Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg‐Gly‐Asp (RGD)‐modified Ad, adeno‐associated viral serotype 2 (AAV2), and self‐complementary AAV2 (scAAV2) vectors within full‐thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real‐time RT‐PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p ≤ 0.026). Ad and Ad‐RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full‐thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:149–155, 2010     

AAV based gene delivery to myocardium in rodents and pigs

Objective: To optimize transgene expression levels after AAV‐mediated gene transfer different delivery methods were compared in a rat (A) and porcine (B) heterotopic heart transplantation model. Methods: (A) Heterotopic abdominal heart transplantations were performed in male Lewis rats. After harvesting the donor hearts, the viral vectors were delivered to the graft by the following methods: (1) 0.35 ml saline solution containing AAV2/9‐LacZ (2 × 10¹¹ vector genome, vg) was injected directly into the myocardium (apex) immediately after reperfusion. (2) cardioplegic solution (0.3 ml) containing AAV‐2(HBSD), 2/9‐LacZ vectors was rapidly injected into the aortic root with the pulmonary trunk clamped. Before transplantation the transfected heart was incubated for 20 min in iced cardioplegia. (3) A reperfusion system was applied: For 20 min a cold solution of cardioplegia (5 ml) and AAV‐2(HBSD) or AAV2/9‐LacZ vectors were recirculated through the donor heart. Transplanted grafts were explanted after 3 weeks. To detect and measure marker gene expression X‐gal staining or a luciferase assay was performed. In a second series we compared the effects of the transduction of PD‐L1 to LacZ using the optimum method (intraaortic root injection) in the same heart transplantation model. (B) Heterotopic abdominal HTX was performed in pigs (Landrace, 10–18 kg) following vector application to the donor heart in an in situ‐Langendorff perfusion system (AAV2/GFP and AAV2/Luciferase). Recipients were given tacrolimus (0.3 mg/kg BW), after 21 days the transplanted hearts were explanted for transgene expression analysis. In a second series we compared AAV2/9‐mediated transduction of PD‐L1 and LacZ in the in situ‐Langendorff model and consequent allogeneic heterotopic abdominal heart transplantation. Results: (A) Highest transfection efficiency was observed in the grafts treated with intracoronary infusion of AAV2/9 at the higher dosage, and the expression pattern was global and homogenous in the grafts. hPD‐L1 transduction resulted in no significant difference of survival time and signs of rejection after allogeneic rat heart transplantation. (B) AAV2‐mediated gene delivery was unable to yield sufficient transgene expression after in situ‐Langendorff perfusion of porcine hearts. AAV2/9 based gene transfer of LacZ and hPD‐L1 led to excellent myocardial gene expression lasting up to 2 months. Due to species incompatibility no protective effects were observed in our allogeneic porcine transplantation model. Conclusions: (A) We demonstrated that infusion of AAV vectors into the aortic root with the pulmonary trunk clamped is a simple and efficient method for gene delivery to the donor heart in an allogeneic rat heart transplantation setting. Gene transfer of hPD‐L1 was ineffective to protect against allorejection, on the contrary there was a trend to aggravated rejection. Therefore the exact mechanism of hPD‐L1 in allograft rejection needs further investigation. (B) The in situ‐Langendorff model was developed to allow isolated target organ perfusion with high vector concentrations under physiological conditions. Using this method AAV2/9 mediated gene delivery into porcine hearts proved effective to induce excellent marker gene expression. Expression of hPD‐L1 could also be achieved but was ineffective in pig allotransplantation due to species incompatibility. Double transgenic pig hearts (e.g. Gal‐KO+CD46) can now efficiently be transduced with hPD‐L1 or hCTLA4‐Ig to further optimize long‐term survival after pig‐to‐primate cardiac xenotransplantation.     

Ex vivo intracoronary gene transfer of adeno‐associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants

Heart transplant gene therapy requires vectors with long‐lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno‐associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar–Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR‐, RT‐PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.     

