Specific organ gene transfer in vivo by regional organ perfusion with herpes viral amplicon vectors: Implications for local gene therapy

Specific organ gene transfer in vivo by regional organ perfusion with herpes viral amplicon vectors: Implications for local gene therapy Ari D. Brooks, MD, Bruce Ng, BA, David Liu, MD, Michael Brownlee, MD, Michael Burt, MD, PhD, Howard J. Federoff, MD, PhD, and Yuman Fong, MD, New York, Bronx, and Rochester, NY

Background. Many gene therapy strategies would benefit from efficient, regional organ delivery of therapeutic genes. Methods. Regional perfusions of lung, liver, or bladder were performed to determine if rapid and efficient gene transfer can be accomplished in vivo, and to determine if in vivo gene transfer can be limited to the organ of interest. In addition, herpes simplex virus tumor necrosis factor (HSVtnf), carrying the human tumor necrosis factor α gene was used as a treatment for methylcholanthrene sarcoma in a syngeneic lung metastases model in Fisher rats. Results. A 20-minute perfusion using HSV carrying β-galactosidase (HSVlac) produced significant expression of this marker gene isolated to the target organs, without organ-specific tissue injury or inflammation. Regional perfusion of organs with HSV carrying the cytokine gene tumor necrosis factor α also resulted in high-level local organ production of this cytokine (2851 ± 53 pg/g tissue in perfused lung versus 0 for the contralateral lung). For the current vector construct, expression of the gene of interest peaked between 2 and 4 days and was undetectable by 2 weeks after perfusion. In animals undergoing perfusion as treatment for pulmonary sarcoma, there was no difference between tumor counts in lungs perfused with HSVlac (17 ± 6) or HSVtnf (22 ± 8), but either treatment resulted in lower tumor counts than controls (111 ± 24 nodules per lung, P <.02). Conclusions. Regional organ perfusion using herpes viral vectors is an effective and well-tolerated in vivo method of transiently delivering potentially toxic gene products to target organs in directing gene therapy. Regional lung perfusion with HSV amplicons reduces tumor burden in a rat model of pulmonary metastases, though HSVtnf cannot be demonstrated to augment the cytopathic effect of the HSV amplicon alone in the current model. (Surgery 2001;129:324-34.) From the Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY; the Department of Medicine, Albert Einstein College of Medicine, Bronx, NY; and the Department of Neurology, Medicine, Microbiology, and Immunology, University of Rochester, Rochester, NY

VIRAL GENE THERAPY STRATEGIES can benefit from methods to target organs of interest as a means of minimizing the cost of vectors used and of limiting potential toxicities of vector or genes of interest. In Supported in part by grants F32CA68802 (A. D. B.), RO1CA76416 (Y. F.), RO1CA72632 (Y. F.), and RO1CA61524 (Y. F.) from the National Institutes of Health and MBC-99366 (Y. F.) From the American Cancer Society. Accepted for publication September 3, 2000. Reprint requests: Yuman Fong, MD, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/56/111697 doi:10.1067/msy.2001.111697

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attempts to deliver genes coding for very toxic proteins such as tumor necrosis factor α (TNF-α) during isolated organ cancer therapy, the need to limit expression of this cytokine in the target organ is obvious.1 When using angiogenic proteins such as vascular endothelial growth factor for genetic revascularization2 or anti-angiogenic proteins such as endostatin for cancer therapy,3 limiting expression to sites of interest may also minimize toxicities elsewhere. A delivery system that can be used in vascular perfusion would potentially have clinical relevance for many targets. The possibility of isolated organ vascular delivery, however, depends on a vector system that reliably transgresses the vascular endothelium and rapidly and efficiently produces gene transfer.

