Injection Of Recombinant Adenovirus Research Articles

FBXW7 suppresses HMGB1-mediated innate immune signaling to attenuate hepatic inflammation and insulin resistance in a mouse model of nonalcoholic fatty liver disease

BackgroundInnate immune dysfunction contributes to the development and progression of nonalcoholic fatty liver disease (NAFLD), however, its pathogenesis is still incompletely understood. Identifying the key innate immune component responsible for the pathogenesis of NAFLD and clarifying the underlying mechanisms may provide therapeutic targets for NAFLD. Recently, F-box- and WD repeat domain-containing 7 (FBXW7) exhibits a regulatory role in hepatic glucose and lipid metabolism. This study aims to investigate whether FBXW7 controls high-mobility group box 1 protein (HMGB1)-mediated innate immune signaling to improve NAFLD and the mechanism underlying this action.MethodsMice were fed a high-fat diet (HFD) for 12 or 20 weeks to establish NAFLD model. Hepatic overexpression or knockdown of FBXW7 was induced by tail-vein injection of recombinant adenovirus. Some Ad-FBXW7-injected mice fed a HFD were injected intraperitoneally with recombinant mouse HMGB1 to confirm the protective role of FBXW7 in NAFLD via inhibition of HMGB1.ResultsFBXW7 improves NAFLD and related metabolic parameters without remarkable influence of body weight and food intake. Moreover, FBXW7 markedly ameliorated hepatic inflammation and insulin resistance in the HFD-fed mice. Furthermore, FBXW7 dramatically attenuated the expression and release of HMGB1 in the livers of HFD-fed mice, which is associated with inhibition of protein kinase R (PKR) signaling. Thereby, FBXW7 restrains Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) signaling in HFD-fed mouse livers. In addition, exogenous HMGB1 treatment abolished FBXW7-mediated inhibition of hepatic inflammation and insulin resistance in HFD-fed mouse livers.ConclusionsOur results demonstrate a protective role of FBXW7 in NAFLD by abating HMGB1-mediated innate immune signaling to suppress inflammation and consequent insulin resistance, suggesting that FBXW7 is a potential target for therapeutic intervention in NAFLD development.

Abstract 5089: Antitumor activities elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1

Abstract Interleukin (IL)-28A/IFN-λ2 and IL-29/IFN-λ1 have been demonstrated to elicit direct and indirect antitumor actions. In this study, we constructed an adenovirus vector expressing either IL-28A/IFN-λ2 (AdIL-28A) or IL-29/IFN-λ1 (AdIL-29) to evaluate the therapeutic properties of intratumoral injection of recombinant adenovirus to apply for the clinical implementation of cancer gene therapy. Despite the lack of antiproliferative effect on MCA205 and B16-F10 cells, a retarded growth of established subcutaneous tumors was observed following multiple injections of either AdIL-28A or AdIL-29 when compared with AdNull. In vivo cell depletion experiments displayed that both NK cells and CD8+ T cells play a major role in AdIL-28A-mediated tumor growth suppression. A significant increase in the number of infiltrating CD8+ T cells into the tumors treated with either AdIL-28A or AdIL-29 was observed. Moreover, specific antitumor cytotoxic T lymphocyte reactivity was detected in spleen cells from animals treated with either AdIL-28A or AdIL-29. In IFN-γ deficient mice, the antitumor activity of AdIL-28A was completely impaired, indicating that IFN-γ is critically involved in the tumor growth inhibition triggered by AdIL-28A. IL-12 provided an additive antitumor effect when combined with AdIL-28A. These results indicate that AdIL-28A and AdIL-29 could be successfully utilized as an alternative cancer gene therapy. Note: This abstract was not presented at the meeting. Citation Format: Muneo Numasaki, Hiroki Tsukamoto, Yoshihisa Tomioka. Antitumor activities elicited by direct intratumoral administration of a recombinant adenovirus expressing either IL-28A/IFN-λ2 or IL-29/IFN-λ1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2017-5089

Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1±8.1mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

A20 Rescues Failed Liver Regeneration in db/db Mice: Renewed Hope for Successful Use of Steatotic Liver Grafts.

