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.

Alzheimer’s disease (AD) is a neurodegenerative disease that causes a reduction in brain-derived neurotrophic factor (BDNF) in multiple animal models, and delivery of BDNF provides neuroprotective effects in those disease models. BDNF regulates a wide range of biological functions in the human body, and therapeutic delivery requires effective delivery routes and targeting specificity.

Adeno-associated viral (AAV) vector systems have recently become popularized for targeted gene delivery. AAVs are non-pathogenic in humans, provide long-term gene expression,

and do not integrate into the host genome. Despite the advancements in viral gene therapy over the past decades, AAV vector systems are limited by the lack of regulation.

Here, we developed a novel regulatable dual AAV vector system for therapeutic delivery of BDNF that can be turned off by oral administration of tamoxifen to adult mice. Our dual AAV vector system contains two co-dependent AAV vectors. One vector carries a pancellular promoter and the BDNF transgene. The second vector carries a drug-inducible Cre recombinase enzyme (Cre) that we hypothesize will result in degradation of the AAV genome and elimination of gene expression.

We found that our dual vector system provides a modest reduction in AAV gene expression following administration of tamoxifen. Further optimization of the system could lead to the first regulatable AAV system suitable for clinical applications. This regulation could act as an “off” switch for eliminating gene expression in the event of adverse side-effects. Moreover, this regulation could allow for transient gene delivery for treating medical conditions such as traumatic brain injury and spinal cord injury. The ability to tightly regulate AAV gene expression is potentially applicable to all 105 on-going clinical trials involving AAV gene therapy.

Introduction

Methods

Results

Discussion