Tissue reactions of adenoviral, adeno-associated viral, and liposome-plasmid vectors in tendons and comparison with early-stage healing responses of injured flexor tendons

PURPOSE: Delivery of growth factor genes that may substantially increase the healing rate of injured digital flexor tendons is a new application of gene therapy. Adenoviral, adeno-associated viral (AAV), and liposome-plasmid vectors have been used to deliver genes to tendons, but the tendon reactions to these vectors--particularly in contrast to the healing responses in the injured tendons--were unknown. This study was designed to compare the tissue reactions of the earlier-mentioned vectors in tendons with the healing responses of injured flexor tendons. METHODS: Forty-two flexor digitorum profundus tendons of 6 New Zealand white rabbits were used. Eighteen tendons were divided into 3 groups of 6 each and injected with different vectors: adenoviral vector, AAV2-luciferase vector, or pCMV-beta vector with liposome. Another 12 tendons were cut and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal flexor tendons were harvested as controls. RESULTS: The tissue reactions of the liposome-plasmid vector in tendons were the most prominent among the 3 vectors tested. The adenoviral vector elicited a moderate degree of tissue reaction. The AAV2 vector caused remarkable reactions in epitenon but almost no reactions in endotenon. Early-stage tissue reactions were more robust in the injured tendons. Compared with early-stage inflammatory and healing responses, the reactions elicited by these vectors were less severe. CONCLUSIONS: The 3 gene delivery systems tested elicit less severe tissue reactions in flexor tendons compared with early-stage inflammatory changes in injured tendons. Adenoviral and AAV vectors elicit less severe tissue reactions than liposome-plasmid vectors. The AAV2 vector appears to cause almost no reaction in endotenon. In terms of tissue reactions, the adenoviral and AAV2 vectors, in particular AAV2, are suitable gene delivery systems for future gene transfer to the tendon in vivo.     

腺相关病毒载体转导基因至愈合肌腱及载体组织反应的研究

目的 探讨腺相关病毒(AAV)载体转导碱性成纤维细胞牛长因子(bFGF)基因对肌腱愈合的影响,并观察腺病毒、AAV以及脂质体一质粒三种基因治疗载体应用于肌腱所产生的组织反应.方法 取13只成年白色来亨鸡的双侧巾趾趾深屈肌腱(26根),随机分为实验组和对照组,每组13根,实验组肌腱完全切断后注射AAV2-bFGF并以改良Kessler法修复,对照组不注射AAV2-bFGF,仪以改良Kessler法修复.第4周未行免疫组织化学染色,第8周术测定趾屈曲功.将6只成年新西兰白兔的趾深屈肌腱(36根)分成二组,每组12根,分别注射10μL的腺病毒、AAV2和脂质体.质粒载体,术后第3、7、14天分别取肌腱,进行石蜡切片、HE染色.结果 AAV2-bFGF可以在术后4周显著地提高肌腱bFGF的表达,而且不增加趾屈曲阻力(粘连形成).所测屈曲功第8周末实验组为(0.052±0.031)J,对照组为(0.049±0.035)J,两组问差异无统计学意义(t=0.31266,P=0.8984).脂质体-质粒载体组肌腱组织反廊重于腺病毒载体组,AAV2载体组肌腱组织反应最轻,在腱外膜处有组织反应,而腱内膜区域几乎无组织反应.结论 用AAV2载体转bFGF基因至肌腱能有效增加愈合肌腱的bFGF.在三种所研究的载体中,腺病毒和AAV2载体引起的组织反应比脂质体.质粒载体轻.AAV2引起的组织反应最轻.AAV2可能会成为肌腱的转基因良好载体.     

