Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Isolation, Expansion, and Endoscopic Delivery of Autologous Enteric Neuronal Stem Cells in Swine.

The enteric nervous system (ENS) is an extensive network of neurons and glia within the wall of the gastrointestinal (GI) tract that regulates many essential GI functions. Consequently, disorders of the ENS due to developmental defects, inflammation, infection, or age-associated neurodegeneration lead to serious neurointestinal diseases. Despite the prevalence and severity of these diseases, effective treatments are lacking as they fail to directly address the underlying pathology. Neuronal stem cell therapy represents a promising approach to treating diseases of the ENS by replacing the absent or injured neurons, and an autologous source of stem cells would be optimal by obviating the need for immunosuppression. We utilized the swine model to address key questions concerning cell isolation, delivery, engraftment, and fate in a large animal relevant to human therapy. We successfully isolated neural stem cells from a segment of small intestine resected from 1-month-old swine. Enteric neuronal stem cells (ENSCs) were expanded as neurospheres that grew optimally in low-oxygen (5%) culture conditions. Enteric neuronal stem cells were labeled by lentiviral green fluorescent protein (GFP) transduction, then transplanted into the same swine from which they had been harvested. Endoscopic ultrasound was then utilized to deliver the ENSCs (10,000-30,000 neurospheres per animal) into the rectal wall. At 10 and 28 days following injection, autologously derived ENSCs were found to have engrafted within rectal wall, with neuroglial differentiation and no evidence of ectopic spreading. These findings strongly support the feasibility of autologous cell isolation and delivery using a clinically useful and minimally invasive technique, bringing us closer to first-in-human ENSC therapy for neurointestinal diseases.

Transplanted ENSCs form functional connections with intestinal smooth muscle and restore colonic motility in nNOS-deficient mice.

BACKGROUND: Enteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function. METHODS: Enteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS-/- mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1-2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility. RESULTS: Transplanted ENSCs formed nitrergic neurons in the nNOS-/- recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS. CONCLUSIONS: These results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

Schwann Cells in the Aganglionic Colon of Hirschsprung Disease Can Generate Neurons for Regenerative Therapy.

Cell therapy offers the potential to replace the missing enteric nervous system (ENS) in patients with Hirschsprung disease (HSCR) and to restore gut function. The Schwann cell (SC) lineage has been shown to generate enteric neurons pre- and post-natally. Here, we aimed to isolate SCs from the aganglionic segment of HSCR and to determine their potential to restore motility in the aganglionic colon. Proteolipid protein 1 (PLP1) expressing SCs were isolated from the extrinsic nerve fibers present in the aganglionic segment of postnatal mice and patients with HSCR. Following 7-10 days of in vitro expansion, HSCR-derived SCs were transplanted into the aganglionic mouse colon ex vivo and in vivo. Successful engraftment and neuronal differentiation were confirmed immunohistochemically and calcium activity of transplanted cells was demonstrated by live cell imaging. Organ bath studies revealed the restoration of motor function in the recipient aganglionic smooth muscle. These results show that SCs isolated from the aganglionic segment of HSCR mouse can generate functional neurons within the aganglionic gut environment and restore the neuromuscular activity of recipient mouse colon. We conclude that HSCR-derived SCs represent a potential autologous source of neural progenitor cells for regenerative therapy in HSCR.

Ednrb -/- mice with hirschsprung disease are missing Gad2-expressing enteric neurons in the ganglionated small intestine.

Hirschsprung disease is most often characterized by aganglionosis limited to the distal colon and rectum, and mice lacking the Endothelin receptor type B (Ednrb) faithfully recapitulate this phenotype. However, despite the presence of enteric ganglia in the small intestine, both human patients and Ednrb-/- mice suffer from dysmotility and altered gastrointestinal function, thus raising the possibility of enteric nervous system (ENS) abnormalities proximal to the aganglionic region. We undertook the present study to determine whether abnormalities with the ENS in ganglionated regions may account for abnormal gastrointestinal function. We performed single-cell RNA sequencing on ENS cells from the small intestine of Ednrb-/- mice and compared the results to a published single-cell dataset. Our results identified a missing population of neurons marked by the enzyme Gad2, which catalyzes the production of γ-Aminobutyric acid (GABA), in the small intestine of Ednrb-/- animals. This result was confirmed by immunostaining enteric ganglia from Ednrb-/- mice and their wild-type littermates. These data show for the first time that ganglionated regions of the Hirschsprung gut lack a neuronal subpopulation, which may explain the persistent gastrointestinal dysfunction after surgical correction of Hirschsprung disease.