Increased incidence of obesity and consequently of extensive liver steatosis is a major contributor to scarcity of adequate liver grafts for transplantation. Livers with extensive macrosteatosis are susceptible to ischemia reperfusion injury and fail to properly regenerate. The aim of this study was to determine the impact of A20, an anti-inflammatory, anti-apoptotic and pro-regenerative protein in hepatocytes, and a regulator of lipid metabolism on outcome of partial hepatectomy (PH) in leptin receptor deficient db/db mice. These obese, hyperinsulinemic mice develop macrovesicular liver steatosis, causing defective liver regeneration and heightened death following PH. Methods. We overexpressed A20 in livers of 8-10 weeks old db/db and lean mice by i/v injection of recombinant adenovirus (rAd). Saline and rAd.β-galactosidase (rAd.β-gal) treated mice were used as controls. Five days after rAd. injection, we performed 2/3 PH. Surviving mice were sacrificed 48 hours later. Resected (0h) and remnant livers (48h) were recovered for histology, immunohistochemistry and RNA extraction. Morphometry was analyzed by hematoxylin and eosin staining and proliferation by immunostaining for the cell proliferation marker, Ki67. All animal experiments met the IACUC criteria for humane care. Results: Overexpression of A20 significantly reduced early lethality in db/db mice following PH. No lethality was observed in A20 treated db/db within 48 hours of PH, as compared to 33% incidence of death in saline and rAd.β-gal control groups. The survival advantage of A20-treated db/db correlated with high hepatocyte proliferation 48h post-PH, as evidenced by Ki67 immunostaining. This was in striking contrast with almost no proliferation noted at this time in control livers, predicting even more lethality in upcoming days. All lean mice, regardless of treatment survived PH and showed a significant increase in liver Ki67 immunostaining 48h after PH. Conclusion: This is the first demonstration that overexpression of A20 restores adequate liver regeneration and alleviates lethality of this procedure in mice with severe hepatic steatosis. Additional work is currently performed to uncover the molecular basis of this advantage. Regardless of mechanisms, these data support the hope of A20-based therapies to improve success of marginal liver grafts due to steatosis.

Multicenter, open-end, randomized controlled study using P53 gene recombinant adenovirus injection (rAd-p53) combined with chemotherapy for orofacial carcinoma (OFC): Phase IV clinical trial—Progress report and conclusion.

6080 Background: The gene therapy product (Gendicine, rAd-p53) had pre-market studies done around 1998 indicated its efficacy in the treatment of squamous cell nasopharyngeal carcinoma in multi-modality treatment regimen. Gendicine has been used successfully in treating over 40 cancer types since then. With the advancement in optimization of combined treatment methods over the years, there was a need for a medium term efficacy study. This report summarizes our phase IV study. Methods: Patient selection, intervention and allocation were done according to GCP and supported by statistical analysis. Orofacial cancer (OFC) patients of advanced stages (III/IV) from multiple hospitals (n=30) in China were recruited for this open-end, randomized, controlled study (2009.7--2012.06). The 2 groups were rAd-p53+chemotherapy (GT+CT) (n=743) and CT alone (n=232). Patient exclusion rate were 20.81% (n=128) and 13.9% (n=28) respectively. Virus particles were delivered intra-tumorally at a dose of 109 (tumor cell/cm2 tumor area) x area x100 (virus multiplicity of infection, MOI), every 3d totally 10 times on d 1-28 inclusive. In synchrony with GT, 5-fluorouracil (5-FU, 250 mg/m2/d), on d 7-11 and d 25-29 along with carbocisplatin (KP, 400 mg/m2/d, i.v.), and with methotrexate (MTX, 50mg/m2 /d) on d 7, 14, 25, 32 were administered. The control group received 5-FU, KP and MTX. Results: For advanced OFC treated with GT+CT, the efficacy and QoL were significantly (p<=0.001) improved, the result was in accordance with our phase II/ III studies. Conclusions: This study proved rAd-p53 when used as an adjunct is safe and efficacious. The side-effect was mostly self-limiting low-grade to mild fever. The combined treatment modality had complete remission (CR) improved by 21.38%, relief ratio (RR) by 11.53% and clinical benefit ratio by 20.82% over GT-CT. Long-term follow-up will be continued.

