Gene Therapy (1997) 4, 331–338  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors OA Brown 1 , RM Santer 2 , AF Shering 1 , AT Larregina 1 , AE Morelli 1 , TD Southgate 1 , MG Castro 1 and PR Lowenstein 1 1 Molecular Medicine Unit, Department of Medicine, University of Manchester School of Medicine, Manchester; 2 School of Molecular and Medical Biosciences, University of Wales Cardiff, UK We have designed a system in which to test gene transfer We encountered a distinctive staining of cells arranged in into gut neurons consisting of an organ culture of neonatal two concentric circles corresponding in location to the rat small intestine. The tissue was exposed to herpes sim- myenteric and submucosal plexuses. Cells in these areas plex- and adenovirus-derived vectors: (1) a temperature- were of similar size and morphology to neonatal enteric sensitive herpes simplex virus-1 (HSV1) vector (tsK-gal) neurons, as visualized by NADPH-diaphorase histochemi- containing the lacZ gene encoding -galactosidase (-gal), stry and immunocytochemical staining with antibodies to under the transcriptional control of the HSV1 immediate– the neuronally expressed proteins PGP 9.5, or neurofila- early 3 (IE3) promoter; (2) RAd35, an E1 - /E3 - replication- ments. Double labelling with antibodies recognizing neuro- deficient adenovirus expressing lacZ under the control of filaments and -galactosidase revealed that most cells a truncated HCMV major IE promoter; and (3) RAd122, an infected by tsK were neurons, while the RAd35 and 122 E1 - /E3 - replication-deficient adenovirus expressing the vectors only infected non-neuronal cells. We thus demon- lacZ under the control of the RSV LTR. Forty-eight hours strate that both HSV1- and adenovirus-derived vectors can after the vector was added to the organ culture, we be used to transfer genes to the gut in vitro, but they trans- detected -gal using immunohistochemistry or X-gal histo- duce different populations of target cells. chemistry in tissue sections examined by light microscopy. Keywords: autonomic nervous system; development; neuronal targeting; smooth muscle teric and submucous plexus of the small intestine is in a Introduction network of cells which is almost two dimensional, parti- The enteric nervous system in its entirety forms a remark- cularly when the gut is distended. 1,6 At postnatal day 2 ably vast and distributed neuronal network, which can (P2) the migration of neurons into the gut is complete be accessed and probed without the physical barriers that and they are present at a concentration of 84 345 neurons protect the central nervous system. 1,2 Viral vectors per cm 2 in the small intestine which is almost seven times derived from recombinant herpes simplex and aden- their concentration in the adult rat. 6 Rodent enteric neu- ovirus have been shown to deliver genes into various rons have the capacity to divide until 1 month after types of post-mitotic neurons both of the central and per- birth. 7 Also at this age, the enteric neurons have yet to ipheral nervous system. The characteristics of gene trans- aggregate into the well-defined ganglia of the myenteric fer are particular to each delivery system, both displaying plexus and form their intimate relationships with enteric advantages and disadvantages. However, in vitro both interstitial (glial) cells. At this stage the outer longitudinal are able to transduce large numbers of cells and achieve muscle layer is approximately 25 m thick and comprises high levels of expression of transgenes (see Refs 3–5 for smooth muscle cells separated by large extracellular reviews). spaces, a fact that was thought would facilitate infection The arrangement of enteric neurons in both the myen- from the serosal surface. The enteric nervous system constitutes a convenient system in which to study the effect of neurotrophins (eg Correspondence: Professor PR Lowenstein, Molecular Medicine Unit, GDNF) on neuronal phenotype development, or the Room 1.302 Stopford Building, Department of Medicine, University of effect of growth factors on age-induced changes in neu- Manchester School of Medicine, Oxford Road, Manchester M13 9PT, UK; ronal numbers as well as a system in which the effect of and Dr RM Santer, School of Molecular and Medical Biosciences, Univer- virus infection on neuron–target cell interactions could sity of Wales Cardiff, PO Box 911, Cardiff, CF1 3US, UK be conveniently evaluated. 7,8 We would like to examine OA Brown and AF Shering contributed equally to this work Received 15 January 1996; accepted 19 November 1996 the expression of transgenes in vitro in the absence of the