Intravenous AAV9 efficiently transduces myenteric neurons in neonate and juvenile mice

Abstract

Gene therapies for neurological diseases with autonomic or gastrointestinal involvement may require global gene expression. Gastrointestinal complications are often associated with Parkinson's disease and autism. Lewy bodies, a pathological hallmark of Parkinson's brains, are routinely identified in the neurons of the enteric nervous system (ENS) following colon biopsies from patients. The ENS is the intrinsic nervous system of the gut, and is responsible for coordinating the secretory and motor functions of the gastrointestinal tract. ENS dysfunction can cause severe patient discomfort, malnourishment, or even death as in intestinal pseudo-obstruction (Ogilvie syndrome). Importantly, ENS transduction following systemic vector administration has not been thoroughly evaluated. Here we show that systemic injection of AAV9 into neonate or juvenile mice results in transduction of 25–57% of ENS myenteric neurons. Transgene expression was prominent in choline acetyltransferase positive cells, but not within vasoactive intestinal peptide or neuronal nitric oxide synthase cells, suggesting a bias for cells involved in excitatory signaling. AAV9 transduction in enteric glia is very low compared to CNS astrocytes. Enteric glial transduction was enhanced by using a glial specific promoter. Furthermore, we show that AAV8 results in comparable transduction in neonatal mice to AAV9 though AAV1, 5, and 6 are less efficient. These data demonstrate that systemic AAV9 has high affinity for peripheral neural tissue and is useful for future therapeutic development and basic studies of the ENS.

Proof‐of‐concept: neonatal intravenous injection of adeno‐associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine

“…This is in line with Gombash et al, who recently employed AAV vectors containing a self-complementary genome encoding GFP under a CB promoter for the transduction of the myenteric plexus. 6 Hence, the ratelimiting second-strand synthesis required in the case of single stranded AAV, does not affect the construct's expression in enteric neurons. The advantage of using single stranded AAV vectors lies in the longer construct length (4.7 kb) they can hold compared to self-complementary AAV (about 2.4 kb).…”

“…14,15 Enteric glia lacked transgene expression, but these cells could be targeted with other AAV serotypes or GFAP promotor-driven constructs, as these strategies have been proven successful in earlier work. 6,12 To our knowledge, the paper by Gombash et al is the only other work that neurochemically identifies AAV-transduced myenteric neurons of the mouse. 6 Our results further revealed significantly lower transduction efficiency in the submucosal plexus of the distal colon, compared to the ileum.…”

“…[1][2][3] Recombinant adenoassociated viruses vectors (AAVs) belong to the most promising candidate vector systems in gene therapy and preclinical research 1,3 , but hitherto little is known about the transduction efficiency of the ENS by AAV. Rahim et al 4 and Schuster et al 5 have preliminarily indicated transduction of the myenteric plexus of the mouse with AAV9, and a more recent paper by Gombash et al 6 has detailed myenteric plexus transduction by self-complementary AAV9 in neonate and juvenile mice with green fluorescent protein (GFP) being expressed under a chicken-b-actin/cytomegalovirus (CB) hybrid promoter. However, data on submucosal plexus transduction are currently lacking, as is quantification of the neuronal subtypes transduced by AAV.…”

Optical Tools to Investigate Cellular Activity in the Intestinal Wall

“…In case of binary expression systems, a ubiquitous promoter (eg, cytomegalovirus) can be used to optimize expression levels while cellular specificity is achieved by the control element driving Cre recombinase. Apart from transgenic animals, viral approaches can also be used either by injecting viral vector in the bloodstream 128 or by delivering vector intraluminally. 127 Here the combination of viral tropism and cell type specific promoters can help to yield expression in a subset of intestinal cells.…”

“…The successful transduction of enteric neurons with adeno-associated viral vectors that has been reported by a few new studies could be a solution. 127,128 An overview of possible strategies to deliver optical probes to intestinal tissues is summarized in Figure 3.…”