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A - Viral Vector Development -> AAV Vectors – Preclinical and Proof-of-Concept Studies

6: The Porcine Model for In Utero Gene Therapy

Type: Oral Abstract Session

Presentation Details
Session Title: AAV Gene Therapy in Large Animal Models
Location: Room 204
Start Time: 5/16/2022 11:15
End Time: 5/16/2022 11:30

IntroductionIn utero gene therapy and gene editing technology (IUGT) offer the potential to treat genetic diseases before the onset of pathology and take advantage of the fetal properties of small size, access to progenitor cell populations, and an immature immune system that are barriers to postnatal therapies. Although proof-of-principle and toxicity studies have demonstrated the safety and feasibility of IUGT in small animal models, studies in large animal models are less prevalent. The nonhuman primate (NHP) model provides obvious advantages but is limited by cost, availability, husbandry, and a limited number of fetuses per pregnancy. The pig is a large animal model with more similarity to humans than small animals in terms of anatomy, development, and immunology, and has the added benefit of a shorter gestation and multiparity over NHPs, facilitating faster evaluation of multiple variables. Furthermore, there are an increasing number of pig models to recapitulate human genetic diseases, highlighting the relevance of this preclinical model. As such, we initiated studies to determine the feasibility and safety of AAV mediated IUGT in the fetal pig model. MethodsAn adeno-associated virus serotype 9 (AAV9) designed to carry the green fluorescent protein (GFP) transgene under a CAG promoter (AAV9.GFP) was intravenously administered to 104 day gestation (term = 115 days) Yucatan mini-pigs. Specifically, after a maternal laparotomy and exposure of the uterus, a uterine incision was made and the umbilical vessels identified. The umbilical vein was cannulated and the viral vector was delivered in a total of 10 milliliters. The hysterotomy was closed in two layers to obtain a water-tight seal and the uterus returned to the maternal abdomen, which was closed prior to recovery from anesthesia. Fetuses were injected with either phosphate buffered saline (PBS) (n=1), low dose AAV9.GFP (4.5 x 10^13 vg/kg; n=1) or high dose AAV9.GFP (9 x 10^13 vg/kg; n=2). One week after injection, the sow was euthanized and fetuses were harvested for analyses. ResultsAt the time of harvest, the sow was healthy and all fetuses were viable with a strong umbilical arterial pulse. Fetal weights were within normal limits for gestational age (715-950g). The livers of all fetuses that had been injected with AAV9.GFP demonstrated strong GFP fluorescence on stereomicroscopy (Figure 1a) and histology (Figure 1b). On flow cytometry, 24.0-52.4% of non-hematopoietic liver cells were GFP positive, while there were no GFP positive cells in the livers of the PBS injected fetus or mom (0.0-0.1%) (Figure 1c). Quantitative RT-qPCR for GFP mRNA demonstrated expression in multiple organs including low level expression in the heart, lung, and intestine, and a 7,000 fold higher expression in the liver of a high dose recipient compared to the PBS injected control (Figure 1d). Pre- and post-injection liver function tests showed a rise in aspartate aminotransferase (AST) and alanine transaminase (ALT) in all fetal pigs including the PBS injected control (Figure 1e). ConclusionThis study validates that cannulation and injection of the umbilical vein for IUGT in fetal pigs is feasible, safe, and does not result in preterm labor at short-term time points. This supports the porcine model as a worthwhile large animal model for future testing and optimization of pre-clinical in utero gene therapies.

Apeksha Dave, Cara L. Berkowitz, Valerie L. Luks, Pallavi V. Menon, Brandon M. White, Rohan Palanki, Haiying Li, Philip W. Zoltick, William H. Peranteau

CHOP, Philadelphia, PA
 A. Dave: None.

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