Although long-lived preservation of grip strength late in disease would not be possible as the disease progressed into the noninjected upper part of the limb, where we did not target the innervating motor neurons, these results demonstrate regional rescue of motor neurons that received the AAV-2 siRNA construct and provide the proof of principle for use of RNA interference in treating ALS in vivo in adulthood. Although AAV or other viral vectors can be directly injected into nervous system tissue and may be effective in mouse models of neurodegeneration,12
such approaches in humans may be fraught with significant morbidity from the trauma of injections, especially if the target is the spinal cord at multiple levels, such as would be anticipated in ALS. In contrast, we anticipate that reduction in mutant SOD1 by AAV-2–mediated delivery of siRNA after peripheral injection may be an effective therapy for SOD1 familial ALS patients. The safety of a similar approach in humans has already been demonstrated by injection of AAV-2 into muscle as a means of generating clotting factor replacement.13
Indeed, a clinical trial using AAV-2 to deliver a growth factor (insulin-like growth factor-1) is to be initiated in ALS patients. It is likely that knockdown of other proteins by virus-delivered siRNA also will be effective for future therapeutic targets both in sporadic ALS and other neurodegenerative diseases.
Toxicity of SOD1 mutants to motor neurons is noncell autonomous; that is, it does not derive solely from mutant damage directly within motor neurons.14
Indeed, expression of SOD1 mutants only in motor neurons has not produced disease or pathology,15,16
and individual motor neurons surrounded by nonmutant neighbors survive indefinitely despite expressing mutant SOD1 at levels sufficient to generate early-onset disease when expressed ubiquitously.14
Regarding this, our evidence indicates that within the spinal cord, selective reduction of mutant SOD1 in motor neurons is sufficient to significantly slow motor neuron killing, demonstrating, as expected, that mutant damage developed directly within motor neurons contributes in an important way to toxicity. Although no evidence currently is available to support direct damage by mutant SOD1 within muscle cells, we cannot exclude that the viral-delivered siRNA used here might also reduce mutant SOD1 expression in muscle, and that a proportion of the benefit we have documented may arise from decreased mutant expression in muscle. Indeed, disconnection at the neuromuscular synapse is among the earliest abnormalities in mutant SOD1 mice,17
and increased synthesis of insulin-like growth factor-1 by muscles18
can slow toxicity from SOD1 mutants; albeit, it is unclear whether this latter benefit derives from insulin-like growth factor-1 action on the neuron or the muscle. Future efforts using viruses that are not retrogradely transported are necessary to determine whether reducing SOD1 mutant synthesis exclusively within muscles will be beneficial.