Injection of Recombinant Adenovirus for Delivery of Genetically Encoded Calcium Indicators into Astrocytes of the Cerebellar Cortex: Figure 1.

The cerebellar cortex contains two astrocyte types: the Bergmann glia of the molecular layer and the velate protoplasmic astrocytes of the granule cell layer. In vivo, these cell types generate both subcellular calcium transients and trans-glial calcium waves. This protocol outlines a method for in vivo calcium imaging in cerebellar astrocytes, using the injection of a replication-incompetent recombinant adenovirus for gene transfer of the fluorescent calcium indicator protein (FCIP) G-CaMP2. The adenovirus contains a cytomegalovirus (CMV) immediate-early (IE) promoter which confines expression of G-CaMP2 to astrocytes. Expression is sufficiently high to allow calcium signals to be recorded in Bergmann glial processes as well as the processes and somata of velate protoplasmic astrocytes. To obtain structural information, G-CaMP2 fused with the brighter chromophore DsRed allows three-dimensional (3D) reconstruction of cells. G-CaMP2 expression lasts for at least 3 wk, enabling long-term functional imaging in both anesthetized and awake animals.

Intratumor injection of oncolytic adenovirus expressing HSP70 prolonged survival in melanoma B16 bearing mice by enhanced immune response

Heat shock proteins (HSPs) possess potent antitumor ability to stimulate immune response. We postulated that intratumor injection of oncolytic adenovirus over-expressing HSPs might be able to exert antitumor activity by inducing antitumor immune response in immunocompetent hosts in addition to the oncolytic activity. In this study, two recombinant oncolytic adenoviruses, Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a, were constructed with or without the HSP expression cassette. The HSP expression and cytopathic killing effect in mouse B16 melanoma and human PLC/PRF/5 hepatoma cells were measured after in vitro infection of adenoviruses. Survival rate of immunocompetent C57/BL mice was observed following intratumor injection of recombinant adenoviruses in B16 melanoma xenograft models. To detect the evidence of immune responses, hematoxylin and eosin staining and RT-PCR for IFN-γ expression in tumor tissues were performed. The Ad.CMV.HSP.IRES.E1a induced significantly higher HSP expression level than Ad.CMV.IRES.E1a in both B16 and PLC/PRF/5 cells. The cytocidal efficacy of Ad.CMV.HSP.IRES.E1a and Ad.CMV.IRES.E1a in PLC/PRF/5 cells was much higher than that in B16 cells, where the two adenoviruses showed similarly very weak oncolytic effect in vitro. However, Ad.CMV.HSP.IRES.E1a improved animal survival rate and exhibited more potent anti-tumor efficiency than Ad.CMV.IRES.E1a in B16 xenograft models. The enhanced efficacy might be mainly attributed to the HSP-mediated immune activity, as evidenced by the up-regulated expression of IFN-γ and local heavier intratumor inflammatory cell infiltration. These results indicated that the recombinant oncolytic adenovirus over-expressing HSPs possessed powerful in vivo anti-tumor efficacy and might be a valuable approach for cancer immune gene therapy.

