471. In Vivo Potency Assay for AAV-Based Gene Therapy Vectors

Abstract

One hurdle in the translation of adeno-associated virus (AAV) vectors for the treatment of human disease is the challenge in demonstrating drug function in a potency assay, a requirement in the clinical translation to product. In vitro measurements of AAV potency suffer from poor efficiency of infectivity and are insensitive measures of potency. To address this issue, we have developed a robust in vivo assay for the measurement of vector potency. Using as the model vector the AAVrh.10 serotype coding for human frataxin (FXN, a mitochondrial protein essential for cellular function and that is deficient in Friedreich's ataxia), and based on the knowledge that intravenous administration of all AAV vectors primarily transduce the liver, we have developed a reproducible in vivo potency assay that can be used to set quality control standards for vector production. The assay is based on administration of the AAV vector (2.5 × 1010 genome copies) administered intravenously to 6 to 8 wk old Balb/c male mice with the liver harvested 2 wk later following PBS perfusion. Liver homogenates are processed to assess vector genome copies, transgene mRNA, and expressed protein. In order to establish acceptance criteria for assays of vector genome and mRNA levels, separate specifications were set for DNA sample load using the mouse housekeeping gene Tfrc, and RNA sample load using mouse 18S RNA, both based on data from quantitative PCR analysis of mouse livers (n=35) assayed in duplicate. Specifications were set as the median ± 2 standard deviations based on these assay results; for the potency assay results to be accepted, each of these specifications must be met by the test sample. For the AAVrh. 10 vector expressing FXN(AAVrh.10hFXN), liver vector genome levels (a measure of reproducibility of delivery) were 8.2 × 104 ± 2.2 × 104 genome copies/µg genomic DNA (n=10 mice, mean ± SD), liver human FXN mRNA levels (a measure of vector potency at the transcription level) were 2.2 × 103 ± 0.7 × 103 copies/µg total RNA (n=10 mice, mean ± SD) and liver human FXN protein levels (ELISA; a measure of vector potency at the protein level) were 54.8 ± 17.52 ng/mg protein (n=10 mice, mean ± SD). From this data, we established the following quality control specifications for AAVrh.10hFXN vectors: vector genome range 6 × 104 - 1 × 105 (copies/µg DNA), hFXN mRNA level range 1.6 × 103 - 2.9 × 103 (copies/µg RNA) and the hFXN protein levels 37 - 72 (ng/mg protein). This approach is adaptable to any AAV vector, providing an in vivo quality control for vector function.