Graduate Students Alphabetical by Graduate Student last name 1. Development of a mouse model to study SMN splicing and replacement therapy Presenting author: Bebee, Thomas Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein due to loss of the Survival Motor Neuron-1 (SMN1) gene. Humans have a duplicate gene, SMN2, that generates low levels of SMN protein due to a C>T point mutation that skips exon 7. We generated a mouse model that recapitulates SMN2 altered splicing by engineering the exon 7 C>T mutation into the homologous mouse Smn gene. The C>T mutation in Smn induces skipping of exon 7 in multiple tissues in the mouse, reduces SMN protein levels, and leads to a mild form of SMA exhibited by reduced hind limb grip strength, rearing, and activity. Increasing SMN expression by correcting splicing or increasing transcription of SMN2 are attractive therapeutic options in SMA patients. However, the timing of SMN replacement will be crucial in the treatment of SMA. Studies in zebrafish predict SMN function during embryogenesis may be important for axonal pathfinding, while mouse models and SMA disease progression in humans suggest that post-natal treatment may be sufficient to protect motor neurons. AAV9 encoding SMN has shown that early postnatal SMN expression in spinal cord motor neurons is sufficient to rescue survival though complete rescue of normal development was not observed, indicating that earlier treatment may be required for full restoration of SMN function and disease correction. To address the question of optimal therapeutic timing for SMA we have developed a temporally inducible transgenic mouse in which the expression of human SMN cDNA is under the control of tamoxifen inducible Cre-recombinase. In our inducible mouse, induced transgene recombination following tamoxifen treatment was validated in 4 week old weanling mice, PND1.5 treated neonatal mice, and E13.5 treated embryos. The temporal induction of transgene recombination and SMN expression was further validated in these mice in various tissues including the brain and spinal cord. When crossed to SMA mouse models our inducible SMN mouse model will allow for the temporal induction of SMN at varying time-points during development, both in utero and postnatally, to evaluate the time-point in which SMN replacement is required for SMA correction. The therapeutic window determined from these experiments can then be used in our Smn C>T mouse model to validate the use of drug therapies targeting splicing correction in the treatment of SMA mice, and in turn SMA patients. 2. Muscle enhancement in SMA Presenting author: Bevan, Adam K. Purpose: Spinal Muscular Atrophy (SMA) is a devastating neurological disease marked by lower motor neuron death and the subsequent atrophy of skeletal muscle. SMA patients can present with various severity of disease; SMA type 1 is very severe, usually leading to respiratory failure by 2 years, while SMA type 3 is non-fatal but patients are weak and are often wheelchair-dependent by their teenage years. While this disease primarily affects only spinal motor neurons, enhancement of skeletal muscle may be a good approach to treat patients with less-severe disease to increase their quality of life. We have therefore tested the muscle enhancing effects of follistatin gene therapy in severe (“Δ7”) and mild (“C/C”) mouse models of SMA. Methods: Newborn Δ7 (SMN2+/+, Δ7SMN+/+, Smn-/-) and C/C mice (SMN2+/+, hybSmn+/+, Smn-/-) were injected with 1e+11 particles of AAV9 carrying the follistatin transgene (AAV9.CBA.FS344) via the facial vein. Behavioral tests were performed starting at 6 days of age and continued until either 1) death in Δ7 mice or 2) at least 60 days of age in C/C mice. Muscle-related physiological measures were also performed in C/C mice at 200 days of age including muscle mass and in situ and ex vivo force generation. Transgenic follistatin expression was also confirmed by body mass, serum follistatin concentration and muscle fiber diameter measurements. Results: Both Δ7 and C/C mice showed early, positive effects of muscle enhancement, though no significant extension of survival was observed in the Δ7 group. In C/C mice, follistatin treatment had a positive effect on righting ability and, as predicted, significantly increased lean body mass and grip strength. Importantly, the ex vivo force generated by stimulation of the nerve was greater in follistatin-treated C/C mice despite having a pathological