Interleukin-4 gene transfer into rat pancreas by recombinant adenovirus

Objective. Adenovirus-mediated gene transfer technology may provide a novel approach in the treatment of pancreatic diseases. In the rat model of chronic pancreatitis induced by dibutyltin dichloride (DBTC), Th1 lymphocytes are known to be involved in the mediation of inflammation. We therefore investigated whether local expression of the Th2 cytokine interleukin (IL)-4 might modulate the inflammatory response. To address this question, we have established a protocol of efficient gene transfer into rat pancreas. Material and methods. Recombinant adenovirus constructs carrying the Escherichia coli β-galactosidase gene (Adβ-gal) or the rat IL-4 gene (AdrIL-4) were injected into the left gastric artery of healthy LEW.1W rats. Expression of β-Gal and IL-4 in pancreatic cells was analyzed by X-Gal staining and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. After optimization of the transduction protocol, effects of the IL-4 gene transfer on pancreatic inflammation and fibrosis were studied in DBTC-treated rats. Results. Seven days after Adβ-gal injection, β-gal-positive cells were detectable in the rat pancreas. RT-PCR analysis using RNA from pancreata of AdrIL-4-treated rats indicated that IL-4 was expressed for at least 14 days after adenovirus application. Expression of the IL-4 transgene was accompanied by a transient increase of the IL-10 mRNA level in the pancreas. In DBTC-treated rats, adenovirus-mediated transfer of the IL-4 gene modified the pattern of infiltrating inflammatory cells in the pancreas. Importantly, a decrease of CD4+ helper cells was observed. Conclusions. Our data suggest that the injection of recombinant adenoviruses into the left gastric artery is a promising approach to achieving expression of therapeutic transgenes in the pancreas.

Peripheral CD8+ T Cell Tolerance Against Melanocytic Self-Antigens in the Skin Is Regulated in Two Steps by CD4+ T Cells and Local Inflammation: Implications for the Pathophysiology of Vitiligo

Experimental evidence has suggested a role for CD8+ cytotoxic T lymphocytes (CTL) in the pathophysiology of vitiligo, a pigmentation disorder with focal loss of melanocytes in the skin. The discovery of tyrosinase-related protein 2 (TRP2) as a model melanocytic self-antigen recognized by CD8+ CTL in C57BL/6 mice allowed us to analyze the requirements for CD8+ T cell-mediated autoimmune destruction of melanocytes in an experimental model. Using two different genetic methods for the induction of cellular immunity in vivo, gene gun bombardment of the skin and injection of recombinant adenovirus, we show that peripheral tolerance of CD8+ T cells recognizing a single TRP2-derived H2-Kb-binding peptide is regulated in two steps. In the induction phase, stimulation and expansion of TRP2-specific CD8+ T cells in vivo depend on CD4+ T cell help. In the effector phase, autoimmune destruction of melanocytes in the skin depends on local inflammation. Our results suggest that accidental stimulation of CD8+ CTL recognizing major histocompatibility complex class I-binding peptides derived from melanocytic proteins in the context of an inflammatory skin disease may play an important role in the pathophysiology of vitiligo.

Altered intracellular sorting signals do not influence the efficacy of genetic melanoma vaccines incorporating helper determinants in mice

A genetic melanoma vaccine consisting of cDNA encoding the model self-antigen tyrosinase-related protein 2 (TRP2) fused in-frame to the immunogenic enhanced green fluorescent protein (EGFP) was able to break immune tolerance and stimulate CD8+ T cells in vivo. In the present study we investigated whether alteration of the intracellular antigen localization as a result of the linkage with immune-enhancing helper proteins affects the resulting immune response. Expression plasmids and recombinant adenoviruses were constructed encoding various fusion proteins with different intracellular sorting signals which direct the antigen to the cytosol, the endoplasmic reticulum or the endosomal compartments. Genetic immunization of C57BL/6 mice was performed with all constructs using particle-bombardment of the skin and injection of recombinant adenoviruses. The resulting immune response was analyzed using ELISPOT and tumor rejection assays. Induction of TRP2-specific CD8+ T cells in vivo and autoimmune-mediated destruction of melanocytes in the bombarded area of the skin were observed with all constructs expressing fusion proteins between TRP2 and EGFP. Importantly, injections of the different recombinant adenoviruses all mediated protective immunity against transplanted B16 melanoma cells. Altered intracellular sorting signals do not significantly influence the efficacy of genetic melanoma vaccines incorporating helper determinants in our model system. These results further support the concept that linkage of immunogenic helper sequences can be successfully applied for antigen-specific immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical applications.

Experimental study of adenovirus vector mediated-hVEGF165 gene on prevention of restenosis after angioplasty.

This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

Cellular characterization of the tropism of recombinant adenovirus for the adrenal glands.

