To better understand spinal muscular atrophy (SMA), scientists must conduct studies using more than just cells in a culture dish. It is best to recapitulate the human body using a large animal model, but many scientists use mice due to their availability. To fill the gap of large animal models needed for SMA, researchers at Ohio State University Wexner Medical Center explored gene knockdown in pigs in the article “A Large Animal Model of Spinal Muscular Atrophy and Correction of Phenotype,” published in Annals of Neurology.
“We wished to determine whether reduction of survival motor neuron (SMN) in postnatal motorneurons resulted in SMA in a large animal model, and whether SMA could be corrected after development of muscle weakness,” wrote Sandra I. Duque, PhD, lead author of the study. The team could not simply deplete motorneurons of SMN through prenatal genetic manipulation, as large animals have only one SMN gene (humans have two, SMN1 and SMN2), and any SMN mutation is therefore lethal.
Working around this, the team first created short hairpin RNA (shRNA1) molecules, which are able to integrate into the genome and affect protein expression. In this case, the team used shRNA1 that targeted SMN. They delivered the shRNA1 by injection into the cisterna magna of the brain.
As a result of the procedure, SMN protein was reduced by over 75% in the pigs. In under four weeks, the pigs developed progressive hindlimb muscle weakness. They had difficulty standing for long periods of time, had abnormal gait and stance, and could not walk up a carpeted incline with ease. Eventually, the pigs lost strength in their front legs and could no longer stand independently.
These symptoms are consistent with the clinical phenotype of human SMA. Quantitatively, electrophysiological biomarkers used to assess SMA in humans (compound muscle action potential (CMAP), motor unit number estimation (MUNE), and electromyography (EMG)) showed fibrillation potentials in the pigs. Pigs were otherwise normal and showed no loss in the ability to swallow or breathe normally.
After creating a clinically-relevant model of SMA, the researcher put their model to use to try to rescue the SMA phenotype. They injected a “rescue vector” that combatted the effects of shRNA1 injection. Pigs receiving the rescue vector 24 hours after injection (presymptomatically) with the shRNA1 against SMN did not develop SMA, although one pig developed mild weakness that did not translate into electrophysiological changes.
Since this delivery timeline is too unrealistic to be used clinically, the researchers also identified the effects of delivering a rescue vector following early symptom onset. Pigs injected with rescue vector had improved electrophysiological tests compared to untreated pigs. “Thus, for clinical trial design, early symptomatic restoration of SMN should result in an improved phenotype that does not progress and recovery of CMAP, indicating better function of the available motorneurons,” concluded the authors. This animal model may also be of use to developing medicines to treat SMA, such as drugs similar to those developed by Isis to treat this disease.