IL-10 Gene Recombinant Lentiviral Vector Into The Inner Ear Through Different Approaches For Local Treatment Of Autoimmune Sensorineural Hearing Loss In Guinea Pigs

Objective:It was evaluated that therapeutic effects of experimental autoimmune sensorineural hearing loss with transferring GFP recombinant replication-defective lentiviral vector of interleukin IL-10 gene in guinea pigs. The transfection and expression of therapeutic genes locally in inner ear and middle ear tissue following locally injection of lentiviral vectors were searched for. At the same time, we tried to explored a safe, efficient and simple method and approach for delivering gene into inner ear with comparing the therapeutic effects with gene vector Injected through three different ways.Methods:The purified 58kD antigen of guinea pig inner ear tissue were used to immune guinea pigs and autoimmune sensorineural hearing loss were induced in 60 animals. All of these animals were divided into six groups by matched-pairs design. Group A, group B and group C were the experimental groups. The viral vector with therapeutic gene were injected into scala tympani in group A, infiltrated through the round window membrane in grope B and injected into endolymphatic sac in group C. Group D, group E and group F were the control groups. The physiological perilymph were injected into scala tympani in grope D, infiltrated through the round window membrane in grope E and injected into endolymphatic sac in group F. Before and after gene therapy (7 days), the serum specific antibody levels were tested by ELISA test. Before and after treatment (7 days), theâ…¢wave thresholds of auditory brainstem response (ABR) were determined. Temporal bone paraffin section was made and observed under the light microscope, and ultrathin sections of spiral ligament and basement membrane were made and observed under electron microscopy and fluorescence microscope. The enzyme immunohistochemistry tests were made to detect lentiviral transfection and gene product expression.Results:After immunization, the levels of serum specific antibody against 58kD antigen in every group were higher than that before immunization, the difference was significant. But comparing between before and after locally lentiviral vector injecting, it showed no significant difference. Comparing with the pre-immune, The mean ABRâ…¢wave threshold of the all experimental groups showed rising before and after locally lentiviral vector injecting, and the differences were significant. But the thresholds of all experimental groups were lower than ones of all control groups. The results of Fluorescence microscope observation and immunohistochemistry showed that the expression sites of the fluorescent of lentiviral vector: Group A: stria vascularis, spiral ligament, Corti device, spiral ganglion, cochlear axis around small blood vessels in group A and spiral ganglion, spiral ligament, Corti device, vascular patterns , small blood vessels around modiolar part, vestibular membrane in group B, and endolymphatic sac, endolymphatic duct, the vestibular macula, crista ampulla in group C.Conclusions:The immunization with purified 58kD antigen of conspecific inner ear tissue can successfully result in autoimmune sensorineural hearing loss in guinea pigs. Recombinant replication-defective lentiviral vector carrying with IL-10 gene were transmitted into the inner ear via different channels, and transfected and expressed gene products (IL-10) in the inner ear location. Auditory function tests and histomorphological observation of the inner ear showed that the hearing function could be effectively improved and the tissue damage induced by autoimmune inflammation could be alleviated after local gene therapy through three kinds of approaches transmitted into inner ear, and the surgical performing did not significantly affect hearing function.