Efficiency And Pathways Of RAAV2-retro Transduction Of Spinal Lower Motor Neurons From The Periphery

With the rapid development of viral tools,various viral tools have begun to be used for gene therapy of various clinically intractable diseases,including motor neuron diseases.Motor neuron diseases is a series of neurodegenerative diseases mainly with progressive changes of upper motor neurons and lower motor neurons.The complex motor dysfunction that occurs with motor neuron diseases is often severely life-threatening.With the continuous discovery of motor neuron diseases related gene targets,recombinant Adeno-Associated Virus(rAAV)-based gene therapy for motor neuron diseases has achieved initial results.How to further improve the viral transduction efficiency of lower motor neurons and thus improve the efficacy of gene therapy is one of the current research hotspots for the treatment of motor neuron diseases.According to the structural characteristics of the projection of lower motor neurons'axons onto muscles to form neuromuscular junctions,the strategy of using viral vectors to introduce therapeutic genes from peripheral muscles to diseased lower motor neurons in the central nervous system has attracted more and more attention in the gene therapy of motor neuron disease.This strategy is simpler,easier,and more secure than other strategies.However,the distribution of lower motor neurons is diffuse,and the efficiency of introducing therapeutic genes from peripheral to centrally damaged lower motor neurons through viral tools has been low.Therefore,it is urgent to search for more ideal solutions in the selection of rAAV subtypes and rAAV delivery methods.By combining tissue clearing processing technology and three-dimensional imaging technology,this article has studied the transduction characteristics of spinal lower motor neurons by intramuscular injection of rAAV2-retro,compared the infection efficiency of three rAAV2-retro injection methods on spinal lower motor neurons and explored the way that rAAV2-retro transduces the central nervous system from the periphery efficiently through the sciatic nerve and spinal cord injury model.The main research contents and results are as follows:(1)To compare the infection efficiency of different rAAV subtypes,eight commonly used rAAV subtypes were injected into a single muscle respectively to retrogradely infect cervical spinal cord neurons.The results showed that intramuscular injection of rAAV2-retro infected the neurons in the ipsilateral cervical spinal cord with the highest efficiency;(2)The characteristics of retrograde infection of central nervous system neurons by intramuscular injection of rAAV2-retro were analyzed.The results showed that intramuscular injection of rAAV2-retro could efficiently and widely transduce lower motor neurons in the brainstem and spinal cord and sensory neurons in the dorsal root ganglia;(3)The infection efficiency of three rAAV2-retro injection methods on neurons in the spinal cord was compared.The results showed that compared with intravenous injection and lateral ventricle injection,the peripheral intramuscular injection of rAAV2-retro was simpler,safer and had stronger selectivity for the corresponding spinal cord segment and the highest infection efficiency for neurons in the spinal cord;(4)The pathway of rAAV2-retro extensively transducing the central nervous system by peripheral intramuscular injection was explored.The results showed that:intramuscular injection of rAAV2-retro could diffuse into the blood and cerebrospinal fluid,and eventually reached an extensive transduction of neurons in the nervous system.In summary,the injection of rAAV2-retro to peripheral monolithic muscle can efficiently transduce lower motor neurons of the central nervous system.This viral injection transduction method is expected to provide a potential treatment for gene therapy of motor neuron diseases.