667. Effects of Various Physical, Chemical, and Biological Exposures on rAAV Stability and Function

Abstract

With an increasing number of potential recombinant adeno-associated virus (rAAV) gene therapies progressing to clinical trials, much time and money are devoted to ensuring that means of handling the vector do not effect the subsequent gene expression. In this study we hope to answer some of the concerns that arise when clinical (and basic science) trials are being planned.The vector used in this study was a rAAV2/1 encoding a chimpanzee alpha-1 antitrypsin (chimpAAT) gene under the control of a chicken beta actin promoter and CMV enhancer. The vector used in the study was all from the same pooled batches. Prior to intramuscular injection in mice the vector was subjected to 1 of 30 different conditions. The conditions included different storage temperatures (-80C, 4C, room temperature), different numbers of freeze thaw cycles, different diluents (serum (human, mouse and complement depleted), sodium chloride, lactated Ringers and gadolinium), high and low pH, ultraviolet (UV) exposure, high temperature (55C and 72C), varying storage materials prior to injection (polypropylene, polyethylene, polystyrene, stainless steel, glass, and syringe). Five B6.129S7.Rag-/- mice per group were then injected intramuscularly with 1×10^11 vector genomes/mouse. Serum was collected from each mouse weekly for 12 weeks. A total human AAT ELISA (which we previously determined accurately detects chimpAAT) was then performed with the serum.We found that the only conditions that led to little or no chimpAAT expression in the mouse serum were UV exposure for 10min and 72C exposure for 20 min. All other conditions resulted in serum chimpAAT levels between approximately 20,000 and 40,000 ng/ml. Statistical analysis of these results as well as vector titer and tissue vector genome data are still pending.These results are encouraging in that a wide variety of conditions had little effect on the resultant protein levels in the serum. We hope that these results will aid investigators when planning both research and clinical trials as they make decisions on how to handle the vector prior to injection.