Although the mutations associated with ALS affect proteins in seemingly unrelated pathways, they all result in a common endpoint, the degeneration of the upper and lower motor neurons. Therefore, many common gene therapy strategies for ALS have aimed to mitigate the toxicity of known genetic variants by either reducing the expression of the mutant gene or by providing a neuroprotective milieu to increase the survival of functional motor neurons.

During embryogenesis, neurotrophic factors are responsible for guiding the development of the nervous system. In mature neurons, neurotrophic factors support the survival and promote the growth of specific populations of neurons [12]. Therefore, many neurotrophic factors have been used in gene therapy based strategies for ALS , including brain -derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF ) [13], insulin like-growth factor -1 (IGF-1) [6], and vascular endothelial growth factor (VEGF ) [14].

In 2002, Wang et al. demonstrated that AAV2 mediated delivery of GDNF administered intramuscularly into the forelimbs and hindlimbs of an SOD1 mouse allowed for efficient retrograde transport of the GDNF to spinal motor neurons ultimately delaying disease onset and prolonging survival by 16.6 days compared to control ALS mice [13]. In a seminal paper by Kaspar et al., AAV -mediated delivery of IGF-1 via intramuscular administration in a mouse SOD1 model was shown to delay disease onset and prolong life compared to vehicle and AAV-GDNF treated mice. Both the delay of disease onset and increased survival was observed when IGF-1 was administered either at pre-symptomatic (Day 60) or symptomatic (Day 90) time points [6]. These authors also demonstrated that a vesicular stomatitis virus glycoprotein (VSV-G ) pseudotyped lentivirus mediated delivery of IGF-1 increased survival, however lentivirus based delivery was not as effective as AAV. Nevertheless others have utilized lentivirus vectors to deliver therapeutic agents intramuscularly. Azzouz et al. showed that a Rabies-G (Rab-G) pseudotyped Equine Infectious anemia virus (EIAV)-based lentiviral vector could be effectively used to deliver the neurotrophic factor VEGF to spinal motor neurons. The authors found that EIAV-LV mediated delivery of VEGF increased survival when administered both prior to or at disease onset [14]. Two similar studies utilized an AAV2-based vector to administer IGF-1 intraparenchymally. Lepore et al. delivered AAV2-IGF-1 directly into the lumbar segment of the spinal cord in an ALS SOD1 mouse model [4] whereas Franz et al. delivered AAV2-IGF-1 directly into the cervical segment of the spinal cord in an ALS SOD1 rat model [15]. Compared to an AAV2-GFP control, a delay in disease onset and an increase in survival were not observed; although both groups report there was a reduction in motor neuron loss [15]. An interesting finding in both studies was that only male rodents presented an increase in either hindlimb (lumbar injection) or foregrip (cervical injection) strength thus indicating that there may be a gender-specific disposition to various gene therapy approaches. In a study by Dodge et al., the authors demonstrated that ICV delivery of AAV4-IGF1 or AAV4-VEGF vectors resulted in broad expression of these genes in many regions of both the brain and spinal cord. Furthermore, delivering AAV4-IGF-1 or AAV4-VEGF intraventricularly provided neuroprotection and resulted in increased motor function and prolonged survival compared to control mice by 12 days and 20 days, respectively [8]. Interestingly, delivery of both neurotrophic factors simultaneously didn’t prove to be more efficacious than either IGF-1 or VEGF separately.

RNA interference (RNAi ) is a posttranscriptional regulatory mechanism that specifically targets a messenger RNA for degradation using a short, complimentary RNA [16]. Since many of the mutations associated with ALS result in toxic gain-of-function proteins, RNAi has allowed researchers to silence the disease allele in animal model s of this disease. Approximately 20 % of familial ALS cases are having been shown to result from a toxic gain of function mutation in the Cu, Zn superoxide dismutase (SOD1) gene. Many studies have thus investigated the efficacy of delivering noncoding RNA sequences to knockdown mutant SOD1 transcripts in SOD1 animal models of ALS [17, 18].

Using a RNAi approach, Raoul et al. demonstrated that lentiviral-mediated delivery of a shRNA hairpin to the mutant SOD1 transcript resulted in a decrease in the loss of motor neuron pools in a SOD1 mouse model of ALS [5]. Although this decrease was only seen in the areas adjacent to the injection sites, the authors showed that there was a significant increase in the age at onset. Similarly, Ralph et al. sought to mitigate toxicity of the mutant SOD1 protein by using an RNAi approach to knockdown expression of the mutant SOD1 gene. Delivery of a small hairpin RNA (shRNA) via an EIAV-lentivirus vector targeting mutant SOD1 transcripts resulted in a 30 % increase in life expectancy in SOD1 mice [18]. A study conducted by Towne et al. found that intramuscular delivery of an AAV6-shRNA targeting mSOD1 transcripts was able to transduce motor neurons, reduce mutant human SOD1 transcripts, and protect the muscle from SOD1-mediated atrophy; interestingly, however, intramuscular delivery of the AAV6-shRNA was not able to improve motor function or prolong survival in the SOD1 mouse model [19]. A recent study by Wang et al. demonstrated that an AAV serotype , rAAVrh.10, could provide diffuse transduction of the CNS when delivered intrathecally in the lumbar region of an ALS SOD1 mouse model. Furthermore, the authors demonstrated that delivery of a shRNA to the mutant SOD1 gene could extend survival by prolonging disease progression in an SOD1 mouse model [20].

ALS is a rapidly progressive neurodegenerative disease that ultimately results in paralysis and death. With only one FDA approved therapy that provides modest benefits at best, patients and families have limited options for effective therapeutic treatments. Gene therapy approaches for ALS provide a potentially more effective alternative for therapeutic intervention. It should be noted that many of the gene therapy studies conducted in animal of models of ALS show promising results when gene therapy vectors are delivered at pre-symptomatic or symptomatic time points; however the greatest improvement is seen when animals are treated pre-symptomatically. Advances in the detection of ALS phenotypes, biomarkers of the disease, and family history records will allow for earlier diagnosis of ALS and may provide a unique opportunity to effectively utilize gene therapy approaches.

In this focused mini-review, we have provided an overview of the common strategies, viral vectors, and animal model s used in gene therapy research for ALS . Furthermore, we have provided a detailed protocol for delivering gene therapy vectors to the spinal cord. These previous studies discussed here in addition to others have provided proof-of-principle for the use of gene therapy strategies for treating neurodegenerative diseases such as ALS; however improving the safety and efficacy of these gene therapy strategies will ultimately allow for the translation of gene therapy to a clinical setting.