Therapeutic Transgene Research Articles

Pharmacological Modulation of Humoral Immunity in a Nonhuman Primate Model of AAV Gene Transfer for Hemophilia B

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

Targeted and Armed Oncolytic Poxviruses for Cancer: the Lead Example of JX-594

Oncolytic viruses (OVs) are designed to replicate in, and subsequently lyse cancer cells. Numerous oncolytic virus platforms are currently in development. Here we review preclinical and clinical experience with JX-594, the lead candidate from the targeted and armed oncolytic poxvirus class. JX-594 is derived from a vaccinia vaccine strain that has been engineered for 1) enhanced cancer targeting and 2) has been "armed" with the therapeutic transgene granulocytemacrophage colony stimulating factor (GM-CSF) to stimulate anti-tumoral immunity. Poxviruses have many ideal features for use as oncolytic agents. The development of oncolytic vaccinia viruses is supported by a large safety database accumulated in the smallpox eradication program. In addition, poxviruses have evolved unique capabilities for systemic spread through the blood that can be harnessed for the treatment of metastatic disease. JX-594 demonstrates a high degree of cancer selectivity and systemic efficacy by multiple mechanisms-of-action (MOAs) in preclinical testing. Data from Phase 1 and 2 clinical trials has confirmed that these features result in potent and systemic efficacy in patients with treatment refractory metastatic cancers.

PTU-043 Development of a liver-specific, tumour-selective recombinant adeno-associated virus vector to target hepatocellular carcinoma

IntroductionHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is currently one of the most difficult to treat. Surgical resection and liver transplantation are considered curative therapies but...

230. Correction of Immunity in Murine Rag2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Using a Codon-Optimized RAG2 Therapeutic Transgene

230. Correction of Immunity in Murine Rag2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Using a Codon-Optimized RAG2 Therapeutic Transgene

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively(1). They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans(2). NS1 is the major effector of viral cytotoxicity(3). In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)(4). The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)(4). The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications. We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit(5). The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product(5). In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Abstract 2712: A non-replicating adenovirus expressing Hey1 is cytotoxic to prostate cancer cells

Abstract Androgen receptor (AR) signalling is active in most hormone-refractory prostate cancers (PCa) and suppression of its activity impedes cell proliferation. We demonstrate that Hey1, a co-repressor of AR, is a potential therapeutic transgene for treatment of PCa. A replication-defective adenovirus expressing Hey1 under control of the CMV promoter was generated (Ad5Hey1; E1 and E3 deleted) and found to be cytotoxic to human PCa cells, both AR-positive 22Rv1 and LNCaP and AR-negative DU145. The doses required to kill 50% of cells (EC50) were comparable to the cytotoxic E1A12S gene expressed from an identical vector (AdE1A12S). Ad5Hey1 repressed AR activity in a dose dependent manner in 22Rv1 cells both with and without mibolerone stimulation as determined by luciferase reporter assays. In 22Rv1 cells, the G2/M population increased in an Ad5Hey1 but not AdE1A12S dependent manner 48h after infection to 37.6±5.4% at 1000 particles per cell (ppc). An increase in cyclin B1 expression reflected the Ad5Hey1-dependent increase in G2/M phase. Expression of p53 increased with increasing doses of Ad5Hey1 whereas p21 levels increased at low doses but decreased at higher doses in 22Rv1 and LNCaP cells. At 1000ppc, a larger proportion of cells were in sub-G1 (38.5±7.5%) indicating that Ad5Hey1 induced apoptotic cell death. Apoptotic death was confirmed by caspase 3 activation in both Ad5Hey1 and AdE1A12S-infected cells by flow cytometry. Efficacy of Ad5Hey1 was also evaluated in vivo in combination with docetaxel, currently used in the clinic for treatment of PCa. In LNCaP xenograft in vivo models, Ad5Hey1 alone caused significant tumour growth inhibition with median time to tumour progression of 92d compared with 54d for animals with mock-treated tumours. Time to progression was further increased when Ad5Hey1 was combined with a sub-optimal dose of docetaxel with 75% of animals surviving at 110d compared with 40% of animals with Ad5Hey1 alone and none of the docetaxel treated animals surviving. Similar tumour growth inhibition was observed in models using DU145 xenografts; the combination of Ad5Hey1 with docetaxel significantly prolonged time to progression from 62d (single agents) to 96d when combined (5-8 animals/group). Furthermore, the antiandrogen bicalutamide, used as a therapy for hormone-dependent PCa, enhanced Ad5Hey1 cytotoxicity and decreased the EC50 of Ad5Hey1 alone by 14.5±10.5% and 33.5±2.5% for 5 and 10µM bicalutamide respectively in 22Rv1 cells. These data indicate that Hey1 can act as a cell death enhancer if over-expressed. Data is presented to suggest Hey1 can be expressed from a replication selective adenovirus to improve oncolytic anti-tumour efficacy in vitro and in PCa xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2712. doi:1538-7445.AM2012-2712

