Development of novel targeted lentiviral vectors for gene therapy of motor neuron diseases

Abstract

Motor Neuron Diseases (MND) including Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA) are neurodegenerative diseases that can cause progressive paralysis and premature death, while there are no treatments available up to date. Gene therapy using lentiviruses has been successful at alleviating symptoms and extending survival in murine models of both. Restricting transduction to specific cells, is critical for safe and efficient gene therapy. The aim of this study was to generate novel lentiviral vectors with tropism to motor neurons (MNs) using surface engineering, that involves incorporation of a fusogenic glycoprotein (mutated sindbis G) and an antibody recognizing a cell-surface receptor, onto the lentiviral surface. Antibodies against rat Thy1.1, rat p75NTR (Low Affinity Nerve Growth Factor Receptor) and mouse CAR (Coxsackie and adenovirus receptor) receptors expressed on the presynaptic terminal of the neuromuscular junction (NMJ) were cloned and used to surface engineer high titer lentiviral vectors ( Thy1.1, p75NTR and CAR). These vectors preferentially transduced cell lines expressing these receptors and primary motor neuron cultures compared to non-targeted controls. The ability of these vectors to be transported retrogradely and transduce MNs was demonstrated in vitro in compartmented microfluidic cultures in fixed and live imaging experiments. Tropism of these vectors was assessed in vivo in the rat brain following intrastiatal injections. In vivo intramuscular delivery of CAR targeted lentiviral vectors in tibialis anterior of mice lead to transduction of motor neurons in ventral spinal cord. Transduction of spinal cord was further demonstrated by in vivo bioluminescence imaging studies in mice injected intramuscularly with the CAR targeted vectors. This is the first ever demonstration that surface engineering can confer novel trafficking and transduction characteristics to lentiviral vectors. These targeted lentiviral vectors have superior trafficking, transduction and specificity for MNs than previously used lentiviral vectors, making them good candidates for non-invasive CNS -targeted delivery of therapeutics in MN diseases.