDNF in specific neural populations in a drug-inducible manner for improvement of functional recovery. Transient, drug inducible expression of BDNF is essential to mitigate detrimental effects such as spasticity and neuropathic pain, which stem from constitutive overexpression of BDNF within the spinal cord (Fouad et al. 2013, #16). In Chapters 2 and 3 we show GsDREADD (Designer Receptor Exclusively Activated by Designer Drug) activation of the intracellular cAMP pathway by the artificial ligand CNO (clozapine-n-oxide) leads to strong expression of BDNF (Farrell et al. 2013, #15). In vitro examination of GsDREADD signaling in primary neuronal culture reveals its efficacy for regulated BDNF expression (Chapter 2) in a drug-dependent manner. In vivo implementation of GsDREADD through AAV transduction into the spinal cord displays strong BDNF production local to the site of injection, but appears to be activated regardless of drug-withdrawal (Chapter 3). Lastly, an alternate method for drug-inducible BDNF production was pursued through “DD-BDNF” technique, which utilizes the degradation domain (“DD”) of bacterial dihydrofolate reductase enzyme (DHFR) fused to the proBDNF N-terminal, The destabilizing DD tag prompts rapid and efficient cellular degradation of the fused protein—proBDNF—via the proteasome before it can be fully expressed. DD-constructs are stabilized in the presence of the FDA approved antibiotic drug TMP (Trimethoprim) to allow temporal regulation of gene expression in a drug-dependent manner. A recent report from the Maximov lab (Sando et al. 2013, #49) demonstrated the feasibility of using this “DD” approach to control the expression and activity of Cre recombinase, EGFP or synaptic proteins in the CNS of transgenic animals and in animals treated with viral vectors expressing DD-constructs. Chapter 4 focuses on in vitro pursuit of DD-BDNF as a viable alternative to GsDREADD for driving drug inducible BDNF expression. Thus far, we have preliminary data demonstrating successful drug inducible control of exogenous BDNF protein expression using DD-BDNF and TMP in vitro. We are currently repeating this to verify our results. We seek to identify a time course and dose response profile of TMP dependent DD-BDNF induction prior to in vivo testing and following completion of AAV-CAG-gCOMET-DD-BDNF vector production.