oa Gene therapy mediated neuroprotection of the neuromuscular junction in the spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disease affecting mainly the alpha motor neurons in the ventral horn of the spinal cord leading to muscular atrophy and paralysis. Although SMA is one of the leading genetic causes of infant mortality, no effective treatment is currently available. Gene delivery studies of Glial Derived Neurotrophic (GDNF) in Parkinson's disease (PD) have demonstrated positive impacts in vitro studies, PD models and early phases of clinical studies. In light of that, the objective of this work is to assess the efficiency of GDNF in preventing the neuromuscular pathology in SMA. Our specific aims are to: i) generate GDNF expressing vectors for in vitro and in vivo transduction; ii) Evaluate the impact of GDNF on axonal growth in SMN-deficient motor neurons in vitro; iii) generate a pre-clinical efficacy proof-of-concept in the SMNΔ7 mouse model of SMA. Because lentivirus (LV) is known to be efficient for in vitro transduction, an LV‐SIN‐PGK‐GDNF plasmid was used to express human GDNF. In order to assess the efficacy of the produced viral prep, it was later used to transduce HEK293T cells. Preliminary analysis of protein levels of the GDNF using western blot and ELISA showed an increase in the LV-GDNF transduced cells in comparison to un-transduced cells. Experiments are ongoing to evaluate the effects of the LV-GDNF on the axonal growth in SMN-deficient primary motor neuron cells. For in vivo studies, self-complementary adeno-associated virus of serotype 9 expressing GDNF gene (scAAV-GDNF) was produced as scAAV9 has been shown to have low pathogenicity and remarkable capacity to transduce both neurons and astrocytes in vivo. Preliminary transfection and transduction assays have shown increase in GDNF expression in the HEK293 transfected and HELA transduced cells. However, further work is being performed to confirm these results. Also, pre-clinical efficacy proof of concept will be performed using SMNDelta7 mouse model via scAAV-GDNF injections. This is going to be followed by evaluating the impacts on the survival and the phenotype of the injected mice. To this end, this work represents an essential step to validate the ability of the LV-GDNF and the scAAV-GDNF to transduce cells and produce the desired GDNF. Ongoing work will give insights on the efficacy of using GDNF gene delivery as a therapeutic approach for SMA.