neuromuscular junction. Conclusions: While follistatin had little effect in the rapidly progressing Δ7 model, our data suggests that muscle enhancement may be a very useful therapy for patients with more mild forms of SMA. 3. Gene Therapy and Ovarian Cancer Presenting author: Bolyard, Chelsea M. Ovarian cancer is the leading cause of death due to gynecological malignancy, and a leading cause of death due to cancer in developed countries. Because patients often present in late stages of disease, aggressive multi-modal therapies may provide the most efficacious results. Within our study, we investigate how Adeno-associated virus (AAV) gene therapy can be combined with chemotherapy to treat ovarian carcinoma. Adeno- associated virus can be modified to incorporate targeting ligands into the viral capsid, facilitating AAV transduction into tumor-associated vasculature when administered systemically. These ligands bind receptors up-regulated on the angiogenic vasculature within the tumor microenvironment. AAV transduction can be increased by treatment with certain types of anti-cancer drugs, such as DNA-damaging agents (doxorubicin), histone deacetylase inhibitors (bortezomib) and proteasome inhibitors (depsipeptide). We examine the ability of TNFα-Related Apoptosis-Inducing Ligand (TRAIL) gene therapy to augment chemotherapy-mediated killing of ovarian cancer cells. Chemotherapeutics can also increase tumor cell sensitivity to TRAIL-mediated apoptosis. In this study, we will deliver TRAIL using the vasculature-targeted self-complementary AAV vector, after treatment with chemotherapeutics. Because our in vivo model of gene therapy targets the tumor-associated vasculature, our in vitro work focuses on the paracrine effects of TRAIL. When ovarian cancer cell lines were pre-treated with drugs, their sensitivity to the paracrine effects of TRAIL increased markedly. With this study, we have determined that TRAIL can be delivered within the context of the AAV genome, and that the TRAIL secreted from transduced cells maintains paracrine activity. Furthermore, the effects of chemotherapeutics can be potentiated by subsequent TRAIL treatment. This implies that with a multi-modal therapy, chemotherapeutic doses could be reduced and systemic toxicity minimized. 4. Aspirin Therapy in Neonatal Hyperoxia Presenting author: Britt, Rodney Purpose: Preterm infants are born with immature lungs, often requiring oxygen therapy to maintain homeostatic blood oxygen levels. Infants with prolonged oxygen exposure are at highest risk of developing chronic lung diseases such as Bronchopulmonary Dysplasia (BPD) and pulmonary complications later in life. Oxygen toxicity induces a pro- inflammatory response which is associated with lung injury and impairment of lung development which may persist through life. Aspirin is a nonselective cyclooxygenase (COX) inhibitor which is known to disrupt pro-inflammatory processes through multiple mechanisms. We tested the hypothesis that aspirin therapy will improve lung function and minimize airway hyperresponsiveness in lungs of adult mice following newborn hyperoxia exposure. Methods: Immediately after birth, newborn mice were exposed to room air (RA) or hyperoxia (>95% O2) and were injected daily with vehicle (PBS), 5, or 10 mg/kg aspirin (ASA). On day 7, hyperoxia exposed mice were returned to room air. Pulmonary function tests (PFT) were performed using the SCIREQ Flexivent system on day 28 and day 70. Results: Airway reactivity was assessed by measuring total resistance following intratracheal treatment with 0, 5, 10, 15, 25, and 50 mg/mL acetylmethacholine. Pressure- Volume loops (PV loops) indicated an increase in compliance in PBS/>95% O2 mice which was ameliorated in the hyperoxia exposed pups treated with aspirin. Total resistance was significantly higher in PBS/>95% O2 mice in response toacetylmethacholine challenge compared to room air controls, however both ASA/>95% O2 treatment groups exhibited only mild increase in resistance in response to 50 mg/mL acetylmethacholine (6.18±0.59 vs 2.93±0.35). Conclusions:Aspirin treatment attenuated detrimental effects of hyperoxia on lung function and airway reactivity. Aspirin may be a beneficial therapy for preterm infants who are at risk of developing BPD. 5. rAAV recombination Presenting author: Cataldi, Marcela P. The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single- stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration