Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive loss of upper and lower motor neurons, which ultimately leads to death within 2-3 years of diagnosis. While multiple mechanisms have been proposed to cause motor neuron death, altered signalling of the pro-survival phosphatydyl-inositol-3-kinase (PI3K/Akt) signalling pathway has been shown in spinal cords of human cases and rodent models of ALS. The depletion of the phosphatase and tensin homologue (PTEN), an essential negative regulator of PI3K, promotes neuronal survival and axonal regeneration. The main aim of this study was to investigate the effect of PTEN silencing using viral vectors, on motor neuron survival and neuromuscular degeneration (NMJ) in experimental models of ALS. Lentiviral vectors expressing small interfering RNA (siRNA) against mouse PTEN achieved approximately 60% PTEN knockdown in purified motor neurons from transgenic SOD1G93A embryos and their non-transgenic littermates. PTEN silencing was associated with increased expression of phosphorylated Akt, and enhanced motor neuron survival. To validate the potential neuroprotective effect of PTEN silencing in vivo, wild-type (WT) mice were used to determine the efficiency of adeno-associated virus serotypes 6 (AAV6) and 9 (AAV9), to deliver PTEN siRNA to spinal cord motor neurons after intramuscular (i.m) administration. Despite effective transduction and PTEN knockdown in the injected muscles, low levels of spinal cord motor neuron transduction were observed for both vectors suggesting poor retrograde transport efficiency. Similar findings were seen in transgenic SOD1G93A mice treated with AAV9 vectors expressing PTEN siRNA, which may explain the lack of significant effect seen on spinal cord motor neuron survival. In addition, no significant effect was seen on neuromuscular junction degeneration in the muscles of the injected hind limbs. In conclusion, these results suggest that viral-mediated PTEN silencing may enhance the survival of transgenic SOD1G93A motor neurons by activating the PI3K/Akt pathway. However, the i.m route for AAV-mediated siRNA delivery to the spinal cord is insufficient for effective PTEN silencing. Alternative CNS delivery routes are needed for proof-of principle studies to determine the therapeutic efficacy of PTEN silencing ALS.