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The current study was designed to determine if viral amplicon vectors based on herpes simplex type 1 could deliver potentially therapeutic genes to various organs in an isolated organ fashion. Two amplicon constructs were evaluated: pHSVlac, an amplicon plasmid coding for Escherichia coli β-galactosidase gene, and pHSVtnf, the plasmid coding for human tumor necrosis factor α (TNF-α). These studies were made on the basis of the observation that herpes simplex virus (HSV) is very efficient in gene transfer to nondividing somatic cells.4-7 In vivo data presented in this study indicate that these amplicon vectors are indeed promising vehicles for the delivery of therapeutic genes to target organ site by common surgical or interventional radiologic techniques and may result in efficacy against tumor. MATERIALS AND METHODS Animals. The experimental protocol was reviewed and approved by the Institutional Animal Care Committee of the Memorial Sloan-Kettering Cancer Center. Adult male Fischer 344 rats (200250 g) were housed in a 12:12-hour light-dark cycle environment and provided with water and standard rat chow ad libitum for at least 7 days before use. Tumor cell line. The rat methylcholanthrene (MCA)-induced sarcoma cell line was serially passaged in the flanks of Fischer rats until use. A single cell suspension was produced by mincing the tumor, incubating it in collagenase D (Boehringer Mannheim, Germany) and passing the chunks through a stainless steel mesh filter. Cells were then serially washed and filtered through a 70 µm nylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ). Intravenous injection of 0.5 to 1 × 107 cells resulted in bilateral tumor nodules 14 days later. Herpes viral vector. A replication deficient HSV vector was constructed to include the marker gene lac-Z/β-galactosidase (HSVlac) or the cytokine gene TNF-α. This HSVlac was constructed with an HSV promoter and packaged as described previously.8,9 The human TNF-α gene (R & D Systems, Minneapolis, MN) was cloned directionally into HSV/PRPuc and packaged as previously described.10,8,9 HSV/PRPuc contains the HSV immediate early 4/5 promoter, a multiple cloning site and SV40 A sequence and has been described previously.11-15 The RR1 cells used for packaging HSV amplicons were maintained in Dulbecco modified Eagle medium containing high glucose (4.5 g/L), 10% fetal calf serum (FCS), 1% penicillin/streptomycin, and 400 µg/mL of bioactive gentamycin

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(G418; Gibco-BRL, Gaithersburg, MD) at 37°C, 5% carbon dioxide. RR1 cells are baby hamster kidney cells stably transfected with the HSV IE3 gene. They were obtained from Dr Paul Johnson.14 D30EBA helper virus was prepared by growth on RR1 cells. D30EBA is a strain 17 derived IE3 mutant deleted from codons 83 to 1236 and was obtained from Dr Roger Everett.15 To package amplicon vectors, 3 × 106 RR1 cells were plated in media containing 10% FCS and 4 hours later were transfected by adding 40 µL of Lipofectin (GibcoBRL), waiting 5 minutes, and then adding the amplicon DNA solution dropwise (30 µg at 1 µg/µL in Dulbecco modified Eagle medium). Six hours later, plates were fed with media containing 5% FCS. Approximately 20 hours after transfection, D30EBA virus in 50 to 100 µL was added to achieve a multiplicity of infection of 0.2. Five milliliters of complete media with 5% FCS was added to each plate after 1 hour. Amplicon virus stocks were harvested 2 days later. After overnight storage at –70°C, fresh RR1 cells (4 × 106 cells/60 mm plate) were infected with sonicated and warmed (34°C) virus stock. Two days later, the stocks were harvested and stored for subsequent use. HSVlac virus stocks were titered by an expression assay. In brief, National Institutes of Health 3T3 cells were plated (2 × 105 cells per well of a 24-well plate) and infected with increasing volumes of an HSV amplicon virus stock in duplicate. Twenty-four hours after infection, cells were fixed and stained with the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-gal) by using standard methods.8 The number of X-gal+ (blue) cells were counted. Titers were expressed as the number of blue forming units per milliliter. The D30EBA helper virus in each stock was titered by plaque assay on RR1 cells. HSVtnf was titered by a slot blot assay as described previously.16 For slot blot analysis, viral DNA was extracted from packaged virus by phenol-chloroform twice, ethanol precipitated with single-strand calf thymus DNA as carrier, denatured at room temperature with 0.2 N sodium hydroxide, 0.5 mol/L sodium chloride for 10 minutes and loaded on nylon membrane with a slot blot apparatus. The membrane was then baked for 2 hours at 65°C and probed with a [32P]-labeled 435 base pair SspI and PvuI fragment containing part of the β-lactamase gene from pBR322 (nucleotides 3733-4168). After stringent washing (0.1 × SSC twice for 15 minutes), blots were exposed to x-ray film and various timed exposures were taken and densitometrically scanned (LKB Ultroscan, Uppsala, Sweden). Band densities between HSVlac and other viral stocks were com-