Recombinant adenoviruses are widely used in gene therapy clinical trials. A particular tropism for the adrenal glands has been reported but the precise cellular base for this tropism has not been determined. Recombinant adenoviruses were injected intravenously into Balb/c nu/nu or C57BL/6 mice. Seventy-two hours later, the animals were sacrificed and the adrenal glands and livers collected. The glands were sectioned and analyzed using immunohistochemical methods to detect adenoviral epitopes and transgene expression. Total RNA were extracted from the liver and adrenal glands of some animals and subjected to real-time RT-PCR. The only cell type infected in the adrenal glands of Balb/c nu/nu or C57BL/6 mice is the adrenocortical cells in the zona fasciculata. Quantitatively, the relative level of gene expression in the adrenal gland is comparable but lower than that measured in the liver. Systemic injection of recombinant adenovirus could be used as a procedure to restore adrenal steroidogenesis in clinical gene therapy protocols. In addition, our study suggest that adrenal dysfunction should be considered when criteria are established to assess the safety of gene therapy formulations administered systemically.

Callosal commissural neurons of Dab1 deficient mutant mouse, yotari.

The yotari mouse is an autosomal recessive mutant mouse, caused by mutation of disabled homolog 1 (Dab1) gene. The mutant mouse is recognized by unstable gait and tremor and by early deaths around at the time of weaning. The cytoarchitectures of cerebeller and cerebral cortices and hippocampal formation of the yotari mouse are abnormal. These malformations strikingly resemble those of reeler mouse. In the present study we examined the callosal commissural (CC) neurons of yotari, reeler and normal mice with the injection of recombinant adenovirus into the frontal area 1 (Fr1) to find some possible phenotypes specific for the yotari mouse. The distribution pattern of CC neurons of the yotari was similar to that of the reeler: retrogradely labeled CC neurons were seen throughout all depths of the contralateral Fr1. However, the present statistical analysis revealed that the difference of the mean intracortical position of the CC neurons between the yotari and the reeler is significantly different (Student's t-test), suggesting that the phenotype of the yotari is clearly different from that of the reeler.

Fetal gene therapy: Efficacy, toxicity, and immunologic effects of early gestation recombinant adenovirus

Background/Purpose: Advancements in gene transfer technology and prenatal diagnosis have allowed investigators to consider an in utero gene therapy approach for fatal genetic diseases. The authors sought to develop fetoscopic techniques for gene delivery and investigate the efficacy and safety of recombinant adenoviral vectors in the fetus. Methods: Fetal sheep between 60 and 130 days' gestation (dGA) underwent either fetoscopic intratracheal injection or umbilical vein (UV) injection of recombinant adenovirus, AdCMV-IacZ. At death, fetal organs were examined for β-galactosidase expression, histopathology, and CD45 immunostaining. Fetal serum was compared with preimmune serum for transaminase levels and the presence of antiadenoviral neutralizing antibodies. Results: Fetoscopic intratracheal delivery of AdCMVIacZ in late-gestation sheep fetuses resulted in efficient alveolar gene transfer, but, antiadenoviral immunologic reactions limited the longevity of transgene expression to 14 days. This prompted an examination of whether early gestational exposure could induce tolerance in the fetus to adenoviral and transgene antigens. AdCMVIacZ (1 × 10 11 particles) was injected via UV into fetuses at 60 dGA. Within 3 days, β-galactosidase expression was localized to the fetal liver, adrenal glands, kidneys, and endocardium. Although adrenal expression was nearly constant over 28 days, expression in fetal liver disappeared within 14 to 28 days. Loss of hepatic expression did not appear to be immune mediated because there was no evidence of hepatic inflammation or appearance of antiadenoviral neutralizing antibodies. Fetuses injected with AdCMVIacZ at 60 dGA were reinjected with 1 × 10 13 particles at 125 dGA and antiadenoviral humoral immune responses were recorded. Despite early-gestation adenovirus injection, fetuses still responded to the late-gestation adenoviral exposure, developing antiadenoviral neutralizing antibodies similar to control fetuses. Conclusions: The authors developed fetoscopic access for pulmonary adenovirus delivery in late-gestation sheep. Although initial alveolar transduction was highly efficient, antiadenoviral immune responses limited the duration of transgene expression. In contrast, early-gestation adenoviral delivery did not elicit antiadenoviral immune responses despite achieving efficient transduction of many fetal tissues. Furthermore, early-gestation adenovirus delivery did not affect late-gestation antiadenoviral immune responses. These findings suggest that the early-gestation sheep fetus is not amenable to adenoviral tolerance induction by UV injection and that it is incompetent of immunologic response to adenovirus. For the purposes of in utero gene therapy, recombinant adenovirus may be applied optimally to genetic diseases requiring transient in utero expression.