The efficacy and safety of gene transfer into the porcine liver in vivo by HVJ (Sendai virus) liposome

BACKGROUND: Gene transfer systems using viral vectors are efficient; however, most viral vectors also tend to evoke immunologic reactions, thereby clinically causing serial side effects. HVJ-liposome vector is a hybrid vector consisting of liposome and an inactivated Sendai virus (Hemmagglutinating Virus of Japan [HVJ]), which has been reported to be less immunogenic and can also be repeatedly administered. We examined the usefulness of this vector for hepatic gene therapy in a pig model. METHODS: Genes encoding beta-galactosidase and luciferase were used as reporter genes. The pigs were injected with the reporter gene loaded-HVJ-liposome into the portal vein under total vascular exclusion of the liver. The transfection efficiencies were then assessed by beta-galactosidase staining, a luciferase assay, and RT-PCR for LacZ mRNA. Biochemical and histologic analyses were performed to evaluate tissue toxicity after gene transfer. RESULTS: The luciferase gene expression in the liver reached its highest level at 7 days after transfection. It continued to be detected up to 28 days after transfection, while all pigs remained healthy throughout the observation period. The transfection efficiency was 15% in the hepatocytes according to beta-galactosidase staining. Extrahepatic transgene expression was slightly observed in the lung and kidney, but not in the spleen or ovary. CONCLUSIONS: These data suggest for the first time that the use of the HVJ-liposome vector is a safe and feasible modality for liver-directed gene transfer in pigs, and it might therefore be suitable for clinical gene therapy trials.     

Transduction efficiency of adenoviral vectors into human glioma cells increased by association with cationic liposomes

Replication-deficient adenoviral vectors are promising agents for human gene therapy of the greater transduction efficiency than other vectors. However, there are distinct disadvantages, including high immunogenicity, which limits the administration to human organs, particularly the brain. Injection of adenoviral vectors into the human brain causes inflammatory responses and induces cerebral edema. The combined effect of adenoviral vectors and cationic liposomes in vitro was investigated in an effort to reduce the immune reaction against the antigens of adenoviral vectors. No toxicity of adenoviral vector-associated liposomes was observed within optimal lipid concentration. The transduction efficiency of the adenoviral vectors containing the beta-galactosidase gene increased almost 10-fold when associated with the cationic liposomes. Furthermore, greater cytotoxicity was induced when the adenoviral vector containing herpes simplex virus-thymidine kinase gene was combined with cationic liposomes than with only the adenoviral vector. These results suggest that the combination of adenoviral vectors and cationic liposomes allows the doses of adenoviral vectors to be reduced while maintaining transduction efficiency.     

Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model

The objective of this study was to compare the efficacy of adenoviral and lentiviral regional gene therapy in a rat critical sized femoral defect model. The healing rates and quality of bone repair of femoral defects treated with syngeneic rat bone marrow cells (RBMCs) transduced with either lentiviral vector (Group I) or adenoviral vector (Group II) expressing bone morphogenetic protein-2 (BMP-2) gene were assessed. RBMCs transduced with the adenoviral vectors produced more than 3 times greater (p < 0.001) BMP-2 when compared to RBMCs transduced with lentiviral vectors in an in vitro evaluation. Serial bioluminescent imaging demonstrated short duration luciferase expression (less than 3 weeks) in defects treated with RBMCs co-transduced with two adenoviral vectors (Group IV; adenovirus expressing BMP-2 and luciferase [Ad-BMP-2 + Ad-Luc]). In contrast, the luciferase signal was present for 8 weeks in defects treated with RBMCs co-transduced with two lentiviral vectors (Group III; lentivirus expressing BMP-2 and luciferase gene [LV-BMP-2 + LV-Luc]). There were no significant differences with respect to the radiological healing rates (p = 0.12) in defects treated with lentiviral versus adenoviral mediated BMP-2 gene transfer. Biomechanical testing of healed Group I femoral specimens demonstrated significantly higher energy to failure (p < 0.05) when compared to Group II defects. Micro CT analysis revealed higher bone volume/tissue volume fraction (p = 0.04) in Group I defects when compared to Group II defects. In conclusion, prolonged BMP-2 expression associated with lentiviral mediated gene transfer demonstrated a trend towards superior quality of bone repair when compared to adenoviral mediated transfer of BMP-2. These results suggest that the bone repair associated with regional gene therapy is influenced not just by the amount of protein expression but also by duration of protein production. This observation needs validation in a more biologically challenging environment where differences in healing rates and quality of bone repair are more likely to be significantly different.     