Schwann cells in the subcutaneous adipose tissue have neurogenic potential and can be used for regenerative therapies.

Stem cell therapies for nervous system disorders are hindered by a lack of accessible autologous sources of neural stem cells (NSCs). In this study, neural crest-derived Schwann cells are found to populate nerve fiber bundles (NFBs) residing in mouse and human subcutaneous adipose tissue (SAT). NFBs containing Schwann cells were harvested from mouse and human SAT and cultured in vitro. During in vitro culture, SAT-derived Schwann cells remodeled NFBs to form neurospheres and exhibited neurogenic differentiation potential. Transcriptional profiling determined that the acquisition of these NSC properties can be attributed to dedifferentiation processes in cultured Schwann cells. The emerging population of cells were termed SAT-NSCs because of their considerably distinct gene expression profile, cell markers, and differentiation potential compared to endogenous Schwann cells existing in vivo. SAT-NSCs successfully engrafted to the gastrointestinal tract of mice, migrated longitudinally and circumferentially within the muscularis, differentiated into neurons and glia, and exhibited neurochemical coding and calcium signaling properties consistent with an enteric neuronal phenotype. These cells rescued functional deficits associated with colonic aganglionosis and gastroparesis, indicating their therapeutic potential as a cell therapy for gastrointestinal dysmotility. SAT can be harvested easily and offers unprecedented accessibility for the derivation of autologous NSCs from adult tissues. Evidence from this study indicates that SAT-NSCs are not derived from mesenchymal stem cells and instead originate from Schwann cells within NFBs. Our data describe efficient isolation procedures for mouse and human SAT-NSCs and suggest that these cells have potential for therapeutic applications in gastrointestinal motility disorders.

Tamoxifen administration alters gastrointestinal motility in mice.

BACKGROUND: Tamoxifen is widely used for Cre-estrogen receptor-mediated genomic recombination in transgenic mouse models to mark cells for lineage tracing and to study gene function. However, recent studies have highlighted off-target effects of tamoxifen in various tissues and cell types when used for induction of Cre recombination. Despite the widespread use of these transgenic Cre models to assess gastrointestinal (GI) function, the effect of tamoxifen exposure on GI motility has not been described. METHODS: We examined the effects of tamoxifen on GI motility by measuring total GI transit, gastric emptying, small intestinal transit, and colonic contractility in wild-type adult mice. KEY RESULTS: We observed a significant delay in total GI transit in tamoxifen-treated mice, with unaltered gastric emptying, accelerated small intestinal transit, and abnormal colonic motility. CONCLUSION: Our findings highlight the importance of considering GI motility alterations induced by tamoxifen when designing protocols that utilize tamoxifen as a Cre-driver for studying GI function.

Enteric mesenchymal cells support the growth of postnatal enteric neural stem cells.

Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb-/- mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb+/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb-/- mice generated functional neurons with similar calcium responses to Ednrb+/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb-/- recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.

Pan-enteric neuropathy and dysmotility are present in a mouse model of short-segment Hirschsprung disease and may contribute to post-pullthrough morbidity.

PURPOSE: Hirschsprung disease (HSCR) is characterized by distal intestinal aganglionosis. While surgery is lifesaving, gastrointestinal (GI) motility disorders persist in many patients. Our objective was to determine whether enteric nervous system (ENS) abnormalities exist in the ganglionated portions of the GI tract far proximal to the aganglionic region and whether these are associated with GI dysmotility. METHODS: Using Ednrb-null mice, a model of HSCR, immunohistochemical analysis was performed to evaluate quantitatively ENS structure in proximal colon, small intestine, and stomach. Gastric emptying and intestinal transit were measured in vivo and small and large bowel contractility was assessed by spatiotemporal mapping ex vivo. RESULTS: Proximal colon of HSCR mice had smaller ganglia and decreased neuronal fiber density, along with a marked reduction in migrating motor complexes. The distal small intestine exhibited significantly fewer ganglia and decreased neuronal fiber density, and this was associated with delayed small intestinal transit time. Finally, in the stomach of HSCR mice, enteric neuronal packing density was increased and gastric emptying was faster. CONCLUSIONS: ENS abnormalities and motility defects are present throughout the ganglionated portions of the GI tract in Ednrb-deficient mice. This may explain the GI morbidity that often occurs following pull-through surgery for HSCR.

Hypoganglionosis in the gastric antrum causes delayed gastric emptying.

