Abstract Title
Immunity and the Outcome of Gene Therapy: Route of Delivery Matters.

Research Category
Gene Therapy

Author(s)
Bill Bremer
Ellyn Peterson
Danielle Griffin
Chrystal Montgomery
Louis Chicoine
Louise Rodino-Klapac
Tom Preston
Chris Walker

Abstract
Progressive muscle weakness in patients with Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Dystrophin gene replacement using rAAV vectors is a promising approach to restore muscle function. However, in preliminary studies, direct injection of vector into skeletal muscle elicited dystrophin-specific T cell immunity in patients with DMD gene deletions. Because of this immune response, and the limitation of direct i.m. delivery of vector to correct a systemic defect in muscle function, we undertook studies of transgene expression and immunity in rhesus macaques after intravascular delivery of vector. Local i.m. injection of a vector encoding an eGFP model protein confirmed the priming of a functional T cell response and failure to sustain transgene expression observed in human subjects. Surprisingly, intravascular delivery of the vector to an entire lower limb of animals resulted in sustained transgene expression in multiple muscles through two years of follow-up. eGFP-specific cytotoxic CD8+ T cells expanded rapidly after intravascular vector delivery, but they lacked effector functions (cytotoxicity and production of cytokines) required to terminate transgene expression. These CD8+ T cells continuously infiltrated muscle despite robust and sustained eGFP expression in myocytes. The eGFP CD8+ T cells did not develop an exhausted phenotype that facilitates persistence of human virus infections. Instead, they maintained an effector phenotype inconsistent with a long-term absence of function. Studies to identify a potentially unique molecular mechanism of T cell silencing are underway. A detailed understanding of how T cell effector functions are shut-off will provide insight into the potential for spontaneous restoration of function, and identify novel molecular targets to reinforce blockade of an unwanted immune response.