Preferential adenovirus-mediated transduction of cells at the sites of laser photocoagulation in the rat eye

PURPOSE. This study aimed to investigate the feasibility of recombinant adenovirus-mediated gene transfer into cells implicated in the development of choroidal neovascularization (CNV). METHODS. A rat model of CNV which used laser photocoagulation was developed. Gene delivery into the laser spots was investigated following subretinal injection of recombinant adenoviruses, AdRSVlacZ, AdCMVlacZ or AdCMVgfp. Immunohistochemical analysis was performed using a proliferating cell nuclear antigen antibody and a cytokeratin-specific antibody to identify the cell types transduced by the recombinant adenoviruses. RESULTS. At 7 days post-injection, lacZ expression was detected in 51.6 ± 13.2% and 71.2 ± 19.3% of laser spots in AdRSVlacZ- and AdCMVlacZ-injected eyes, respectively. By 28 days post-injection, lacZ expression was only present in AdCMVlacZ-injected eyes. In vivo fundus fluorescent photography of AdCMVgfp-injected eyes detected gfp expression in 79.9 ± 12.7% and 35.6% ± 19.7% of laser spots at 4 and 7 days post-injection, respectively. Although fundus fluorescent photography did not detect the gfp signal at 10 days post-injection, fluorescent microscopy revealed a gfp signal in 81.3 ± 6.0% of laser spots. Immunohistochemical analysis detected retinal pigment epithelial (RPE) cells as the most predominant proliferating cell type in the laser spots, although several other proliferating cell types were also identified. X-gal staining showed that the majority of transduced cells were those present in the laser spots. CONCLUSIONS. It is proposed that following laser photocoagulation, proliferating RPE cells are susceptible to adenovirus-mediated gene delivery and that they may be suitable targets for the delivery of antiangiogenic factors by recombinant adenoviruses in order to inhibit developing CNV membranes.

Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue in mice.

Gene transfer to cerebral blood vessels has been accomplished in rats and dogs by injection of replication-deficient adenovirus into cerebrospinal fluid. In this study we examined transgene expression after injection of adenovirus into the cerebrospinal fluid of mice. Responses were observed in ICR mice and C57BL/6 mice, which are outbred and inbred strains, respectively. We injected replication-deficient recombinant adenovirus expressing nuclear targeted beta-galactosidase, driven by either the Rous sarcoma virus promoter (AdRSV-betaGal) or the cytomegalovirus promoter (AdCMV-betaGal), into the cisterna magna of anesthetized ICR and C57BL/6 strains of mice. The brains were examined from 1 to 21 days after injection by chemiluminescent enzyme activity assay or histochemical staining. After injection of AdRSV-betaGal, expression of beta-galactosidase in ICR mice peaked on day 7 and returned to basal by day 14. Expression of beta-galactosidase in C57BL/6 mice was maximal on days 7 to 14 and was minimal by day 21 after injection of AdRSV-betaGal. After injection of AdCMV-betaGal in C57BL/6 mice, peak expression of transgene occurred on day 1 and was greatly diminished by day 3. Transgene expression was observed primarily on the ventral surface of the brain, with preferential expression in leptomeninges and adventitia along the major cerebral arteries of that region. Injection of recombinant adenovirus in the cisterna magna resulted in transgene expression in leptomeninges and perivascular tissue of cerebral blood vessels in two strains of mice. The CMV promoter elicited rapid but short-lived expression of the transgene, while the RSV promoter elicited slower, more sustained transgene expression. Expression of AdRSV transgene was prolonged in C57BL/6 mice compared with ICR mice. This approach for gene transfer may be useful to study cerebral vascular biology in genetically altered strains of mice.

