Effects of keratinocyte and hepatocyte growth factor in vivo: implications for retrovirus-mediated gene transfer to liver

We have previously shown that intravenous administration of keratinocyte growth factor (KGF) induces hepatocyte proliferation, allowing for efficient and noninvasive in vivo gene transfer with high-titer retroviral vectors in mice. The distinctive periportal distribution of transduced cells led us to investigate the ability of virus-sized particles to perfuse the liver adequately after growth factor treatment. We found that perfusion was adequate, and that transduction was limited to the periportal region because only those cells were stimulated to divide. Cells in this region also showed increased expression of Ram-1, the receptor for the murine Moloney leukemia virus (MoMLV) amphotropic envelope, after KGF treatment. In further studies we found that recombinant hepatocyte growth factor (HGF) induces a different population of hepatocytes to divide and upregulate Ram-1. The differential pattern of induction suggested that combining KGF and HGF would improve gene transfer efficiency further. Indeed, simultaneous delivery of both growth factors leads to an overall increase in the number of proliferating cells. Importantly, when coupled with MoMLV delivery, efficiency of gene transfer increased. These results confirm the utility of growth factors for noninvasive hepatic gene transfer in mice, and demonstrate how experiments to define the mechanism of transduction can be taken advantage of to develop improved gene transfer protocols.     

Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.     

In vivo cell lineage analysis during chemical hepatocarcinogenesis using retroviral-mediated gene transfer

We have studied the proliferation of cells in two models of chemical hepatocarcinogenesis. The cells were genetically labeled in vivo using retrovirally mediated transfer of the Escherichia coli beta-galactosidase marker gene coupled to a nuclear localization signal (nls-lacZ gene). In the first carcinogenic model, rats were fed a choline-deficient diet containing 2-acetylaminofluorene, and their livers were perfused with recombinant retrovirus at the onset of oval cell proliferation. The second model was based on the administration of diethylnitrosamine coupled with a partial hepatectomy and is thought to induce cancer with no involvement of oval cells. Analysis of beta-galactosidase expression in the liver at various times after gene transfer revealed the presence of large clusters of positive cells in both models. Moreover, the beta-galactosidase-positive cells displayed morphologic, antigenic, and enzymatic profiles consistent with a hepatocyte phenotype. Our results, therefore, provide evidence for a strikingly similar clonal proliferation of apparently normal hepatocytes during the course of 2-acetylaminofluorene- as well as diethylnitrosamine-induced liver carcinogenesis.     

Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain

The use of in vivo electroporation in combination with a chemotherapeutic agent (electrochemotherapy) for the treatment of liver tumors was examined. Induced rat hepatomas were treated with a 0.5 unit intratumor bleomycin dose followed by rectangular direct current pulses. Six pulses were administered during treatment using a needle array electrode that rotated the applied electric field around the tumors. A 84.6% objective response rate resulted for tumors that received both bleomycin and electric pulses. Control groups that received partial or no treatment showed less than 10% objective response rates. These results indicate that electrochemotherapy can be translated from the treatment of cutaneous cancers to the treatment of liver tumors and other internal tumors.     

Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, and transgene expression from adenovirus vectors can provide in vivo delivery of proteins. On the basis of this knowledge, we hypothesized that local administration of a replication-deficient adenovirus vector expressing complementary DNA for VEGF (AdVEGF) would induce collateral vessel formation in the setting of ischemia that could protect against subsequent acute vascular occlusion. METHODS: Hindlimb ischemia was induced in Sprague-Dawley rats by means of unilateral ligation of the common iliac artery immediately followed by administration of 4 x 10(9)-plaque-forming units VEGF, the control vector AdNull, or phosphate-buffered saline solution into the iliofemoral adipose tissue and thigh muscles. Untreated rats with common iliac ligation were used as an additional control group. RESULTS: Local VEGF expression was observed for 5 days in AdVEGF-treated rats but not in controls. Three weeks after ligation and vector administration, the ipsilateral femoral artery was ligated for a model of an acute vascular occlusion in the setting of preexisting ischemia. Blood flow to the ischemic hindlimb relative to the contralateral hindlimb evaluated with color microspheres demonstrated significantly increased blood flow in the AdVEGF-treated rats compared with each control group (p < 0.0001). Relative blood flow assessed by means of 99mTc-sestamibi radionuclide scans also demonstrated increased blood flow to the ligated hindlimb of AdVEGF-treated rats compared with each control group (p < 0.002). AdVEGF-treated rats also demonstrated increased vascularity in the ligated limb compared with each control group as assessed by means of angiography (p < 0.0001) and histologic quantification of blood vessels less than 80 microm diameter in local adipose tissue and capillaries per muscle fiber (p < 0.0002). AdVEGF treatment prevented a rise in femoral venous lactate femoral venous concentrations 1 hour after femoral artery ligation in control rats (p < 0.04). CONCLUSIONS: An adenovirus vector expressing VEGF complementary DNA is capable of stimulating an angiogenic response that protects against acute vascular occlusion in the setting of preexisting ischemia, suggesting that in vivo gene transfer of VEGF complementary DNA might be useful in prophylaxis of advancing arterial occlusive disease.     

