Respiratory directed gene therapy prolongs survival in an ALS mouse model
Authors: ElMallah Mai K , Keeler‐Klunk Allison , Zieger Marina , McCall Angela L , Pucci Logan , Brown Robert H , Mueller Christian 
Published: June 2021

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease with no current cure. Patients with ALS die 3–5 years after diagnosis from respiratory failure. Approximately 5–10% of ALS is familial and 20% of familial ALS is due to mutations in the gene encoding Cu/Zn superoxide‐dismutase 1 (SOD1). The most commonly used ALS mouse model is the SOD1G93A mouse which recapitulates human paralysis and respiratory disease. Adeno‐associated viral (AAV) gene therapy has the potential to treat this form of ALS by delivering artificial microRNA (miRNA) that silences SOD1 (AAV‐miRSOD1) to the diseased motor units. We hypothesized that respiratory directed AAV‐miRSOD1 can ameliorate respiratory insufficiency in this ALS mouse model and prolong survival.Adult 60 day old SOD1G93A mice were injected with AAV‐miRSOD1 via both an intralingual (IL) and intrathoracic (IT) injection. Littermate control non‐transgenic (NTG) and SOD1G93A mice were administered PBS through IL and IT injections. Breathing and strengths assessments were performed at 90 and 120 days then every 2 weeks until end point. Breathing was assessed by whole body plethysmograpy (WBP) during normoxia (inspired oxygen fraction (FiO2) = 0.21; nitrogen balance) for baseline measures and during a “respiratory” challenge with 7% hypercapnia (FiO2 = 0.21; FiCO2 = 0.07; nitrogen balance). Respiratory mechanics were assessed in anesthetized, ventilated animals using a flexiVent (Scireq) at end stage. Post mortem histological assessments of NMJ, and axonal integrity, as well as molecular studies of vector genome biodistribution and SOD1 knock down were performed.AAV‐miRSOD1 treated SOD1G93A mice survived an average of 39 days longer than untreated SOD1G93A littermates (p<0.001). Disease onset was delayed so strength and breathing were maintained for longer in the AAV treated animals. Specifically, the peak inspiratory flow and minute ventilation was comparable to NTG animals until end stage. Furthermore, treated animals were able to maintain their strength well beyond the time that the untreated littermate SOD1G93A mice reached end stage. Significant SOD1 knock down occurred in the diaphragm and tongue of the AAV treated animals. Respiratory mechanics at end stage were similar between treated and untreated SOD 1 groups. NMJ integrity was improved in the diaphragms of AAV treated animals at end stage compared to the untreated animals with a higher proportion of de‐innervated NMJs seen in the PBS injected compared to the AAV injected mice. Hypoglossal and phrenic axonal integrity was also improved. In conclusion, respiratory directed AAV gene therapy using microRNA to silence SOD1 delays disease onset, delays onset of respiratory insufficiency and improves survival in SOD1G93A mice.Support or Funding InformationR21 NS098131‐02 (MKE)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.