Abstract 4065: A simple biological imaging system for detecting adenovirus receptor expression in tumor cells using a telomerase-specific replication-competent adenovirus

Abstract Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunocytochemistry. We recently generated a green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401, which induces ectopic GFP expression in tumor cells, but not in normal cells. In this study, we evaluated whether induction of GFP expression by OBP-401 infection is associated with CAR expression in tumor cells. OBP-401-mediated GFP induction was further examined in xenograft tumor tissues that have different levels of CAR expression and in surrounding normal tissues. We used six human sarcoma cell lines that have different levels of CAR expression and CAR-positive normal human lung fibroblasts (NHLF). Flow cytometry analysis confirmed that cells of four of the sarcoma cell lines (OST, U2OS, NOS-10 and MNNG/HOS) as well as the NHLF cells showed detectable CAR expression, whereas cells of the NMFH-1 and OUMS-27 sarcoma cell lines had no detectable CAR expression. To determine suitable conditions for OBP-401 infection, all tumor and normal cells was infected with OBP-401 at multiplicity of infections of 1, 10 and 100 plaque forming units/cell. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 hours after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. We further examined the potential of the OBP-401-mediated method for the detection of CAR expression in tumor and normal tissues, we used this method to analyze CAR expression of human xenograft tumor tissues. OBP-401 infection induced GFP expression in OST tumor tissues, but not in OUMS-27 tumor tissues or in normal muscle tissue. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4065. doi:1538-7445.AM2012-4065

265 DEVELOPMENT OF A LIVER-SPECIFIC, TUMOUR-SELECTIVE RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR TO TARGET HEPATOCELLULAR CARCINOMA

Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is currently one of the most difficult to treat. Surgical resection and liver transplantation are considered curative therapies but they are feasible for only a small number of patients. Other therapeutic options, such as radiofrequency ablation and arterial chemoembolization, are effective only in small tumours. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for gene transfer-based therapeutic approaches for HCC because of their safety profile and remarkable tropism for the liver, with >90% of the vector particles being detectable in hepatocytes following a single systemic administration in non-human primates. Methods Sequence complementarity to miR-122a (122aT) was cloned downstream of luciferase under the control of a new liver specific promoter/enhancer element, HLP, and flanked by the AAV-inverted-terminal-repeats (ITRs). AAV8 capsid-pseudotyped AAV8-HLP-LUC±122aT vector stocks were prepared by 293T-based three-plasmid transient transfection, and used for: in vitro transduction of HCC cell lines HUH-7 (miR-122a-positive) and SNU-387 (miR-122a-negative); in vivo tail vein injection of tumour-bearing SCID and B6 mice. Results A single tail-vein injection of rAAV8 in tumour-bearing mice resulted in selective transduction of the liver as well as of HCC, but not neuroblastoma xenografts. We cloned the 122aT into our expression cassette to improve the transgene expression for HCC, taking advantage of the differential expression of miR-122a (abundant in normal liver but down regulated in most HCC). Normalised transgene expression with AAV8-HLP-LUC-122aT was at least 26.5-fold higher in SNU-387 when compared to HUH-7 cell lines. Tail-vein injection of rAAV8-HLP-LUC resulted in high transgene expression in liver, but mice transduced with rAAV8-HLP-Luc-122aT had little or no luciferase expression despite the proviral DNA presence. Subcutaneous HCC xenografts in mice showed a strong luciferase signal following tail-vein injection of both of these vectors. Conclusion Inclusion of 122aT in the expression cassette allows regulation of rAAV mediated transgene expression in HCC cell lines and normal liver, and reduces expression in miR-122a positive cells (eg, normal hepatocytes). This novel vector, therefore, has the potential to deliver therapeutic transgenes to HCC in preference to normal hepatocytes in the liver, thus limiting toxicity. Competing interests None declared.