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Fig 1. Diagram of regional perfusion of lung. The left pulmonary artery of the rat is cannulated and inflow of blood occluded by clamping. The lung is rinsed with 1 cm3 normal saline solution before perfusion with HSV. The left pulmonary vein is then clamped and the lung perfused with HSV. After a continuous perfusion or after the viral solution is left indwelling for 20 minutes, the cannula is removed, the arteriotomy repaired, and the clamps removed.

pared and the titer of HSVtnf calculated from the density relative to HSVlac, given that this latter amplicon was titered by an expression assay (blue forming units on NIH 3T3 cells). The titers of viral stocks were expressed as particles per milliliter. Titers of amplicon stocks were between 0.8 and 2 × 106 particles/mL. D30EBA titers in stocks ranged between 5 × 106 to 6 × 107 plaque forming units per milliliter. Recombination for wild-type revertants was monitored by plaque assay on Vero cells and occurred at a frequency of 1 × 10-6. Regional perfusions Lung perfusions. Lung perfusions were performed according to a modification of a previously described method.17 Briefly, the left lungs of Fischer rats have only 1 pulmonary artery and 1 pulmonary vein. Animals received anesthesia of pentobarbital (50 mg/kg). The animals were intubated and placed on a ventilator. Through a left thoracotomy, the left hilar vessels were isolated (Fig 1). The pulmonary artery was cannulated with a polypropylene tubing (PE10; Clay Adams, Parsippany, NJ). The pulmonary vasculature was flushed with 1 cm3 of perfusion fluid before beginning perfusion. This flush in essence washed the majority of blood from the pulmonary vasculature. Then the pulmonary vein was clamped and a venotomy made for drainage of effluent. The viral preparation was introduced in 2 methods into the lung. Animals either received 1 × 106 or 5 × 106

viral particles infused in a volume of 0.5 cm3 and left indwelling for 20 minutes (indwelling group) in an isolated lung (pulmonary vein clamped) or received 5 × 106 particles administered by a slow infusion during 10 minutes (perfusion group) at a rate of 100 µL/min. The indwelling and the continuous perfusion systems were compared because we postulated that the rapid gene transfer that can be accomplished by amplicon HSV may achieve all its effects in the first pass in a continuous system. If this is true, delivery by interventional radiology would be achievable. Perfusion was followed by a flush with phosphate buffered saline (PBS). At that time, the pulmonary vein clamp was removed, the artery decannulated and repaired with a single 9-0 suture. Air was evacuated from the left thorax by a chest tube after thoracic closure. Animals subjected to perfusion with media alone (control) or with virus by each of the 2 methods were killed at 24, 48, and 96 hours after treatment for analysis. The number of animals for each time point, treatment condition, and vector (HSVlac or HSVtnf) consisted of 3 animals per group. Liver perfusions. Liver perfusions were as previously described for use with HSVlac.4 The pyloric vein was cannulated with a polypropylene PE10 tubing. Liver blood inflow was occluded by clamping the portal vein and hepatic artery. The liver was then flushed with 1 mL normal saline solution. The hepatic vascular outflow was then occluded by

