Ex vivo adenovirus-mediated gene delivery leads to long-term expression in pancreatic islet transplants

BACKGROUND: Replication-deficient adenovirus, one of the most efficient vectors in gene therapy, has been limited by transient transgene expression due to its episomal location and loss during cell division, as well as a host immune response against viral proteins. METHODS: Murine pancreatic islets were infected ex vivo with ad5-cytomegalovirus (CMV)-beta-galactosidase and transplanted into diabetic recipients with normalization of glucose metabolism. RESULTS: High levels of beta-galactosidase activity were detectable histologically for at least 20 weeks after transplant, and beta-galactosidase and viral mRNA were also present that long. Sera from transplanted animals did not significantly inhibit ad5-CMV-interleukin-2-Ig infection of HeLa cells in vitro, whereas sera from intravenously delivered ad5-CMV-beta-galactosidase drastically diminished HeLa cell infection, suggesting the presence of reduced levels of antibodies in transplanted animals as compared with intravenously infected animals. Immunofluorescent staining of islet isografts infected with ad5-CMV-beta-galactosidase revealed the presence of CD8+ and CD4+ T lymphocytes at all time points, however, no islet destruction was seen. CONCLUSIONS: Treatment of islet isografts ex vivo with ad5-CMV-beta-galactosidase results in prolonged transgene expression, possibly due to an attenuated immune response against adenovirus.     

Chronic chlorpropamide therapy of noninsulin-dependent diabetes augments basal and stimulated insulin secretion by increasing islet sensitivity to glucose

To determine the effect of chronic sulfonylurea therapy on islet function in noninsulin-dependent diabetes mellitus (NIDDM), studies were performed in 18 untreated NIDDM patients before and after 12-16 weeks of chlorpropamide therapy. Fasting plasma glucose (FPG) fell with chlorpropamide therapy from 249 +/- 16 to 157 +/- 8 mg/dl (mean +/- SEM; P less than 0.001), and basal insulin increased from 17 +/- 2 to 24 +/- 3 microU/ml (P less than 0.001). The percent change in basal insulin correlated with the pretreatment FPG (r = 0.62; P less than 0.01) and inversely with the change in FPG during chlorpropamide (r = -0.57; P less than 0.025). Thus, patients with the highest pretreatment FPG showed the largest relative increase in basal insulin and the largest fall of FPG with chlorpropamide therapy. In nine patients, arginine-stimulated acute insulin responses (AIR) were studied at each of three plasma glucose (PG) levels both before and during chlorpropamide treatment. AIR at FPG was not different before and during treatment. However, when PG during treatment was matched by glucose infusion to the pretreatment FPG, the AIR was clearly increased during chlorpropamide therapy (176 +/- 65 vs. 49 +/- 11 microU/ml; P less than 0.02). When AIR is plotted against PG for each individual, the slope of the regression line generated (slope of glucose potentiation) is a measure of that patient's islet sensitivity to glucose. The logarithm of the slope of glucose potentiation correlated inversely with FPG (r = -0.92; P less than 0.001). Chlorpropamide treatment increased the slopes of potentiation from 0.26 +/- 0.11 to 1.47 +/- 0.70 (P less than 0.01). We conclude that chronic chlorpropamide therapy augments both basal and stimulated insulin secretion in NIDDM and that this may be an important mechanism of the drug's hypoglycemic effect. The data support the hypothesis that the hyperglycemia of NIDDM is related to islet insensitivity to glucose and that chlorpropamide treatment improves this impairment.     

Role of integrin expression in adenovirus-mediated gene delivery to the intestinal epithelium

Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.     

Transgenic overproduction of islet amyloid polypeptide (amylin) is not sufficient for islet amyloid formation

Islet amyloid polypeptide forms islet amyloid deposits in non-insulin-dependent diabetes mellitus. We have generated transgenic mice which express human islet amyloid polypeptide in their pancreatic beta cells yet do not develop islet amyloid deposits despite producing levels of the amyloidogenic human peptide 2 - 3 fold higher than the native (mouse) peptide. To determine whether marked overproduction of islet amyloid polypeptide is a potential cause of islet amyloid formation, we increased expression of this transgene by producing homozygous transgenic animals and by making heterozygous mice experimentally insulin resistant with nicotinic acid. Pancreatic content of islet amyloid polypeptide-like immunoreactivity in homozygous and nicotinic acid-treated mice was 2-fold (25 +/- 7 fmol/microg; n = 6) and 3.5-fold (47 +/- 20 fmol/microg; n = 3) higher, respectively, than that of untreated heterozygous animals (13+/-2 fmol/microg; n = 11; both p < 0.05). Despite this marked increase in production of islet amyloid polypeptide, neither group of mice developed gross islet amyloid deposits even after 16 months of age. We conclude that overproduction of islet amyloid polypeptide, even as produced by extreme insulin resistance, is not in itself sufficient for islet amyloid formation.     