Gene therapy for rat hepatocarcinoma (HCC) using intratumorous injection of recombinant adenoviruses carrying herpes simplex virus thymidine kinase gene (HSVTK)

Gene therapy for rat hepatocarcinoma (HCC) using intratumorous injection of recombinant adenoviruses carrying herpes simplex virus thymidine kinase gene (HSVTK)

Characterization of the immune response after local delivery of recombinant adenovirus in murine pancreas and successful strategies for readministration.

The pancreas is an ideal organ for adenoviral gene therapy because of the high level of gene transfer that can be achieved and because of the many diseases that can potentially be treated using this technology. In this report, we characterize the immune response to direct pancreatic injection of adenovirus and we overcome some of the limitations it imposes by using immunosuppression. Direct injection of recombinant adenovirus into the pancreas leads to the production of neutralizing antibodies and to sensitized splenocytes which engage in increased cytotoxic, lymphoproliferative, and cytokine release activity when reexposed to adenovirus. Transgene expression is transient and the vector cannot be readministered. Deletion of CD4+ T helper cells improves expression over time (40% of pancreatic cells express transgene at day 28 vs. 5% in controls), and allows the vector to be readministered in the pancreas, albeit, inefficiently, when compared to naive animals. Similarly, blockade of CD40 ligand, which preserves the CD4+ T helper cell population, also improves expression over time (30% of pancreatic cells express transgene at day 28), and allows the vector to be readministered. With both approaches, neutralizing antibodies are decreased and the remaining splenocytes do not engage in activated immune responses. Thus, local delivery of the adenoviral vector induces a systemic response that prevents pancreatic readministration, even with direct injection. Blockade of CD40 ligand and T helper cell depletion are transient regimens that induce systemic immunosuppression. Until the development of newer strategies that selectively suppress adenoviral immune responses, these are viable alternatives for enhancement of pancreatic adenoviral delivery.

Chapter 20 DNA- and Adenovirus-Mediated Gene Transfer into Cardiac Muscle

This chapter discusses and compares two methods of gene transfer into the rodent heart: direct plasmid DNA injection and injection of recombinant adenovirus. The predominant use of direct injection into the heart has been to analyze promoter elements in vivo using reporter genes driven by the promoter element of interest. The second general use of in vivo gene transfer is to examine the consequences of expression of a given gene on the structure or function of the heart. This could involve expression of a mutant protein or ectopic expression of an isoform of a protein normally found in another setting. For these purposes DNA injection would not be efficient enough for most forms of phenotypic modification. Therefore, focus has shifted on the use of recombinant adenoviruses. Adenoviruses efficiently infect a large number of replicating and nonreplicating cell types. Their ability to infect nonreplicating cells makes them ideal for use in the heart, because adult cardiac myocytes are terminally differentiated.

Invited Review. The potential for gene therapy in duchenne muscular dystrophy and other genetic muscle diseases

Dystrophin cDNAs have been introduced into skeletal muscle fibers of dystrophin-deficient mice (mdx) through direct DNA injection in plasmid expression vectors and by replication-defective recombinant adenovirus vectors. The introduced genes appear to protect those muscle fibers from necrosis in which they become expressed. By direct injection of dystrophin cDNA in plasmid expression vector, only 1-2% of adult mdx muscle fibers of the injected muscle expressed dystrophin. On the other hand, by recombinant adenovirus injection into very young mdx muscle, a better efficiency has been reported. We have discussed several putative and proven factors that may contribute to the thus far demonstrated relatively low efficiency of dystrophin gene transfer. These include poor uptake of gene constructs by muscle fibers, degradation of the injected DNA, and poor access of gene constructs to the nuclear compartment. Neutralization or elimination of these factors could improve the efficiency of gene transfer so that it might, in the future, qualify as an effective therapy for DMD and some other genetic diseases of muscle.