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Fig 2. Diagram of regional perfusion of liver. The pyloric vein is cannulated and the inflow to the liver (portal vein and hepatic artery) clamped. The liver is rinsed with 1 cm3 normal saline solution before perfusion with HSV. The inferior vena cava (IVC) is clamped above and below the liver. The HSV solution is then injected in the liver and left indwelling for 20 minutes. The cannula is then removed, the pyloric vein ligated, and all occluders removed.

clamping the suprahepatic and infrahepatic inferior vena cava. We then infused 5 × 106 viral particles into the pyloric vein catheter and left indwelling in the liver for 20 minutes (Fig 2). After 20 minutes, all vessels were unclamped, the pyloric vein cannula was removed, and the vein ligated. The animals were resuscitated with 5 cm3 of normal saline solution intraperitoneally. Three animals per group per time point were perfused with HSVlac, HSVtnf, or normal saline solution (controls), and killed at 24, 48, and 96 hours for analysis. Bladder perfusions. While the rats were under pentobarbital sodium anesthesia (50 mg/kg), the abdomen was entered through a low midline incision. The bladder was cannulated with a 24 g angiocath and decompressed by drainage of all urine. It was then filled with 5 × 106 viral particles. After 20 minutes, the viral solution was removed and the bladder was repaired with a chromic suture. Three animals per group per time point were perfused with HSVlac, HSVtnf, or normal saline solution (controls), and killed at 24 and 96 hours for analysis. Experimental design β-Galactosidase expression. Groups of animals underwent regional lung, liver, or bladder perfusion with the HSVlac virus solution by using the methods outlined previously. At the time of death, blood and organs were harvested for analysis.

Blood samples were collected in heparinized tubes by vena caval puncture. The plasma was isolated by centrifugation at 4°C and stored at –70°C until analysis. For the lung perfusion studies, both lungs were removed, inflated with Tissue-tek imbedding medium (Miles Inc, Elkhart, IN) before frozen sections were performed. Livers and bladders were harvested, blotted to remove blood, and imbedded in Tissue-tek imbedding medium and stored at –70°C until sectioning. Five- to 10-µm sections were performed. Tissues transduced with HSVlac were histochemically stained with the chromogenic substrate 5-bromo-4-chloro-3-indoly β-D-galactoside (X-gal), which forms a dense blue precipitate in the presence of β-galactosidase.18 Staining was performed under conditions to control pH to minimize background cellular staining. Tissue sections were fixed for 5 minutes with 1% glutaraldehyde with 5 mmol/L EDTA, washed twice with PBS, then incubated with X-gal solution (X-gal [1 mg/mL] in PBS containing 1 mmol/L magnesium chloride, 5 mmol/L K3Fe(CN)6, and 5 mmol/L K4Fe(CN)63H2O). The histologic sections were counterstained with hematoxylin. The histologic sections were then evaluated for blue staining. Four representative sections from disparate areas of each organ were examined. The number of blue stained

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Fig 3. Histochemically stained sections of tissues harvested 72 hours after regional perfusion. Shown are representative histochemical section of control tissues A1 (lung), B1 (liver), C1 (bladder), and HSV-perfused tissues A2 (lung), B2 (liver), and C2 (bladder). Sections were stained with X-gal according to the protocol elaborated in the methods section. Counterstain was with hematoxylin. Original magnification, × 20.

cells was divided by the total number counted to determine the percent of staining. Human TNF-α expression. Animals underwent regional organ perfusion with the HSVtnf as outlined previously. At designated time points, animals were killed by pentobarbital overdose and their blood collected by abdominal aortic puncture. Lungs, heart, liver, and kidneys were harvested, weighed, and homogenized in NP-40 cell lysate buffer. The supernatants were collected and stored at –70°C for analysis. The TNF-α levels were measured by a commercially available enzyme-linked immunosorbent assay kit that is specific for human TNF-α (R&D Systems, Minneapolis, MN) and results read on a spectrophotometric plate reader (EL312e; Bio-Tek Instruments, Winooski, Vt). HSVtnf-perfusion as treatment for MCA sarcoma. On day 0, 30 rats were injected with 5 × 106 MCA sarcoma cells by means of the right internal jugular vein. On day 5, animals were treated with HSVtnf by using 2 different treatment protocols. Indwelling treatment. Initially, 68 animals were