Enhanced neuroblastoma transduction for an improved antitumor vaccine.

BACKGROUND: A recent clinical trial of an antineuroblastoma vaccine used adenovirus serotype 5 (Ad5) vectors to transduce autologous tumor cells with the gene encoding IL-2. A method to improve transduction efficiency was sought to enable the use of lower viral titers, especially when in situ adenoviral-mediated tumor cell transduction is considered. MATERIALS AND METHODS: A chimeric adenoviral delivery vector was utilized in which the fiber head from adenovirus serotype 3 was incorporated into the backbone of Ad5. Since the fiber head protein is responsible for viral attachment to target cells, a different spectrum and range of infectivity might result. Both the chimeric (Av9LacZ4) and Ad5 (Av1LacZ4) vectors were constructed to carry a beta-galactosidase transgene. The relative transduction efficiency of these two vectors was then evaluated in five tumor-derived short-term neuroblastoma cultures and four established neuroblastoma cell lines. Enzyme activity was assessed using three different methods: in situ staining, flow cytometric analysis, and a quantitative assay. RESULTS: A significant improvement in transduction efficiency of the short-term neuroblastoma cultures with the new chimeric adenovector was demonstrated. A similar improvement in transduction efficiency was not observed in the established cell lines, suggesting that the cell surface receptor for the Ad 3 serotype had been lost in vitro. Increased transduction of tumor cells with N-myc amplification was also observed. CONCLUSIONS: The newly constructed chimeric adenoviral vector transduces short-term neuroblastoma cultures more efficiently than the standard Ad5 vector. This vector will permit the use of lower viral titers and may be useful in other adenoviral-based gene-therapy protocols. Increased transgene expression in N-myc-amplified cells offers possible selectivity for in situ gene delivery.     

Pseudotyped adeno‐associated viral vector tropism and transduction efficiencies in murine wound healing

Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno‐associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency profile for specific cells in the cutaneous wounds. We compared transduction efficiencies of cells in the epidermis, cells in the dermis, and the fascicles of the panniculus carnosus by AAV2/2 and three pseudotyped AAV vectors, AAV2/5, AAV2/7, and AAV2/8 in a murine excisional wound model. AAV2/5 and AAV2/8 result in significantly enhanced transduction of cells both in the epidermis and the dermis compared to AAV2/2. AAV2/5 transduces both the basilar and supra‐basilar keratinocytes. In contrast, AAV2/8 transduces mainly supra‐basilar keratinocytes. Both AAV2/7 and AAV2/8 result in more efficient gene transfer to the muscular panniculus carnosus compared to AAV2/2. The capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the murine wound healing model. Both AAV2/5 and AAV2/8 administration result in significantly enhanced gene transfer. To further characterize cell specific transduction and tropism profiles of the AAV pseudotyped vectors, we performed in vitro experiments using human and mouse primary dermal fibroblasts. Our data demonstrate that pseudotyping strategy confers a differential transduction of dermal fibroblasts, with higher transduction of both human and murine cells by AAV2/5 and AAV2/8 at early and later time points. At later time points, AAV2/2 demonstrates increased transduction. Interestingly, AAV2/8 appears to be more efficacious in transducing human cells as compared to AAV2/5. The pseudotype‐specific pattern of transduction and tropism observed both in vivo and in vitro suggests that choice of AAV vectors should be based on the desired target cell and the timing of transgene expression in wound healing for gene transfer therapy in dermal wounds.     

Transduction of pancreatic islets with pseudotyped adeno-associated virus: effect of viral capsid and genome conversion

BACKGROUND: Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. METHODS: Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. RESULTS: Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. CONCLUSIONS: The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.     