Sensitization to morphine induced by viral-mediated gene transfer

Repeated administration of morphine sensitizes animals to the stimulant and rewarding properties of the drug. It also selectively increases expression of GluR1 (an AMPA glutamate receptor subunit) in the ventral tegmental area, a midbrain region implicated in morphine action. By viral-mediated gene transfer, a causal relation is shown between these behavioral and biochemical adaptations: Morphine's stimulant and rewarding properties are intensified after microinjections of a viral vector expressing GluR1 into the ventral tegmental area. These results confirm the importance of AMPA receptors in morphine action and demonstrate specific locomotor and motivational adaptations resulting from altered expression of a single localized gene product.     

Eradication of rat malignant gliomas by retroviral-mediated, in vivo delivery of the interleukin 4 gene

Overexpression of interleukin 4 (IL-4) can impair the tumorigenicity of glioma cells, but direct evidence of its antitumor efficacy after in vivo gene transfer into malignant gliomas has not been provided. To test this, we first injected into the brain of Sprague Dawley rats a 1:1 mixture of C6 rat glioblastoma cells and psi2.L4SN20 or E86.L4SN50 retroviral producer cells (RPCs), secreting 20 and 50 ng of IL-4/5 x 10(5) cells/48 h, respectively. Twenty-seven and 56% of rats receiving injections with these low- or medium-level IL-4 RPCs, respectively, survived tumor injection, whereas control rats died in about 1 month. E86.L4SN50 RPCs coinjected with 9L gliosarcoma cells into syngeneic Fischer 344 rats yielded similar results. A novel IL-4 RPC clone expressing higher levels of IL-4, E86.L4SN200, coinjected with 9L cells increased to 75% the fraction of long-term survivors and induced tumor regression in 50% of rats when injected into established 9L gliosarcomas. Cured rats developed an immunological memory because they rejected a challenge of wild-type 9L cells into the contralateral hemisphere. Magnetic resonance imaging was used to monitor 9L and C6 gliomas and gave direct evidence for tumor rejection in treated rats. Immunohistology showed inflammatory infiltrates in IL-4-treated tumors in which CD8+ T lymphocytes were more abundant, although CD4+ T lymphocytes, B lymphocytes, and macrophages were also present. Overall, these findings suggest that IL-4 gene transfer is a new, promising approach for treating malignant gliomas.     

Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.     

Gene therapy by in vivo interferon-gamma gene transfer to murine bladder tumor

For the clinical application of the cytokine gene therapy, the antitumor effects of systemic administration of Interferon-gamma (IFN-gamma) and those of in vivo direct IFN-gamma gene transfer to the tumors of mouse bladder carcinoma (MBT2) were compared. After the subcutaneous inoculation of MBT2 cells into mice, 10(2), 10(3) or 10(4) units of IFN-gamma were injected intraperitoneally (i.p.) or subcutaneously (s.c.). Neither i.p. nor s.c. injection of IFN-gamma resulted in tumor suppression or prolonged the survival time of tumor-bearing mice. The effect of in vivo direct IFN-gamma gene transfer by a retrovirus vector to MBT2 tumors was also evaluated. After the subcutaneous inoculation of MBT2 cells into mice, a virus culture supernatant containing IFN-gamma gene was injected into the same tumor site once a day for 3 days. In 50% of the mice in the treatment groups with IFN-gamma gene induction, no tumor formation was observed. Tumor-free survival and actuarial survival in the treatment groups were significantly longer than those in the control group. These results showed the possibility of in vivo direct IFN-gamma gene transfer into tumors and were encouraging for the execution of tumor cell-targeted IFN-gamma gene therapy against human bladder cancer.     