separated in 4 treatment groups: control-no treatment (n = 15), saline (n = 19), HSVlac (n = 16), and HSVtnf (n = 18). These animals were subjected to indwelling saline or viral treatment (1 × 106 particles). The solution was left indwelling for 20 minutes with the inflow and outflow vessels clamped. The total pulmonary artery clamp time was approximately 35 to 40 minutes. On day 14, all animals were killed and their lungs were harvested and stained with India ink in Fakete’s solution for tumor nodule counting. Lung nodules were counted in a blinded fashion. Several representative stained lungs were paraffin mounted and sectioned for histologic confirmation of tumor nodules. Perfusion treatment. When it was clear that extended vascular clamping and treatment with saline had significant effect on decreasing tumor burden, treatment protocol was changed to a non-outflow occluding perfusion method. On day 5 after tumor implantation, 22 animals underwent regional lung perfusion with HSVlac (n = 6), HSVtnf (n = 8), or PBS (n = 8). The remaining 8 animals served as notreatment controls for this trial. The concentrated

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Surgery Volume 129, Number 3 Table I. Blood chemistries 72 hours after regional organ perfusions Control Regional liver perfusion Regional lung perfusion Regional bladder perfusion

Glucose

T bili

187 ± 4 195 ± 4 189 ± 3 199 ± 9

0.8 ± 0.1 0.7 ± 0.2 0.5 ± 0.1 0.6 ± 0.2

AST

Albumin

82 ± 2 83 ± 8 71 ± 4* 83 ± 7

2.7 ± 0.1 2.7 ± 0.1 2.3 ± 0.1* 2.8 ± 0.1

Transferrin

Triglyceride

180 ± 4 178 ± 5 163 ± 3* 186 ± 4

58 ± 1 56 ± 4 60 ± 6 65 ± 4

Controls were animals not subjected to operation. Mean ± SEM. T bili, Total bilirubin; AST, amino-S-transferase. *P < .05 versus control.

Table II. TNF-α levels in various organs after regional perfusion (5 × 106 particles of HSV TNF) of lung (non-outflow-occluding perfusion), liver (indwelling), or bladder. TNFα tissue levels (pg/g)

Liver perfusion Lung perfusion Bladder perfusion

Liver

Left lung

Right lung

Bladder

Kidney

180 ± 10 BD BD

BD 2400 ± 300 BD

BD BD BD

BD BD 1700 ± 250

BD BD BD

Lung perfusions involved regional perfusion of the left lung. BD, Below detectability.

virus solution, 5 × 106 viral particles per cubic centimeter, was continuously perfused at a rate of 100 µL/min for 10 minutes without outflow occlusion, followed by a 5-minute flush with PBS at the same rate. This resulted in a total pulmonary artery clamp time of 15 minutes. Liver function tests. Plasma samples were analyzed for liver function tests by automated methods (Technicon RA500; Technicon Instruments Corp, Tarrytown, NY). Glucose was analyzed by an automatic glucose analyzer (model 23A; Yellow Springs Instrument Co, Yellow Springs, OH) by using the glucose oxidase reaction.19 Statistical analysis. All data are expressed as mean ± SEM. Statistical analysis was performed by using analysis of variance with Bonferroni’s post hoc testing to compare TNF-α production among groups. Significance was defined as P < .05. RESULTS β-Galactosidase expression. A representative histochemical section of the lung, liver, and bladder perfused with HSVlac is presented in Fig 3. A histochemical section of the contralateral nonperfused lung and liver or bladder perfused with saline are also shown in Fig 3. Significant expression of β-galactosidase is noted in the HSVlac-perfused lung while no blue staining was noted in the contralateral lung. The same dose of HSVlac used in the regional liver perfusions, when delivered intravenously into the tail vein did not produce detectable lac-z expression in the liver (data not shown). No inflammatory infiltrates or other signs of tissue damage were seen. Additionally, no