Nerve growth factor gene therapy using adeno-associated viral vectors prevents cardiomyopathy in type 1 diabetic mice

Diabetes is a cause of cardiac dysfunction, reduced myocardial perfusion, and ultimately heart failure. Nerve growth factor (NGF) exerts protective effects on the cardiovascular system. This study investigated whether NGF gene transfer can prevent diabetic cardiomyopathy in mice. We worked with mice with streptozotocin-induced type 1 diabetes and with nondiabetic control mice. After having established that diabetes reduces cardiac NGF mRNA expression, we tested NGF gene therapies with adeno-associated viral vectors (AAVs) for the capacity to protect the diabetic mouse heart. To this aim, after 2 weeks of diabetes, cardiac expression of human NGF or β-Gal (control) genes was induced by either intramyocardial injection of AAV serotype 2 (AAV2) or systemic delivery of AAV serotype 9 (AAV9). Nondiabetic mice were given AAV2-β-Gal or AAV9-β-Gal. We found that the diabetic mice receiving NGF gene transfer via either AAV2 or AAV9 were spared the progressive deterioration of cardiac function and left ventricular chamber dilatation observed in β-Gal-injected diabetic mice. Moreover, they were additionally protected from myocardial microvascular rarefaction, hypoperfusion, increased deposition of interstitial fibrosis, and increased apoptosis of endothelial cells and cardiomyocytes, which afflicted the β-Gal-injected diabetic control mice. Our data suggest therapeutic potential of NGF for the prevention of cardiomyopathy in diabetic subjects.     

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement. In seeking a TSP for cholangiocarcinoma, Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L promoters were configures in adenoviral vectors, and evaluated in cholangiocarcinoma cells lines (Oz and SkChA-1). Luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest promoter activity, with Cox-2M appearing slightly stronger than Cox-2L. Infectivity enhanced vectors with RGD-motif in the fiber-knob region were also constructed with the luciferase transgene driven by a CMV control and the Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity than the RGD-infected cells. This paradigm was then applied to a therapeutic HSV-TK/GCV model by constructing RGD-enhanced HSV-TK vectors driven by Cox-2M and Cox-2L promoters. In vitro cytocidal effect analysis confirmed that the RGD-modified, cox-2 (M and L) driven vectors showed a stronger cytocidal effect upon gancyclovir administration than the vectors with wild-type fiber. The Cox-2 promoter demonstrates a favorable selectivity profile for cholangiocarcinoma, and RGD-modification further enhances transduction efficiency. This combination has potential to overcome the obstacles to clinical application of adenoviral gene therapy in cholangiocarcinoma. Presented at the Forty-Third Annual Meeting of The Society for Surgery of The Alimentary Tract, San Francisco, California, May 19–22, 2002 (oral presentation).     

Total vascular exclusion safely facilitates liver specific gene transfer by the HVJ (sendai virus)-liposome method in rats

BACKGROUND: Most virus mediated transfection systems are efficient; however, their highly immunogenic properties do tend to cause clinical problems. HVJ-liposome vector is a hybrid vector consisting of liposome and inactivated sendai virus (hemagglutinating virus of Japan HVJ), which has been reported to be have a low immunogenicity, while it can also be repeatedly administered. To enhance the transfection efficiency, especially in the liver, we investigated the efficacy of total vascular exclusion (TVE) during the portal vein injection (PVI) of the vectors. MATERIALS AND METHODS: beta-galactosidase and luciferase expression were used as reporter genes. Wistar rats were injected with HVJ-liposome through PVI without TVE (PVI group, n = 10) or PVI with TVE (PVI + TVE group, n = 10). The control rats were infused with equal volumes of saline through the portal vein (control group n = 12). The transfection efficiencies were assessed by beta-galactosidase staining and a luciferase assay. Biochemical and histological analyses were performed to evaluate the tissue toxicity after gene transfer. RESULTS: The reporter genes expression in the liver dramatically increased after PVI + TVE in comparison to after PVI alone (1.2 x 10(5)versus 1.5 x 10(4) RLU/mg protein, P < 0.05 according to a luciferase assay). Notably, the extrahepatic "leaky" transgene expression could be minimized by PVI + TVE, whereas the general condition remained unchanged according to both the biochemical parameters and histological findings. CONCLUSIONS: The present data indicate that PVI + TVE may thus facilitate the liver-specific gene delivery using the HVJ-liposome method and this modality might also be applicable to other gene transfer systems.     