In Vivo Evaluation of a Cancer Therapy Strategy Combining HSV1716-Mediated Oncolysis with Gene Transfer and Targeted Radiotherapy

Oncolytic herpes viruses show promise for cancer treatment. However, it is unlikely that they will fulfill their therapeutic potential when used as monotherapies. An alternative strategy is to use these viruses not only as oncolytic agents but also as a delivery mechanism of therapeutic transgenes to enhance tumor cell killing. The herpes simplex virus 1 deletion mutant HSV1716 is a conditionally replicating oncolytic virus that selectively replicates in and lyses dividing tumor cells. It has a proven safety profile in clinical trials and has demonstrated efficacy as a gene-delivery vehicle. To enhance its therapeutic potential, we have engineered HSV1716 to convey the noradrenaline transporter (NAT) gene (HSV1716/NAT), whose expression endows infected cells with the capacity to accumulate the noradrenaline analog metaiodobenzylguanidine (MIBG). Thus, the NAT gene-infected cells are susceptible to targeted radiotherapy using radiolabeled (131)I-MIBG, a strategy that has already shown promise for combined targeted radiotherapy-gene therapy in cancer cells after plasmid-mediated transfection. We used HSV1716/NAT as a dual cell lysis-gene delivery vehicle for targeting the NAT transgene to human tumor xenografts in vivo. In tumor xenografts that did not express NAT, intratumoral or intravenous injection of HSV1716/NAT induced the capacity for active uptake of (131)I-MIBG. Administration of HSV1716/NAT and (131)I-MIBG resulted in decreased tumor growth and enhanced survival relative to injection of either agent alone. Efficacy was dependent on the scheduling of delivery of the 2 agents. These findings support a role for combination radiotherapy-gene therapy for cancer using HSV1716 expressing the NAT transgene and targeted radionuclide therapy.

X-Linked Inhibitor of Apoptosis Protein-Mediated Attenuation of Apoptosis, Using a Novel Cardiac-Enhanced Adeno-Associated Viral Vector

Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector.

Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: A new concept for improvement of non-viral therapeutic gene delivery

The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of ‘sticky points’ dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II topoisomerases. The technology for such self-entanglement can be an avenue for the improvement of gene delivery with high-molecular-weight naked DNA using therapeutically important methods associated with considerable shear stress. Priority applications include in vivo muscle electroporation and sonoporation for Duchenne muscular dystrophy patients, aerosol inhalation to reach the target lung cells of cystic fibrosis patients and bio-ballistic delivery to skin melanomas with the vector DNA adsorbed on gold or tungsten projectiles.

Semireplication-competent vesicular stomatitis virus as a novel platform for oncolytic virotherapy

Among oncolytic viruses, the vesicular stomatitis virus (VSV) is especially potent and a highly promising agent for the treatment of cancer. But, even though effective against multiple tumor entities in preclinical animal models, replication-competent VSV exhibits inherent neurovirulence, which has so far hindered clinical development. To overcome this limitation, replication-defective VSV vectors for cancer gene therapy have been tested and proven to be safe. However, gene delivery was inefficient and only minor antitumor efficacy was observed. Here, we present semireplication-competent vector systems for VSV (srVSV), composed of two trans-complementing, propagation-deficient VSV vectors. The de novo generated deletion mutants of the two VSV polymerase proteins P (phosphoprotein) and L (large catalytic subunit), VSVΔP and VSVΔL respectively, were used mutually or in combination with VSVΔG vectors. These srVSV systems copropagated in vitro and in vivo without recombinatory reversion to replication-competent virus. The srVSV systems were highly lytic for human glioblastoma cell lines, spheroids, and subcutaneous xenografts. Especially the combination of VSVΔG/VSVΔL vectors was as potent as wild-type VSV (VSV-WT) in vitro and induced long-term tumor regression in vivo without any associated adverse effects. In contrast, 90% of VSV-WT-treated animals succumbed to neurological disease shortly after tumor clearance. Most importantly, even when injected into the brain, VSVΔG/VSVΔL did not show any neurotoxicity. In conclusion, srVSV is a promising platform for virotherapeutic approaches and also for VSV-based vector vaccines, combining improved safety with an increased coding capacity for therapeutic transgenes, potentially allowing for multipronged approaches.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-012-0863-6) contains supplementary material, which is available to authorized users.