staining for β-galactosidase could be detected in any other organ not perfused with HSVlac. Lungs treated by 1 × 106 viral particles for a 20minute dwell showed characteristic blue staining in 2% of the cells in the left lung at 24 hours. Lungs treated with 5 × 106 HSVlac in a 20-minute dwell expressed β-galactosidase in 5% to 7% of cells. Continuous perfusion with 5 × 106 particles produced more intense staining, particularly of the perihilar area of the lung, with 20% of the lung cells stained. The majority of stained cells were type I and type II pneumocytes and pulmonary macrophages. Fibroblasts in the pleura and hilum also were stained. There was no evidence of staining in the endothelial cells in the large or mediumsized vessels within the lung. None of the animals had blue staining in the right lung. By biochemical parameters, regional perfusion of any of the 3 organs studied had minimal effects on the general well-being of these animals (Table I). There was no significant hepatitis. Albumin levels dropped only in the animals subjected to regional lung perfusion. No rise in the acute phase protein transferrin could be noted in any group. Human TNF-α expression. High levels of human TNF-α were seen in the left lung on day 1 (2400 ± 300 pg/g tissue). On day 2, the local levels of TNF-α were still detectable at 200 ± 40 pg/g. TNF-α production decreased rapidly and was less than the 20 pg/g limits of detection by day 4. There was no human TNF-α detected in the right lungs, heart, liver, or serum of any animal. Control animals were negative for human TNF-α by enzyme-linked immunosorbent assay.

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Fig 4. Results of treatment by indwelling virus. Sarcoma lung nodules 2 weeks after intravenous injection of 5 × 106 cells. Animals underwent left regional lung perfusion on day 5 with a 20-minute dwell of 1 × 106 viral particles of HSVlac (n = 16), HSVtnf (n = 18), 0.5 mL saline (n = 19), or no treatment (n = 15).

In additional studies, animals with regional perfusions of lung (n = 3), liver (n = 3), or bladder (n = 3) using HSVtnf were killed at 24 hours and tissues assayed for human TNF-α (Table II). TNF was only detected in the target organ of the perfusion. The regional delivery of proteins of interest can clearly be produced by local organ gene transfer by using HSV amplicon vectors. Treatment with HSVtnf. In the indwelling studies, saline infusion alone significantly decreased left lung tumor burden. This is likely the result of the long ischemia time. Consequently, no increased efficacy of viral perfusion could be demonstrated (Fig 4). In the perfusion studies (Fig 5), the no-treatment group (100 ± 17 tumor nodules) and saline control group (111 ± 24 tumor nodules) mean left lung tumor counts were not significantly different from each other (P = 1.0, Fig 5). The HSVlac (17 ± 6 tumor nodules) and HSVtnf (22 ± 8 tumor nodules) groups both had significantly lower tumor counts than the no-treatment (P < .02) and saline control groups (P < .005) but were not different from each other (P = NS). Right-sided tumor counts (NoTx, 100 ± 15; saline, 92 ± 28; HSVlac, 94 ± 27; HSVtnf, 92 ± 19) were not significantly different among groups. DISCUSSION Gene transfer methods currently in clinical and preclinical studies can be grouped in 2 types: (1) physical methods including lipid-mediated trans-

