| Research BackgroundLimb spasticity is one of the most common complications after spinal cord injury.It has been reported that 12%–37% of patients with acute spinal cord injury have spasms,and the incidence of limb spasms in patients with chronic spinal cord injury is 65%–78%.At present,there is still no effective method to treat the cause of spasms,only to reduce spasm symptoms.Therefore,the development of a new antispasmodic therapy and a rehabilitation model is essential for the functional recovery and quality of life of patients with spinal cord injury.A single treatment may improve a particular function,but a combination of treatments based on personalized medicine is expected to achieve better results.Neurotrophic factor 3(NT3)can not only reduce the excitability of motor neurons,but also is a neurotrophic factor necessary for the survival of sensory neurons and motor neurons.Studies have shown that the input of afferent signals from surrounding muscles is essential for restoring motor function and rebuilding the neural circuits of the injured spinal cord,and this may be because the surrounding muscle spindles synthesize NT3.At the same time,the overexpression of NT3 in the muscles rebalances excitatory and inhibitory inputs.However,whether it can normalize the spinal cord reflex in rats after spinal cord injury has not been confirmed.In addition,exercise is considered to be the simplest,safest,and most effective treatment method in clinical practice.It can improve motor function by balancing sensory input and exercise output,thereby improving the quality of life of patients.At the same time,intensive training can normalize the proprioceptive reflex of the spinal cord.A single treatment method may be able to improve a certain function,but it cannot be comprehensive,and a combination of certain therapies based on individualized medicine will be expected to achieve better results.Virus-based gene therapy can specifically deliver the therapeutic gene to the diseased site so that it can be stably expressed locally or throughout the body for a long time.Therefore,it is currently widely used in the diagnosis and treatment of central system diseases.Among them,adeno-associated virus is considered to be the most promising gene therapy vector due to its safety and high efficiency.Among the many serotypes of AAV vectors,AAV9 type adeno-associated virus is considered to be the "standard" vector for the treatment of central system diseases.However,the efficiency of AAV9 type adeno-associated virus vector crossing the blood-brain barrier has not yet reached expectations,leading to its poor efficacy in the treatment of central system diseases.Increasing the dose of the virus or adjusting the route of administration,such as intrathecal administration,are two attempts to solve the above problems.However,the former will cause excessive exposure of other peripheral organs in the body and produce toxic reactions,while the latter will increase significant complications.risk.So people focused their attention on another possible way-by modifying the capsid structural protein of the AAV vector to change its transfection efficiency.Research purposesConstruct a recombinant adeno-associated virus(AAV)encoding human NT3,and explore the protective effect of a combination therapy of recombinant adeno-associated virus(AAV-NT3)encoding human NT3 and exercise intervention on muscle spasm after spinal cord injury.Explore the mechanism of this combination therapy to reduce spasticity,providing a theoretical basis for the prevention and treatment of muscle spasm after spinal cord injury in clinical work.In addition,constructing recombinant AAV with higher delivery efficiency,and exploring the delivery efficiency of different routes of administration to the central nervous system such as the brain and spinal cord and the toxicity of peripheral tissues,Playing a guiding and reference significance to the application of gene therapy in central nervous system diseases.Research methods1.Construction and verification of adeno-associated virus encoding human NT3:insert green fluorescent protein(f-GFP),neurotrophic factor 3(NT3)and helper plasmids into an AAV vector driven by a human CMV promoter,and then package and produce recombinant AAV by HEK293 cells.The Western Blot was used to detect the changes of NT3 protein expression in rat hindlimb muscles and spinal dorsal root ganglia after virus injection.2.The protective effect of AAV-NT3 and exercise intervention on muscle spasm after spinal cord injury in rats: the spasm frequency of rats was evaluated by swimming test and H reflex of rat hindlimb muscles,and the hindlimb motor function of rats was evaluated by BBB score.3.The mechanism study of AAV-NT3 and exercise intervention on the protection of muscle spasm after spinal cord injury in rats: the use of immunofluorescence staining to explore the plastic changes of spinal cord motor neurons and interneurons,and the use of Western Blot to detect recombinant potassium chloride Cotransporter 2(KCC2transporter)expression.4.Construction of a new centrally targeted gene vector AAV-CPP.16: insert the peptide sequence into the capsid protein genome of the AAV vector for modification,produce a variety of different adeno-associated virus gene vectors,and inject them through the tail vein of mice and the 3D in vitro model of the blood-brain barrier for screening.5.Transduction efficiency of the new centrally targeted gene vector AAV-CPP.16:through intravenous injection and lateral ventricle injection,two mainstream central nervous system administration routes,to explore whether AAV-CPP.16 can be highly effective expressed in the spinal cord and its exposure toxicity to peripheral organs.Results1.Successfully constructed NT3 recombinant adeno-associated virus and successfully transfected it into rats;Compared to the control group and AAV-GFP group,the expression level of NT3 in hind limb muscles and root ganglia(DRG)increased significantly in the rats injected with AAV-NT3.2.Swimming test and RDD of H reflex showed that gene therapy,exercise intervention and combination therapy of AAV-NT3 can reduce the frequency of muscle cramps,but only AAV-NT3 and combination therapy can increase the BBB score of hind limbs.3.Combination therapy can significantly increase the number and area of spinal cord motor neurons,but AAV-NT3 gene therapy alone does not have this effect;Combined therapy can significantly increase the area of spinal cord cholinergic interneurons and GABAergic interneurons quantity;The expression of KCC2 in the spinal cord of AAV-NT3 gene therapy and combination therapy groups increased significantly.4.Compared with the control group wt AAV9,the percentage of RFP-positive cells in the brain of mice injected with AAV-CPP.16 was significantly increased,and the fluorescence intensity expressed in the 3D in vitro model of the blood-brain barrier was significantly increased.5.After intravenous administration and lateral ventricle administration,compared with AAV9,the expression of AAV-CPP.16 in the spinal cord was increased,and a higher proportion of motor neurons overlapped with GFP-labeled cells.6.After intravenous injection,compared with AAV9,AAV-CPP.16 significantly increased the proportion of GFP-positive cells in spinal cord DRG and peripheral tissues and organs except liver and heart;after intraventricle injection,AAV9 and AAV-.CPP.16 hardly expresses GFP protein in peripheral tissues and organs.Conclusion1.AAV-NT3 gene therapy,exercise intervention and combined therapy can all reduce muscle spasm after injury;Compared with gene therapy or exercise intervention alone,combined therapy has a certain curative effect trend,but the difference is not significant.2.AAV-NT3 gene therapy has limited effect on motor neurons,and exercise intervention alone cannot improve the motor function of the hind limbs.The combined treatment of the two can not only reduce the muscle spasm after spinal cord injury by changing the excitability of spinal motor neurons and interneurons and increasing the content of KCC2 in the spinal cord,but also significantly improve the motor function of the hind limbs.3.Compared with wt AAV9,AAV-CPP.16 has higher blood-brain barrier penetration efficiency and brain cell transfection efficiency in various strains of mice and cynomolgus monkeys,and as the virus dose increases,its efficiency has also been significantly improved.4.After intravenous or lateral ventricle administration,compared with AAV9,the efficiency of AAV-CPP.16 gene delivery to the spinal cord and motor neurons of adult mice is significantly improved.5.Intravenous injection of AAV-CPP.16 will not bring obvious DRG and liver toxicity,but it will cause a large amount of exposure to peripheral tissues;while in the lateral ventricle route of administration,the extremely high delivery efficiency of AAVCPP.16 will not It causes obvious DGR toxicity,and it has almost no exposure toxicity to peripheral tissues. |
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