cDNA cloning, gene expression and secretion of chitinase in winged bean

The role of nitric oxide (NO) in the control of arteriovenous anastomoses (AVAs) has not been studied in vivo in a thermoregulatory end organ. In this study, the effect of local inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME) on the microvasculature in the rabbit ear (n=12) was observed in vivo through a chronically implanted ear microvascular chamber. Ear cutaneous blood perfusion (CBP), total auricular arterial flow (TAF), and ear temperature were monitored simultaneously with the direct microvascular observations. Results revealed that intrafacial artery infusion of L-NAME produced significant vasoconstriction of arterioles, AVAs, and venules (p < 0.05). A decrease of ear blood perfusion also was demonstrated by changes of CBP, TAF, and surface temperature. The data provide evidence that basal generation of NO influences the vascular resistance in the thermoregulatory end organ. Moreover, endogenous NO production may be more important in regulating the AVA flow than is flow in other parts of the rabbit ear microvasculature. The effects of NO inhibition on ear microvasculature were not abolished by superior cervical ganglionectomy, indicating that NO production in the rabbit ear is not a neurally mediated mechanism. Further study with a short-term rabbit ear preparation showed that inhibition of NO production with L-NAME enhanced microvascular constrictive responses to extraluminal application of norepinephrine. NO thus appears to play a role of basal vasodilator in opposition to the basal adrenergic vasoconstrictor tone in the rabbit ear.     

Immunoreactivity and expression of amylin in gastroenteropancreatic endocrine tumors

Amylin was isolated from human insulinomas, but there has been only preliminary data regarding whether this peptide can also be detected in other types of gastroenteropancreatic endocrine tumors. In the present study, immunohistochemical staining of 87 gastroenteropancreatic endocrine tumors demonstrated amylin immunoreactivity in 21.8% of the neoplasmas. Thirteen of 15 insulinomas, three of 21 gastrinomas, two of 29 nonfunctioning tumors, and one of 18 carcinoids were amylin-immunoreactive. Seventeen of the 19 amylin-immunoreactive tumors were primarily located in the pancreas, but two tumors were found in the intestine. Measurements of amylin messenger RNA expression in a few tumors revealed amylin synthesis in these tumors. Amylin immunoreactivity did not correlate with invasion and metastasis. However, the rate of curative resections was significantly higher in amylin-immunoreactive tumors. These results demonstrate for the first time that amylin immunoreactivity is not restricted to insulinomas and can also occur rarely in endocrine tumors of the intestine.     

Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

Potent and selective inhibition of gene expression by an antisense heptanucleotide

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.     

Selective expansion of alveolar macrophages in vivo by adenovirus-mediated transfer of the murine granulocyte-macrophage colony-stimulating factor cDNA

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1. 9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.     

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.     

Reversal of CPT-11 resistance of lung cancer cells by adenovirus-mediated gene transfer of the human carboxylesterase cDNA

To evaluate the concept that transfer of the human carboxylesterase (CE) gene will overcome the drug resistance of a solid tumor to CPT-11 (irinotecan), we used an adenovirus vector (AdCMV.CE) carrying human CE cDNA to infect CPT-11-resistant A549 human adenocarcinoma cells (A549/CPT) in vitro and in vivo and evaluated cell growth over time. The A549/CPT cells, selected by stepwise and continuous exposure of parental A549 cells to CPT-11 over 10 months, had a 6-fold resistance to CPT-11 and 42% CE activity in comparison with parental A549 cells. AdCMV.CE infection resulted in an increase in functional CE protein in resistant cells in vitro that was sufficient to convert CPT-11 to its active metabolite, SN-38, and effectively suppressed resistant cell growth in vitro in the presence of CPT-11. When AdCMV.CE was directly injected into established s.c. resistant A549-based tumors in nude mice receiving CPT-11, there was a 1.8-fold reduction in tumor size at day 20 compared to that of controls (P < 0.05). These observations suggest that adenovirus-mediated gene transfer of the human CE gene and concomitant administration of CPT-11 may have potential as a strategy for local control of acquired CPT-11 resistance of solid tumors.     

Chronic overproduction of islet amyloid polypeptide/amylin in transgenic mice: lysosomal localization of human islet amyloid polypeptide and lack of marked hyperglycaemia or hyperinsulinaemia

Type 2 (non-insulin-dependent) diabetes mellitus is characterised by hyperglycaemia, peripheral insulin resistance, impaired insulin secretion and pancreatic islet amyloid formation. The major constituent of islet amyloid is islet amyloid polypeptide (amylin). Islet amyloid polypeptide is synthesized by islet beta cells and co-secreted with insulin. The ability of islet amyloid polypeptide to form amyloid fibrils is related to its species-specific amino acid sequence. Islet amyloid associated with diabetes is only found in man, monkeys, cats and racoons. Pharmacological doses of islet amyloid polypeptide have been shown to inhibit insulin secretion as well as insulin action on peripheral tissues (insulin resistance). To examine the role of islet amyloid polypeptide in the pathogenesis of Type 2 diabetes, we have generated transgenic mice with the gene encoding either human islet amyloid polypeptide (which can form amyloid) or rat islet amyloid polypeptide, under control of an insulin promoter. Transgenic islet amyloid polypeptide mRNA was detected in the pancreas in all transgenic mice. Plasma islet amyloid polypeptide levels were significantly elevated (up to 15-fold) in three out of five transgenic lines, but elevated glucose levels, hyperinsulinaemia and obesity were not observed. This suggests that insulin resistance is not induced by chronic hypersecretion of islet amyloid polypeptide. Islet amyloid polypeptide immunoreactivity was localized to beta-cell secretory granules in all mice. Islet amyloid polypeptide immunoreactivity in beta-cell lysosomes was seen only in mice with the human islet amyloid polypeptide gene, as in human beta cells, and might represent an initial step in intracellular formation of amyloid fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1