fection,20 electroporation,21 calcium phosphatemediated gene transfer, and the gene gun,22 or (2) viral vector-mediated gene transfer.20,21,23 The longest experience and the most successful studies to date have been in the use of retroviral vectors,24,25 which are the basis for a number of ongoing clinical trials that use these vectors for ex vivo gene transfer vehicles.26 Retroviral vectors, however, are limited in that they require replicating cells for infection and, therefore, are inefficient in most somatic cells. This poses particular problems for in vivo delivery strategies. For targeting to the liver, for example, gene transfer was successful only in the setting of simultaneous hepatectomy to stimulate liver cell proliferation, and then only with minimal (< 0.5%) efficiency.27,28 This explains the ongoing efforts studying other viral vectors, such as adenovirus or vaccinia virus, for the in vivo delivery of gene therapy.29-31 The immunogenic nature of the adenoviral vectors has proven to be a major obstacle in the local delivery of genes using this vector system.32,33 The current study examines another candidate viral vector system and demonstrates that herpes amplicon vectors are a promising tool for such in vivo gene therapy. These vectors are derived from the herpes simplex virus type 1, a DNA virus efficient in infection of epithelial cells. The amplicon vectors have been rendered replication incompetent but remain capable of infecting a wide array of nonreplicating cells. We have previously demonstrated efficient β-galactosidase mark-

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Fig 5. Results of treatment by perfusion of virus. Sarcoma lung nodules 2 weeks after intravenous injection of 5 × 106 cells. Animals underwent left regional lung perfusion on day 5 with a 10-minute continuous perfusion of 5 × 106 viral particles of HSVlac (n = 6), HSVtnf (n = 8), 0.5 mL saline (n = 8), or no treatment (n = 8).

er gene transfer to hepatocytes in vitro and in vivo.4 The current studies extend these prior observations in demonstrating (1) efficient marker gene transfer in vivo to lung and bladder, (2) therapeutic gene transfer to these same organs, (3) concentrated local production of a secreted cytokine by such gene transfer, and (4) efficacy against tumor growth by local viral transduction. Because of the low efficiency of previous gene transfer methods, initial attempts at regional gene delivery involved transduction of cells in vitro and selection and concentration of transduced cells, which were then transfused or transplanted into an organ to study the effect of the inserted gene on these cells in vivo.34,35 Though capable of modulating the functions of cells in the circulation36 and ideal for production of tumor vaccines,37 the approach of infusion of ex vivo modified cells is cumbersome. HSV amplicon vectors were originally designed for use in treatment of neurologic conditions because of the natural neurotropism of these viruses. It was noted in previous in vitro studies, however, that HSV-mediated gene transfer can be accomplished into a wide variety of tumors38 and somatic cells4,39 and accomplished within 20 minutes to 1 hour of exposure. The rapidity of HSV infection and gene transfer suggested that gene transfer may be achievable during regional perfusions of organs of sufficiently short duration to minimize physiologic stress of vascular isolation to the organ. The current studies demonstrate that such regional perfusions can be performed. Gene transfer during these regional perfusions is effi-

cient. Furthermore, a lack of histochemical or biochemical evidence of gene transfer to other organs confirms that gene delivery during such regional perfusions can be specific. By using well-described surgical techniques of regional lung perfusion17 or regional liver perfusion,40 specific organ gene delivery may be feasible. Of note, we compared continuous perfusion of the left lung with isolated dwell, postulating that the rapid gene transfer can be accomplished in the first pass through the organ. Data demonstrate that continuous perfusions accomplished gene transfer equal to that of an isolated dwell. This implies that gene delivery using HSV amplicons may be possible without complete vascular isolation by using interventional radiologic techniques. Such specific delivery of genes to 1 isolated organ is particularly applicable in cancer gene therapy because many promising gene products under investigation, including interleukin-2 and TNF may be toxic if diffuse production is instigated.1,41 Isolated delivery of these genes to specific target organs can maximize production of these potentially toxic proteins in regions of interest while minimizing the effects elsewhere. Isolated delivery of genes coding for angiogenic proteins can also target ischemic organs for genetic revascularization without risking the oncogenic potential of such growth factors on other organs.42 Anti-angiogenic cancer therapy can also theoretically be delivered specifically to sites of interest to minimize adverse effects elsewhere. Several options have been reported in the literature involving regional or local administration of

