Therapeutic Effect Of PLP_193-151-Specific T Lymphcytes Modified By α-Melanocyte Stimulating Hormone Gene On Experimental Autoimmune Encephalomyelitis

Autoimmune diseases are induced by the immune responses to self-antigens, which can impaire the homologous organ and cause the function obstacles of them. Knowledge about the autoimmune diseases is often acquired via investigations on the animal models, which have the similar aspects on the clinic symptoms and tissue pathology. It is known that experimental autoimmune encephalomyelitis (EAE) is a disease characterized by inflammation and demyelination of the central nervous system (CNS) and served as a prototypical rodent model for human multiple sclerosis. EAE is mediated by CD4+ Thl lymphocytes and divided into active relapse-remitting EAE (REAE) and adoptivetransferring EAE (tEAE).At present, treatments for the abnormalities in the autoimmune diseases are giving non-specific immune inhibitors, which can cause serious side effects, such as general suppression of immune system, infection or tumors. Now, researches on the gene therapy of EAE are in the ascendant. There are two pathways for the gene therapy of EAE. One is systemic gene therapy, which is to construct the recombinant viral vectors or plasmids carrying the therapeutic gene and inject them into blood or transfect autoreactive T cells with them. Another pathway is to transfer the therapeutic gene carried by the viral vectors, plasmid DNA or DNA-liposome vector complexes directly into CNS. Vectors based on retroviruses (RV), adenoviruses (Adv), adeno-associated viruses (AAV), and herpes simplex virus (HSV) are commonly used to express the transgenic products and target the organs.Melanocyte-stimulating hormone (MSH) is a neuroimmunopeptide that plays a crucial role in the regulation of immune and inflammatory reactions. Recent studies have shown that a-MSH might promote the restoration of the injured nerves or spinal cord. The results of our previous experiments have shown that a-MSH had a protective action on EAE mice. The action mechanisms of a-MSH were involved in inhibiting the production and the roles of proinflammatory cytokines, but enhancing the secretion of anti-inflammatory cytokines. a-MSH is an endogenous peptide without evident toxicity. But the half-life of a-MSH is very short and its metabolizing rate is very fast. The defect of a-MSH is to require repeated injections in order to get the satisfactory effects. Webrought forward this study just because of the following reasons. First, a-MSH has the definite anti-inflammatory and the nerves restoration roles. Second, rAAV2 are not nosogenetic for human and able to infect both dividing and non-dividing cells. rAAV2 can be maintained in the host cell, creating the potential for long-term gene expression. Last, specific autoreactive T cells have the property recruited to the inflammatory sites and express the target gene only after stimulated with the same self-antigen. In this study, EAE as an animal model was used to testify the therapeutic effects and related action mechanisms of a-MSH gene modified PLP139-151-specific T cells (MSH-T cells), which were prepared by transfecting PLP139-151-T cells with rAAV2 carrying the a-MSH gene (a-MSH-rAAV2). The aim for this study was to explore the effective gene therapy for the chronic autoimmune inflammation and demyelination diseases within human CNS, and offer the theoretical and experimental foundations for the research on the treatment of the other autoimmune diseases. The results were described in three parts as follows.Part I Preparation of a-MSH-rAAV2DNA sequences that encoding the region of mouse IL-6 signal peptide and a-MSH gene were synthesized, and definitely cloned into the pAAV-IRES-hrGFP expression vector. Confirmed pAAV-IL-6sp-a-MSH-hrGFP plasmids were extracted and purified with endotoxin-free plasmid extracting kit.rAAV2 virus was prepared by co-transfecting HEK-293 cells with pHelper and pAAV-RC and the recombinant expression plasmid pAAV-IL-6sp-a-MSH-hrGFP or pAAV-IRES-hrGFP using calcium phosphate-based protocol. Then rAAV2 virus stock was concentrated through chloroform-PEG8000/NaCl method and purified through ultrafil...