Sustained factor VIII expression after in utero gene therapy

Objective. FVIII deficiency is an excellent gene therapy model because it is a single gene defect with a broad therapeutic window. One 5% correction of FVIII levels can prevent spontaneous bleeding episodes, and a number of experimental animal models exist. Current gene therapy approaches are limited by immune reaction to the gene product and viral proteins, and the need for multiple administrations. In this study, we investigate whether in utero administration of AAV or lentiviral vectors containing the Factor VIII gene would overcome these obstacles and allow sustained expression. Methods. AAV and lentiviral vectors containing a canine Factor VIII construct with an upstream liver-specific promoter were generated. Fourteen-day gestation fetuses of Factor VIII deficient mice were injected intraperitoneally or intravenously with AAV2/8 serotype vector or VSV-g pseudotyped lentiviral vector. Canine Factor VIII levels in plasma have now been assessed at monthly intervals up to 8 months after in utero treatment. FVIII levels are expressed as the percentage of canine plasma Factor VIII activity in knockout mouse serum. Results. In utero injections with AAV and lentiviral constructs resulted in 33 and 68% survival rates to weaning, respectively, with the differences related to known technical issues. Approximately 45% (5/11) of mice injected with the AAV vector had greater than 1% FVIII at 4 weeks, with a mean of 3.8 ± 2.0%. These levels remained stable for up to 8 months after injection. In addition 53% (9/17) of mice given the lentiviral construct showed FVIII levels greater than 1% with a mean of 1.0 ± 0.1%, with levels remaining stable for 6 months. Conclusions. In utero gene delivery of Factor VIII of AAV or Lentiviral vectors results in durable, postnatal plasma levels of Factor VIII protein in a murine model of Hemophilia A. Further optimization of dosage, titer, route of administration (intravenous versus intraperitoneal), and viral constructs are being investigated to achieve higher levels of expression. However, the sustained expression demonstrated in this study supports the promise of prenatal gene transfer.     

Adenovirus encoding soluble tumor necrosis factor alpha receptor immunoglobulin prolongs gene expression of a cotransfected reporter gene in rat lung

OBJECTIVE: Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy. METHODS: F344 rats received intratracheal administration of 1 x 10(9) plaque-forming units of adenoviral vector encoding beta-galactosidase or both adenoviral vector encoding beta-galactosidase and adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin. In the expression study beta-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis. RESULTS: Soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolonged the duration of beta-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in beta-galactosidase expression after readministration of soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase. CONCLUSION: Adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs beta-galactosidase expression but does not increase beta-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor alpha is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.     

A genetically retargeted adenoviral vector enhances viral transduction in esophageal carcinoma cell lines and primary cultured esophageal resection specimens