Expression of keratinocyte growth factor mRNA in ex vivo and in vitro specimens of human cornea and conjunctiva

Treatment of HIV and related malignancies with pharmacologic and biologic agents has not appreciably modified the course of disease. Immunologic impairment remains the critical factor in response. We report the long-term results of a single session of low-flow (0.3 L/min) extracorporeal perfusion hyperthermia on 29 men and 2 women with disseminated Kaposi's sarcoma and profound immunologic impairment. Any antiretroviral drug employed by the patient was stopped 72 hours prior to treatment and withheld during the period of follow-up. Core temperature was raised to 42 degrees C and held for 1 hour with extracorporeal perfusion and ex vivo blood heating to 49 degrees C as the means of temperature control. Of 31 patients, 2 died of complications secondary to treatment (cardiac arrhythmia; CNS bleed). There were two cases of intravascular coagulopathy. Pressure point skin damage may occur despite adequate cushioning. At 30 days posttreatment complete or partial regressions were seen in 20/29 of those treated, with regressions persisting in 14/29 of those treated by 120 days posttreatment. At 360 days, 4/29 maintain tumor regressions with 1 in complete remission (at 26 months). The patient in complete remission remains culture-negative and PCR-negative for HIV. CD4+ counts rose from around 250 to, and remain around, 800 in this man. Selected healed lesions were biopsied to demonstrate tumor absence. Patients were selected for treatment if pretreatment testing of the tumor showed regression in vitro with heat exposure. Analysis of the early and midterm failures showed little sustained rise of the CD4+ cells if presenting total CD4+ counts were below 50 and had been at such low levels for extended periods, although other surrogate markers of HIV activity declined (semiquantitative PCR) during this period and is felt to support the hypothesis of apoptosis proposed in this illness. Analysis of the tumors of the few men not responding demonstrated elevated levels of IL-6 as compared to responders (12 vs < 1 pg/ml). At 120 days 29/31 patients remained alive (expected, 20). At 360 days, 21/31 remained alive (expected, 11). In no patient was HIV activity stimulated with heat exposure. CMV retinitis did clear in some patients treated (both techniques), but treatment alone did not prevent later development of retinopathy. EBV parameters were markedly altered in the short term with heat exposure in some patients. Few patients showed herpes simplex activation. Varicella-zoster virus remitted in some patients. There is utility in the use of systemic hyperthermia to control HIV and related malignancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo

Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.     

Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament

To define the early biological effect of in vivo introduction of the PDGF-B gene on the healing of ligaments, a HVJ-liposome suspension containing platelet-derived growth factor (PDGF)-B cDNA was injected directly into the injured patellar ligament of 14-week-old male Wistar rats. Rats were killed at 1, 4 and 8 weeks for the morphological analysis of angiogenesis by laminin immunohistochemistry and of collagen deposition by Masson's Trichrome staining and collagen I immunohistochemistry. PDGF-B gene transfer caused the enhanced expression of PDGF in healing ligament up to 4 weeks after transfection, leading to an initial promotion of angiogenesis and subsequent enhanced collagen deposition in the wound. Enhanced and accelerated matrix synthesis in the PDGF-B gene introduced healing ligament suggests that this gene transfer technique may be a potentially useful tool for improving soft tissue repair.     

Direct gene transfer to mouse melanoma by intratumor injection of free DNA

The evaluation of commercially available test systems of competitive enzyme-linked immunosorbent assay (ELISA), for Aflatoxin M1 (AFM1) was performed in experimental conditions, through repeated analysis, in samples of milk powder contaminated with known concentrations of the toxin. Recoveries of AFM1 added to milk at levels of 0.10, 0.20, 0.50 and 1.00 ng/ml were 83.0%, 87.5%, 103.0% and 111.8% respectively. Relative standard deviations for the above mentioned concentrations were 65.5%, 31.8%, 10.9% and 13.6%, respectively (n = 10, per spiking level). According to these results the ELISA is appropriate for AFM1 research and/or surveying, mainly for concentrations between 0.20-1.00 ng/ml.