BACKGROUND: Enteric nervous system (ENS) abnormalities have been implicated in delayed gastric emptying but studies exploring potential treatment options are limited by the lack of an experimental animal model. We examined the ENS abnormalities in the mouse stomach associated with aging, developed a novel model of gastroparesis, and established a new approach to measure gastric emptying. METHODS: A modified gastric emptying assay was developed, validated in nNOS -/- mice, and tested in mice at multiple ages. Age-related changes in ENS structure were analyzed by immunohistochemistry. Gastric aganglionosis was generated in Wnt1-iDTR mice using focal administration of diphtheria toxin (DT) into the anterior antral wall. KEY RESULTS: Older mice (>5 months) exhibit hypoganglionosis in the gastric antrum and a decreased proportion of nNOS neurons as compared to younger mice (age 5-7 weeks). This was associated with a significant age-dependent decrease in liquid and solid gastric emptying. A novel model of gastric antrum hypoganglionosis was established using neural crest-specific expression of diphtheria toxin receptor. In this model, a significant reduction in liquid and solid gastric emptying is observed. CONCLUSIONS & INFERENCES: Older mice exhibit delayed gastric emptying associated with hypoganglionosis and a reduction in nNOS-expressing neurons in the antrum. The causal relationship between antral hypoganglionosis and delayed gastric emptying was verified using a novel experimental model of ENS ablation. This study provides new information regarding the pathogenesis of delayed gastric emptying and provides a robust model system to study this disease and develop novel treatments.

Enteric neuronal cell therapy reverses architectural changes in a novel diphtheria toxin-mediated model of colonic aganglionosis.

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Intraganglionic macrophages: a new population of cells in the enteric ganglia.

The enteric nervous system shares embryological, morphological, neurochemical, and functional features with the central nervous system. In addition to neurons and glia, the CNS includes a third component, microglia, which are functionally and immunophenotypically similar to macrophages, but a similar cell type has not previously been identified in enteric ganglia. In this study we identify a population of macrophages in the enteric ganglia, intermingling with the neurons and glia. These intraganglionic macrophages (IMs) are highly ramified and express the hematopoietic marker CD45, major histocompatibility complex (MHC) class II antigen, and chB6, a marker specific for B cells and microglia in avians. These IMs do not express antigens typically associated with T cells or dendritic cells. The CD45+ /ChB6+ /MHCII+ signature supports a hematopoietic origin and this was confirmed using intestinal chimeras in GFP-transgenic chick embryos. The presence of green fluorescent protein positive (GFP+) /CD45+ cells in the intestinal graft ENS confirms that IMs residing within enteric ganglia have a hematopoietic origin. IMs are also found in the ganglia of CSF1RGFP chicken and CX3CR1GFP mice. Based on the expression pattern and location of IMs in avians and rodents, we conclude that they represent a novel non-neural crest-derived microglia-like cell population within the enteric ganglia.

Collagen 18 and agrin are secreted by neural crest cells to remodel their microenvironment and regulate their migration during enteric nervous system development.

The enteric nervous system (ENS) arises from neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the intestinal wall. Many extracellular matrix (ECM) components are present in the embryonic gut, but their role in regulating ENS development is largely unknown. Here, we identify heparan sulfate proteoglycan proteins, including collagen XVIII (Col18) and agrin, as important regulators of enteric neural crest-derived cell (ENCDC) development. In developing avian hindgut, Col18 is expressed at the ENCDC wavefront, while agrin expression occurs later. Both proteins are normally present around enteric ganglia, but are absent in aganglionic gut. Using chick-mouse intestinal chimeras and enteric neurospheres, we show that vagal- and sacral-derived ENCDCs from both species secrete Col18 and agrin. Whereas glia express Col18 and agrin, enteric neurons only express the latter. Functional studies demonstrate that Col18 is permissive whereas agrin is strongly inhibitory to ENCDC migration, consistent with the timing of their expression during ENS development. We conclude that ENCDCs govern their own migration by actively remodeling their microenvironment through secretion of ECM proteins.

Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

Morphine is one of the most widely used drugs for the treatment of pain. However, side effects, including persistent constipation and antinociceptive tolerance, limit its clinical efficacy. Prolonged morphine treatment results in a "leaky" gut, predisposing to colonic inflammation that is facilitated by microbial dysbiosis and associated bacterial translocation. In this study, we examined the role of enteric glia in mediating this secondary inflammatory response to prolonged treatment with morphine. We found that purinergic P2X receptor activity was significantly enhanced in enteric glia that were isolated from mice with long-term morphine treatment (in vivo) but not upon direct exposure of glia to morphine (in vitro). LPS, a major bacterial product, also increased ATP-induced currents, as well as expression of P2X4, P2X7, IL6, IL-1ß mRNA in enteric glia. LPS increased connexin43 (Cx43) expression and enhanced ATP release from enteric glia cells. LPS-induced P2X currents and proinflammatory cytokine mRNA expression were blocked by the Cx43 blockers Gap26 and carbenoxolone. Likewise, colonic inflammation related to prolonged exposure to morphine was significantly attenuated by carbenoxolone (25 mg/kg). Carbenoxolone also prevented gut wall disruption and significantly reduced morphine-induced constipation. These findings imply that enteric glia activation is a significant modulator of morphine-related inflammation and constipation.-Bhave, S., Gade, A., Kang, M., Hauser, K. F., Dewey, W. L., Akbarali, H. I. Connexin-purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation.

The effect of gut microbiome on tolerance to morphine mediated antinociception in mice.

There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1ß expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.

HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release via TLR4 signaling in the enteric nervous system.

The loss of gut epithelium integrity leads to translocation of microbes and microbial products resulting in immune activation and drives systemic inflammation in acquired immunodeficiency syndrome (AIDS) patients. Although viral loads in HIV patients are significantly reduced in the post-cART era, inflammation and immune activation persist and can lead to morbidity. Here, we determined the interactive effects of the viral protein HIV-1 Tat and lipopolysaccharide (LPS) on enteric neurons and glia. Bacterial translocation was significantly enhanced in Tat-expressing (Tat+) mice. Exposure to HIV-1 Tat in combination with LPS enhanced the expression and release of the pro-inflammatory cytokines IL-6, IL-1ß and TNF-α in the ilea of Tat+ mice and by enteric glia. This coincided with enhanced NF-κB activation in enteric glia that was abrogated in glia from TLR4 knockout mice and by knockdown (siRNA) of MyD88 siRNA in wild type glia. The synergistic effects of Tat and LPS resulted in a reduced rate of colonic propulsion in Tat+ mice treated with LPS. These results show that HIV-1 Tat interacts with the TLR4 receptor to enhance the pro-inflammatory effects of LPS leading to gastrointestinal dysmotility and enhanced immune activation.

Specific localization of ß-Arrestin2 in myenteric plexus of mouse gastrointestinal tract.

ß-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of ß-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of ß-arrestin2 within the gastrointestinal wall is not known. In this study we found that ß-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of ß-arestin2 was significantly higher in the ileum than the colon. We identified four variants of ß-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing ß-arrestin2 in the murine myenteric plexus and the co-localization of MOR1 and ß-arrestin2. ß-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, ß-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of ß-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC50: wild type = 5.8 vs. ß-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of ß-arrestin2 in the myenteric plexus within MOR1-expressing neurons and provide a relation for direct intracellular crosstalk between MOR1 receptor activation and ß-arrestin2 signaling in the myenteric neurons. ß-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.

Ribonucleotide reductase inhibitors hydroxyurea, didox, and trimidox inhibit human cytomegalovirus replication in vitro and synergize with ganciclovir.

Ganciclovir (GCV) is a deoxyguanosine analog that is effective in inhibiting human cytomegalovirus (HCMV) replication. In infected cells GCV is converted to GCV-triphosphate which competes with dGTP for incorporation into the growing DNA strand by the viral DNA polymerase. Incorporated GCV promotes chain termination as it is an inefficient substrate for elongation. Because viral DNA synthesis also relies on cellular ribonucleotide reductase (RR) to synthesize deoxynucleotides, RR inhibitors are predicted to inhibit HCMV replication. Moreover, as dGTP competes with GCV-triphosphate for incorporation, RR inhibitors may also synergize with GCV by reducing intracellular dGTP levels and there by promoting increased GCV-triphosphate utilization by DNA polymerase. To investigate potential of RR inhibitors as anti-HCMV agents both alone and in combination with GCV, HCMV-inhibitory activities of three RR inhibitors, hydroxyurea, didox, and trimidox, were determined. In both spread inhibition and yield reduction assays RR inhibitors had modest anti-HCMV activity with 50% inhibitory concentrations ranging from 36±1.7 to 221±52µM. However, all three showed significant synergy with GCV at concentrations below their 50% inhibitory and 50% toxic concentrations. These results suggest that combining GCV with relatively low doses of RR inhibitors could significantly potentiate the anti-HCMV activity of GCV in vivo and could improve clinical response to therapy.