Glucagon-like peptide 1 is a gastrointestinally derived hormone with profound effects on nutrient-induced pancreatic hormone release. GLP-1 modulates insulin, glucagon and somatostatin secretion by binding to guanine nucleotide binding protein-coupled receptors resulting in the activation of adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). In the B-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion channel activity, intracellular Ca2+ handling and exocytosis of the insulin-containing granules. The stimulatory action of GLP-1 on insulin secretion, contrary to that of the currently used hypoglycaemic sulphonylureas, is glucose dependent and requires the presence of normal or elevated concentrations of the sugar. For this reason, GLP-1 attracts much interest as a possible novel principle for the treatment of human type-2 diabetes. Here we review the actions of GLP-1 on islet cell function and attempt to integrate current knowledge into a working model for the control of pancreatic hormone secretion.     

Inhibition of eFGF expression in Xenopus embryos by antisense mRNA

This pilot study analyzed the bone reactions to early loaded titanium plasma-sprayed implants. A total of 24 titanium plasma-sprayed implants (12 in the maxilla and 12 in the mandible) (Primary Healing Implant, Legnano) were inserted into four Macaca fascicularis monkeys with instruments specially designed to obtain a precise fit of the implant in the bone socket. A metal superstructure was cemented into 10 mandibular and 10 maxillary implants 15 days after implant insertion. The four remaining implants were used as controls. Eight months after implant placement, a block section was carried out, the defect was filled with nonresorbable hydroxyapatite, and all 24 implants were retrieved. The implants were treated to obtain thin ground sections that were examined under normal and polarized light. Histologic analysis showed that bone was observed around the implant surface in all implants. Morphometric analysis demonstrated that bone lined 67.2% (SD = 3.1%) of the maxillary implant surface, and 80.71% (SD = 4.6%) of the mandibular implant surface. No differences were found in the percentage of bone-implant contact in the control implants. In the loaded implants, however, the bone around the implants had a more compact appearance. The study demonstrated that it is possible to obtain a high percentage of bone-implant contact in early loaded titanium plasma-sprayed implants.     

Role of kinins in basal and furosemide-stimulated renin secretion

OBJECTIVE: To determine risk factors predictive of outcomes to aid in the cost-effective preoperative evaluation and postoperative management of patients who are undergoing tonsillectomy and adenoidectomy for obstructed breathing during sleep. DESIGN: A historical cohort study with a nested case-control analysis that examined risk factors associated with postoperative respiratory complications. SETTING: Children's Medical Center of Dallas, Dallas, Tex, which is a pediatric referral hospital for secondary and tertiary pediatric care with both private and university-appointed physicians. PATIENTS: A convenience sample of 355 patients who were undergoing tonsillectomy and adenoidectomy for obstructed breathing during sleep throughout a 1-year period. INTERVENTION: None. MAIN OUTCOME MEASURE: The occurrence of postoperative complications, including airway obstruction, apneas with oxygen desaturations, airway interventions (e.g., endotracheal intubation), or administration of supplemental oxygen, as they related to associated medical conditions (e.g., cerebral palsy or prematurity) and diagnostic tests (e.g., chest x-ray film and electrocardiogram). RESULTS: Five associated medical conditions (cerebral palsy; seizures; age, < or = 3 years; congenital heart disease; and prematurity) were identified as important predictors of a complicated postoperative course using stepwise logistic regression analysis. Those children with an abnormal chest x-ray film or electrocardiogram were also identified as having an associated medical condition that was predictive of postoperative complications. CONCLUSIONS: Children with 1 or more of the associated risk factors identified should be considered candidates for postoperative inpatient observation. A preoperative chest x-ray film and electrocardiogram were found to be of little predictive value, and they are probably not cost-effective screening tests for postoperative respiratory complications.     

Regulation of serotonin2A receptor expression by an antisense oligodeoxynucleotide

The regulation of 5-HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5-HT2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT2A receptor sites as measured by the binding of 2-[125I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT2A receptor density and an increase in headshake behavior induced by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT2A receptor expression.     

Studies on inhibition of activated oncogene expression by antisense RNA]

Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e.g. GPIb, GPIIa, GPIIb, GPIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70-90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO-expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB-IVC7 and CLB-SWI6 were used to hold the corresponding antigens GPIV and GPV, human anti-A specifically bound to these proteins derived from A1-platelets; neither GPIV nor GPV derived from A2-, B- or O-platelets bound anti-A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti-A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.     

Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression]

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.