Early-stage Study Of AAV Vector Mediated Gene Therapy For Spinal Muscular Atrophy

Spinal muscular atrophy(SMA)is one of the single-gene genetic diseases with high neonatal mortality rate,which is characterized by high carrying rate and high mortality.Mutation or deletion in SMN1 gene is the mainly cause of SMA.At present,the treatment for SMA is limited,there is only one drug approved by FDA and the price is extremely expensive.Thus,domestic patients are facing with no medicine that are aviliable.Gene therapy brings the possibility of treating for spinal muscular atrophy.In this paper,we carried out the molecular design of gene therapy drugs with optimized gene elements.We constructed 4 novel chimeric promoters with different 5’UTR intron-based on CA promoter.We obtained that CAR promoter could increased expression level of Gluc or green fluorescent protein significantly.We also according to the human codon preference to optimize human SMN1 coding sequences.That could lead to a slightly higher than wild type.However,it could be significantly increased by fused the CAR promoter with codon optimized SMN sequences.We next selected psc AAV-CAR-EGFP to produce r AAV9 virus to study its biodistribution by intravenous(IV)or intracerebralventrical(ICV)injection in mice. Both administration routes could increase green fluorescence in the central nervous system(CNS)and skeletal muscle.However,ICV injection of r AAV9-CAR-EGFP could cause phenomenal EGFP transduction in the whole brain and spinal cord compared with that by IV injection and showed that AAV9 could cross the blood brain barrier(BBB).The IV injection route was more efficient in peripheral organs, especially in skeletal muscle,the heart and liver.In addition,IV injection in adult mice could achieve continuous and strong expression in the liver.However,both injection routes in neonatal mice appeared to decrease transduction and fluorescence significantly in the liver.Finally,based on the biodistribution results of r AAV9,we selected the ICV administration route to treat the SMA type I mice with psc AAV-CA-co SMN1.The results showed that SMA mice could have a significant improvement in bodyweight increasing,and the median survival time increased from 10 days to more than 50 days,that could confirm the efficacy of AAV9-CA-co SMN1.Our reports are the first team testing AAV9 mediated gene therapy drug on Taiwanese SMA mouse model,which phenotype is more severe than △ 7 SMA mouse model.The median survival time of this model achieved more than 50 days (prolong more than 500%)after treatment,and it laid the foundation for the clinical progress of SMA gene therapy.