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

Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno‐associated viral vectors (AAVs).

remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). Methods Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. Key Results The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the INTRODUCTION Viral vector technology in gene delivery to the enteric nervous system (ENS) is poorly exploited, despite important merits of viral vectors in the transduction of the central nervous system in both preclinical research and gene therapy. [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. Here, we further explored the transduction of the ENS in the ileum and colon of the mouse by single stranded AAV8 and AAV9 encoding GFP driven by the immediately early human cytomegalovirus (CMV) promoter after neonatal i.v. injection.

Recombinant AAV vector preparation
Vector production and purification were performed at the Leuven Viral Vector Core as previously described. 7 Adeno-associated viral vectors encoding enhanced GFP (eGFP) reporter protein driven by the CMV promoter were packaged in an AAV8-or AAV9-capsid.   ratio) into HEK293T cells using 25 kDa linear polyethylenimine, viral vector particles were collected from the supernatant and concentrated using tangential flow filtration and iodixanol gradient purification. Gradient fractions of concentrated AAV particles were stored at À80°C. Titers for AAV stocks were controlled by qPCR analysis to determine AAV genome copies (GC/mL) and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Injection procedure
Following hypothermic anesthesia, the respective AAV vectors were administered to C57BL/6 mouse pups on postnatal day one by injection into the superficial temporal vein using a 33G needle (Hamilton, Reno, NV, USA). 20 lL of AAV vector with 1.09 9 10 12 GC/mL AAV8 or 1.19 9 10 12 GC/mL AAV9 was administered. Eight animals from two different litters were injected for each vector. At 35 days of age the injected mice were sacrificed. All animal experiments were approved by the Ethical Committee for Animals of the University of Antwerp and were in line with EU directive 2010/63/EU.

Immunohistochemistry
The ileum and distal colon were fixed in 4% paraformaldehyde. Whole-mounts and cryosections were prepared as described before. 8 After rinsing with PBS, tissues were incubated with corresponding Cy3-conjugated secondary IgG antibodies raised in donkey (1 : 800; Jackson Immunoresearch, West Grove, PA, USA). Antibodies were validated by appropriate negative control experiments as detailed previously. 10,11 In case of VIP and CGRP immunostaining for quantification, axonal transport was blocked with colchicine in organotypic culture enhancing the perikaryal localization of these neuropeptides. The culture medium was composed of 10% FCS, 100 U/mL penicillin-streptomycin, 50 lg/mL gentamycin, 2.5 lg/ mL amphotericin B, 1 lM nifedipine, 0.1 mg/mL colchicine in DMEM:F12 (Life Technologies). Images were acquired using a Zeiss (Oberkochen, Germany) Axiophot fluorescence microscope equipped with an Olympus (Tokyo, Japan) DP70 digital camera (cryosections) or a Zeiss LSM510 confocal microscope (wholemounts).

Quantification & statistical analysis
Whole-mounts from at least three animals were counted (exact n is indicated in the results section). Randomly chosen fields of view amounted to an imaged area of 1.5 mm 2 per whole-mount. The number of counted neurons is listed in Table 1. Images were manually counted using ImageJ and data were further analyzed with Graphpad (La Jolla, CA, USA) Prism 6. Data are represented as mean AE SEM. The influence of the factors 'intestinal region' (ileum or colon) and 'AAV serotype' (AAV8 or AAV9) on the number of transduced (GFP-positive) enteric neurons (HuC/D-positive) was statistically evaluated with two-way ANOVA at a p = 0.05 significance level.

Transduction of the myenteric and submucosal plexuses by AAV8 and AAV9
Green fluorescent protein fluorescence was limited to enteric ganglia and interganglionic connecting nerve strands, with an identical GFP pattern for AAV8 or AAV9 ( Fig. 1A and B). Endogenous GFP fluorescence coincided with anti-GFP immunoreactivity in the whole-mounts, indicating that endogenous GFP fluorescence had not suffered from fixation or staining procedures (data not shown). Adeno-associated viral vector-transduced GFP fluorescence allowed morphological classification into Dogiel subtypes and tracing of individual axons along internodal strands in wholemount preparations (Fig. 1C). The pseudotypes, AAV8 and AAV9, were equally potent in transducing the ileal and colonic enteric plexuses ( Table 2). The myenteric plexus of both regions was transduced to the same extent, with 25-30% of the HuC/D immunoreactive myenteric neurons coexpressing GFP (Fig. 1D-F, Table 2). However, a significantly lower number of submucosal neurons showed GFP fluorescence in the colon (p = 0.0012, two-way ANOVA), although GFP-fluorescent nerve fibers could be readily observed ( Fig. 1G and H, Table 2).

Transduction of the neurochemical classes of enteric neurons by AAV8 and AAV9
All neuronal subtypes were susceptible to AAV transduction, as revealed by neurochemical marker staining (Fig. 2, Table 3). Given the low transduction of the colonic submucosal plexus, subtypes were not quantified for this specific region. The highest transduction efficiency was observed in the CGRP-or CALBimmunoreactive myenteric neurons, with 50 to almost 80% of these neurons expressing GFP. In the other subpopulations the transduced proportion remained closer to the 20-30% range. About 50 ganglia from two animals per AAV serotype were evaluated for the glial cell markers GFAP or S100. None of them yielded any GFP signal in enteric glial cells (Fig. 2I and J).

DISCUSSION
We demonstrate that AAV8 and AAV9, carrying GFP under control of a CMV promoter, efficiently transduce myenteric and submucosal neurons of the mouse small and large intestine when injected i.v. in P1 neonates. 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). Both vectors were equally potent, in contrast to the lower transduction efficiency of AAV8 compared to AAV9 observed in the adult rat colon after intramural injection of AAV-GFP under the CB promoter. 12 It should be noted that the systemic distribution of intravenously injected AAV is not limited to the ENS. 4,5,13 In the light of gene therapy, future efforts should evaluate specificity-enhancing strategies such as ENS-specific promotors, AAV with modified glycan binding ability or micro-RNAs. 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. The reason for this difference is not clear: transduction efficiency can be affected due to anatomical constraints such as villous fenestrated capillaries in the ileum facilitating viral vector access, or pertain to more complex physiological differences such as regional differences in immune response. 16 Alternatively, even though the CMV promoter can be considered as an ubiquitous promoter, lower CMV-driven expression in the submucosal plexus of the distal colon may also account for this difference.
Gombash et al. did not quantify transduction in neurochemically coded neurons, but did report that GFP expression was absent in VIP-or nNOS-positive myenteric neurons after AAV9 transduction, which contrasts with our observations showing nearly 20% of inhibitory (nitrergic) motor neurons expressing GFP. 6 The proportion of transduced intrinsic sensory neurons (CGRP-or CALB-immunoreactive) was substantially larger compared to inhibitory motor neurons, but transduction of the latter was not absent. Moreover, submucosal VIPergic neurons were readily transduced in our study. These discrepant observations might pertain to differences in the promoter (CB vs CMV), although the genetic background of the mice (FVB vs C57BL/6) may also play a role.
This study strengthens the validity of AAV vectors in transducing the myenteric plexus of the mouse and expands AAV application to the submucosal plexus. This application has important preclinical merits on the short term: Cre-inducible transgene-cassettes combined with Cre-expressing mouse lines enables selective manipulation of enteric neurons in vivo. For example, transduction of genetically encoded calcium sensors or channel rhodopsins allows selective imaging and manipulation in live cell experiments. The fact that transduction can be executed in the neonatal time window is important for studying postnatal ENS development during the weaning period. In the longer term, genetic therapies targeting the ENS in clinical applications can be evaluated.