OBJECTIVE: To evaluate if an integrin-retargeted adenoviral vector could establish a more efficient and tumor-specific gene transfer in esophageal carcinoma cells. SUMMARY BACKGROUND DATA: Although preclinical data indicated that adenoviral gene therapy could be a promising novel treatment modality for various malignancies, clinical results are often disappointing. An important problem is the decreased tumoral expression of the Coxsackie and adenovirus receptor (CAR), which mediates adenoviral entry. Retargeting the adenoviral vector to other cellular receptors, by inserting an arginine-glycine-aspartate (RGD) tripeptide in the fiber knob, might overcome this problem. METHODS: Four esophageal carcinoma cell lines and 10 fresh surgical resection specimens were cultured. All were infected with the native adenovirus (Ad) and the retargeted adenovirus (AdRGD), encoding for the reporter genes luciferase or Green Fluorescent Protein to analyze gene transfer efficiency. RESULTS: In all cell lines, an increase in viral expression per cell and an increase in the percentage of transduced cells were seen with the retargeted adenovirus. Also, in the primary cultures of carcinoma cells, a more efficient gene transfer was seen when the retargeted vector was used. This phenomenon was less pronounced in normal cells, indicating that the RGD virus transduces tumor cells more efficiently than normal cells. CONCLUSIONS: This study demonstrates that an RGD retargeted adenovirus infects human esophageal carcinoma cells with enhanced efficiency, while in normal esophageal cells this effect is less pronounced. Therefore, this retargeted vector is expected to have a better performance in vivo, when compared with nonretargeted vectors used for cancer gene therapy so far.     

In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models.

Regional gene therapy techniques are promising methods to enhance bone formation in large bone defects that would be difficult to treat with allograft or autograft bone stock. In this study, we compared in vivo temporal expression patterns of adenoviral- and lentiviral-mediated gene therapy in two bone formation models. Primary rat bone marrow cells (RBMC) were transduced with lentiviral or adenoviral vectors containing luciferase (Luc) or BMP-2 cDNA, or cotransduced with vectors containing Luc and bone morphogenetic protein 2 (BMP-2). In vitro protein production was determined with luciferase assay or ELISA (for BMP-2 production) weekly for 12 weeks. Two bone formation models were used -- a hind limb muscle pouch or radial defect -- in SCID mice. A cooled charged-coupled device (CCD) camera was used to image in vivo luciferase expression weekly for 12 weeks. In vitro, adenoviral expression of BMP-2 and luciferase was detected by ELISA or luciferase assay, respectively, for 4 weeks. Lentiviral expression of BMP-2 and luciferase was sustained in culture for 3 months. Using the CCD camera, we found that adenoviral vectors expressed luciferase expression for up to 21 days, but lentiviral vectors expressed target gene expression for 3 months in vivo in both bone formation models. There was no detectable difference in the amount of bone formed between the adenoviral and lentiviral groups. Lentiviral-mediated delivery of BMP-2 can induce long term in vitro and in vivo gene expression, which may be beneficial when developing tissue engineering strategies to heal large bone defects or defects with a compromised biologic environment.     

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.     

Enhancement of flap survival and changes in angiogenic gene expression after AAV2‐mediated VEGF gene transfer to rat ischemic flaps

Necrosis of surgically transferred flaps due to ischemia is a serious wound problem. We evaluated the improvement of flap survival and changes in angiogenic gene expression profiles after transfer of the VEGF gene by means of adeno‐associated virus type 2 (AAV2) vector to rat ischemic flaps. Thirty rats were divided into one experimental group, one AAV2‐GFP group, and one saline group. AAV2‐VEGF or AAV2‐GFP were injected intradermally into the rat dorsum in the AAV2‐VEGF or AAV2‐GFP group. The saline group received saline injection. A 3 × 10 cm flap was raised in each rat two weeks post‐injection. One week after surgery, flap viability was evaluated. Angiogenesis real‐time PCR array was performed to analyze the expression of angiogenesis‐associated genes. The AAV2‐VEGF treatment significantly improved flap survival (p<0.05). Immunohistochemical staining showed increased VEGF expression in AAV2‐VEGF treated flaps. The PCR array identified remarkable changes in 6 out of the 84 angiogenesis‐associated genes in AAV2‐VEGF treated flaps. Particularly, EGF, PDGF‐A and VEGF‐B genes were up‐regulated in these flaps. In contrast, FGF2 gene expression was down‐regulated. In conclusion, AAV2‐VEGF improves flap survival and affects the expression of a series of endogenous growth factor genes, which likely play critical roles in the enhancement of ischemic flap survival.