Neither approach affected micro-dys.FLAG gene expression in AAVrh.74 antibodypositive or AAVrh.74 antibodynegative study groups. of preexisting immunity (natural infection) to AAV. == Introduction == Duchenne muscular dystrophy (DMD) is the most common, severely debilitating childhood form of muscular dystrophy. The disease is caused by mutations in theDMDgene,1,2which follows an X-linked recessive inheritance pattern. The size of this gene1creates an exceptionally large target for spontaneous germ-line mutations (1 in 10,000 sperm or eggs). Based on pooled data from worldwide newborn screening studies and the most recent study,3the revised estimate of DMD incidence at birth is ~1:5,000 newborn males. Newborn screening can never eliminate the disease, emphasizing the importance of finding an effective treatment. Dystrophin plays a central role in muscle function and integrity; specifically, it provides a scaffold for a number of important proteins that form the CHMFL-ABL/KIT-155 dystrophin-glycoprotein complex, linking the subsarcolemmal cytoskeleton to the extracellular matrix in skeletal muscle and cardiomyocytes.4Mutant dystrophin hinders stability of the dystrophin-glycoprotein complex, weakening the sarcolemmal membrane, leading to muscle cell injury and muscle fiber loss with replacement by connective tissue and fat.4Gene replacement by mini- or micro-dystrophins (micro-dys) delivered CHMFL-ABL/KIT-155 by recombinant adeno-associated virus (rAAV) represents an approach showing promise in proof-of-principle studies in mouse and dog.5,6,7,8,9These smaller dystrophin transgenes were designed to accommodate for the genome packaging limit of AAV (<5 kb) while maintaining much of the functional features of dystrophin. Safetytolerability clinical gene transfer trials for muscular dystrophies have so far been limited to direct intramuscular delivery trials.10,11,12Efficacy will require a different strategy reaching multiple muscle groups best achieved through the circulation. Inmdxmice, we have demonstrated that rAAV can deliver micro-dys9,13using an isolated limb perfusion model through the femoral artery. Outcome parameters indicative of efficacy include reduction in central nucleation, improved tetanic force, and increased resistance to eccentric contraction.9,13These studies set the stage for a more ambitious model in the nonhuman primate (NHP) translatable to the clinic.14This approach was modeled after themdxmice studies with targeted vascular delivery to one or a small group of contiguous muscles.9In the present study, we used the gastrocnemius as the target DGKD muscle for delivery because we could thread the catheter along to the sural artery through femoral access and achieve consistent results that were dose dependent in the macaque.15 In addition to proof of principle for our vascular delivery model, the impetus for the current study was our experience in clinical gene transfer trials of mini-dystrophin and -sarcoglycan.10,11,12In DMD, we found that transduction efficiency was limited by T-cellmediated responses to transgene by two different mechanisms.12The first was a T-cell response mounted against novel epitopes presented by the mini-dystrophin transgene in an area of the patient’s deletion. A second mechanism emerged unexpectedly demonstrating that revertant fibers had presented novel epitopes downstream of patient mutations eliciting antigen-specific T-cell responses before and after gene transfer. In the LGMD2D study, five of the six patients demonstrated robust transgene expression, whereas subject 6 had no measurable expression. The only parameter that was unique to subject 6 was the presence of preexisting binding and neutralizing antibodies to AAV1 before gene CHMFL-ABL/KIT-155 transfer.10Others have reported similar findings of poor transgene expression when vector has been administered in the presence of AAV neutralizing antibody titers,16,17,18and recent gene transfer clinical trials have enrolled subjects negative for anti-AAV antibodies.19,20In a rhesus macaque with endogenous dystrophin, it is difficult to fully address the problem of transgene T-cell immunity; however, the potential impedance of AAV antibodies on transgene expression and ways to circumvent this could be tested directly. Moreover, any confounding responses due to transgene immunity are minimized in this model. We created an experimental paradigm closely simulating the CHMFL-ABL/KIT-155 clinical scenario where we attempted gene transfer in rhesus macaques with preexisting immunity to rAAV rhesus serotype 74 ((rAAVrh.74) anti-AAV binding antibody titers and presumed T-cell immunity to rAAVrh.74), in addition to sero-negative animals. The AAVrh.74 serotype is endogenous to the rhesus macaque and was isolated from mesenteric lymph nodes and.