551. Longitudinal High Resolution 3-D Imaging of AAV Gene Expression In Vivo

Abstract

Adeno-associated viral (AAV) are small non-integrating vectors that are being developed for a number of gene therapy applications. Different AAV serotypes, armed with constitutive or tissue specific promoters, capable of infecting various tissue types have been engineered to enable preferential transduction of the target tissues. In order to rapidly screen and evaluate the best AAV serotype and promoter for high levels of transgene expression in a specific organ, we have been developing the sodium iodide symporter (NIS) as a reporter gene for screening the biodistribution of AAV vectors. NIS is expressed endogenously in thyroid follicular cells where its role is to concentrate iodine for the synthesis of thyroid hormones. For more than 70 years, thyroidal NIS expression has been provided the basis for I-123 gamma camera or SPECT/CT imaging in thyroid disorders and for I-131 tumor ablation of metastatic thyroid cancer. Using NIS imaging, we would be able to longitudinally monitor gene expression in the same animal over time, from rodents, dogs, nonhuman primates and in human subjects. In this study, we have generated a panel of AAV vectors of serotype 1, 2, 5, 8, and 9 and administered the vectors via various routes of delivery. The mice and rats were imaged using SPECT (I-125 or Tc99m pertechnetate) isotopes on a high resolution microSPECT/CT scanner. Results indicate robust AAV gene expression by 2 weeks post vector administration. View Large Image | Download PowerPoint Slide[Figure 1: AAV mediated NIS gene expression in the liver and heart of mice post IV administration of vector]. For example, AAV9-CMV-NIS gave high levels of cardiac gene expression and interestingly, of subcutaneous brown fat (scapular region). In contrast AAV1-CMV-NIS resulted in highest gene expression in the subcutaneous brown fat only whereas AAV-LST-NIS (liver specific promoter) gave only NIS expression in the liver. When the regions of interests were quantitated, we found that AAV-NIS gene expression was maintained over 90 days in immunocompetent rats and over 200 days in immunocompromised mice. Using NIS as the reporter gene, we are able to use the same vector construct and perform similar noninvasive NIS imaging studies in large animal models using the same SPECT isotopes in combination with clinical SPECT/CT scanners. In contrast to traditional biodistribution studies utilizing PCR analysis or luciferase imaging, NIS imaging enables rapid identification of the organs supporting high levels of transgene expression.