Foetal Intraperitoneal AAV8 Injections as a Valid and Promising Tool to Specifically Target Neuronal Subpopulations Within the Gastrointestinal (GI) Tract

Abstract

Adeno‐associated viral vectors (AAV) are a versatile tool for gene transfer to manipulate neuronal function in preclinical research and are potential means for gene therapy. Recent studies showed successful in vivo transduction of the mouse enteric nervous system (ENS) by AAV after neonatal and postnatal injection of the viral vector (1–5). Although the full development of the mouse ENS also continues after birth, the prenatal phase starting from E9,5 till E14 spans the period of the start and complete colonization of the GI tract by neural crest cells. In humans the equivalent period corresponds to week 4–7 of gestation. Defects in ENS development result in congenital gastrointestinal phenotypes, such as Hirschsprung's disease. Therefore, in order to better understand the underlying mechanisms of ENS development and related dysfunctions, genetic studies performed at prenatal stage are essential. To test our transduction technique, AAV8 encoding eGFP driven by the immediately early human cytomegalovirus promoter was injected intraperitoneally into E14 mouse embryos. At postnatal day 35, the transduction of cell types in the gastrointestinal tract was evaluated using fluorescence microscopic imaging of the transgene‐encoded eGFP in combination with a set of neuronal and glial markers. In the distinct gastrointestinal segments, i.e. oesophagus, gastric corpus, ileum and distal colon, eGFP was exclusively found in neurons, stained with HuC/D, indicating a highly cell type specific transduction. Immunostaining revealed viral‐encoded eGFP neurons which were either nitrergic in nature, calbindin‐immunoreactive (ir), calretinin‐ir or CGRP‐ir, indicating that several functional neuronal subpopulations were transduced. A strong eGFP signal was also seen in neuronal fibers, either of intrinsic or possibly also of extrinsic origin as systemic administration of AAVs at foetal stage leads to extrinsic transduction of the nervous system as well (6). eGFP positive glial cells could not be observed. These results demonstrate the specific and successful transduction of the prenatal ENS. However, compared to earlier results from the neonatal and postnatal injections (2–4), the transduction efficiency in the myenteric plexus in prenatal injections is still significantly lower (6% versus 20–30%). Therefore, further research is still indicated to improve the technique (e.g. exploring other AAV serotypes and promoters), but the results presented in this study clearly show the enormous potential of this technique in addition to the transgenic animal breeding.Support or Funding InformationThis project is supported by BOF‐KP grant 32765 of the University of Antwerp.