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gene transfer in vivo. Instillation of a vector solution into the trachea has been reported to result in topical application to the epithelial lining of the lungs.43 Gold particle-DNA complexes have been introduced into tumor cells by using the “genegun.”22 This technique has been demonstrated to result in efficient gene transfer and expression within the “blast” zone, on the surface of the tumor, skin, or solid organ. Intraperitoneal or intrathoracic injection of vector solutions has been shown to transfect the mesothelial lining cells of those compartments.44 These regional techniques may coat an organ or body compartment, but may not deliver genes to the organ parenchyma. Direct injection of vector solution into an organ or tumor has also been used for localized delivery.45-48 The problem with this technique is that it may disrupt the tumor or organ anatomy and may result in uneven gene distribution. A number of authors have therefore studied the use of vascular gene delivery.49,50 Internal carotid artery cannulation has served as a potential portal for virus delivery to the brain.51 A study in sheep looked at the feasibility of gene delivery to coronary vessels with a percutaneous double balloon catheter.52 Finally, Nabel et al53 reported on the use of a pulmonary artery catheter for the delivery of vector to a lung in a human subject. In the current study, we demonstrated gene delivery by HSV amplicons by means of regional organ perfusion to produce high, transient in vivo expression of genes of interest. The possibility of delivering these therapies by vascular infusion makes tissue targeting applicable to all organs and broadens applicability of such vector systems. The transient nature of gene expression resulting from the episomal nature of HSV-mediated gene transfer can be a particular advantage when delivery of potentially toxic genes is contemplated. Equally important is the possibility of limiting the amount and, therefore, the cost of viral vectors needed by regional organ delivery, because financial considerations will be a determinant of feasibility of any novel therapy for clinical use. The current report also demonstrates efficacy of the HSV amplicon in decreasing tumor burden in vivo as delivered by means of local perfusion. These results are not surprising, however, because the HSV amplicon and its helper virus are cytopathic.54,55 In vitro studies in our laboratory and others have demonstrated lysis and apoptosis in cells exposed to the HSVlac (data not shown). More importantly, during a study of in vivo delivery of HSVlac to the rat brain, significant tumor necrosis and inflammation was noted in the HSVlac-infected areas.45 The packaging of HSV amplicons

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requires a helper virus that is replication competent. This recombinant helper virus is modified to remove most nonessential genes; however, it still retains some of its lytic capacity.54 The rat MCA sarcoma, however, is extremely resistant to TNF-α therapy. A previous study performed in our laboratory evaluated the use of TNF-α protein in the same regional lung perfusion model.56 In that study, 420 µg of murine TNF-α was needed to decrease tumor burden while half that dose showed no effect. So it is not surprising, given the high efficacy of HSV vectors, that local delivery of the TNF-α gene to the lung in this model failed to increase the antitumor effect of the amplicon alone. Whether TNF local production independent of direct lytic activities of the virus will be efficacious awaits studies using HSVtnf produced by new viral production methods not requiring helper virus.45,55,57 Liver and the lung are 2 important sites of disease in cancer patients. These 2 sites represent important sites for primary cancers. Owing mostly to the prevalence of chronic hepatitis, a precursor to neoplastic transformation of the liver cell, the liver represents the most common site of cancer for solid organs worldwide.58 In the United States, lung cancer still represents the number one killer of all cancers.59 In addition, these are not only the 2 most common sites of metastatic disease from solid organ primaries, but they also are common sites of isolated metastatic disease.60 As sites of isolated metastatic disease, cancer in these organs effects up to 100,000 patients in the United States yearly.60 Therefore, any effective novel treatment modality directed at cancer of these organs can potentially alter the treatment and outcome of a large patient population. The current studies demonstrate that HSV-mediated gene transfer can be accomplished in vivo rapidly and efficiently. Using a regional perfusion, foreign genes can be delivered into either the liver, lung, or bladder and have expression of such foreign genes isolated to the target organ and with minimal toxicity. This delivery system therefore holds promise for the delivery of genes coding potentially toxic compounds and encourages study of this method of delivery for genes of antineoplastic potential.

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