Parkinson's Disease: Gene Therapies

With the recent development of effective gene delivery systems, gene therapy for the central nervous system is finding novel applications. Here, we review existing viral vectors and discuss gene therapy strategies that have been proposed for Parkinson's disease. To date, most of the clinical trials were based on viral vectors to deliver therapeutic transgenes to neurons within the basal ganglia. Initial trials used genes to relieve the major motor symptoms caused by nigrostriatal degeneration. Although these new genetic approaches still need to prove more effective than existing symptomatic treatments, there is a need for disease-modifying strategies. The investigation of the genetic factors implicated in Parkinson's disease is providing precious insights in disease pathology that, combined with innovative gene delivery systems, will hopefully offer novel opportunities for gene therapy interventions to slow down, or even halt disease progression.

Retroviral Integrations in Gene Therapy Trials

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis.

A simple detection system for adenovirus receptor expression using a telomerase-specific replication-competent adenovirus

Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunohistochemistry. Here, we show a simple system for detection and assessment of functional CAR expression in human tumor cells, using the green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 h after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor therapy.

Baculovirus‐mediated vascular endothelial growth factor‐DΔNΔC gene transfer induces angiogenesis in rabbit skeletal muscle

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.

Gene Therapy Research at the Frontiers of Viral Immunology

Gene Therapy Research at the Frontiers of Viral Immunology

Histone Deacetylase Inhibitors Restore Cell Surface Expression of the Coxsackie Adenovirus Receptor and Enhance CMV Promoter Activity in Castration-Resistant Prostate Cancer Cells

Adenoviral gene therapy using the death receptor ligand TRAIL as the therapeutic transgene can be safely administered via intraprostatic injection but has not been evaluated for efficacy in patients. Here we investigated the efficacy of adenoviral TRAIL gene therapy in a model of castration resistant prostate cancer and found that intratumoral injections can significantly delay tumor growth but cannot eliminate established lesions. We hypothesized that an underlying cause is inefficient adenoviral delivery. Using the LNCaP progression model of prostate cancer we show that surface CAR expression decreases with increasing tumorigenicity and that castration resistant C4-2b cells were more difficult to transduce with adenovirus than castration sensitive LNCaP cells. Many genes, including CAR, are epigenetically silenced during transformation but a new class of chemotherapeutic agents, known as histone deacetylase inhibitors (HDACi), can reverse this process. We demonstrate that HDACi restore CAR expression and infectivity in C4-2b cells and enhance caspase activation in response to infection with a TRAIL adenovirus. We also show that in cells with high surface CAR expression, HDACi further enhance transgene expression from the CMV promoter. Thus HDACi have multiple beneficial effects, which may enhance not only viral but also non-viral gene therapy of castration resistant prostate cancer.

The assessment of adeno‐associated vectors as potential intrinsic treatments for brainstem axon regeneration

Adeno-associated virus (AAV) vector-mediated transgene expression is a promising therapeutic to change the intrinsic state of neurons and promote repair after central nervous system injury. Given that numerous transgenes have been identified as potential candidates, the present study demonstrates how to determine whether their expression by AAV has a direct intrinsic effect on axon regeneration. Serotype 2 AAV-enhanced green fluorescent protein (EGFP) was stereotaxically injected into the brainstem of adult rats, followed by a complete transection of the thoracic spinal cord and Schwann cell (SC) bridge implantation. The expression of EGFP in brainstem neurons labeled numerous axons in the thoracic spinal cord and that regenerated into the SC bridge. The number of EGFP-labeled axons rostral to the bridge directly correlated with the number of EGFP-labeled axons that regenerated into the bridge. Animals with a greater number of EGFP-labeled axons rostral to the bridge exhibited an increased percentage of those axons found near the distal end of the bridge compared to animals with a lesser number. This suggested that EGFP may accumulate distally in the axon with time, enabling easier visualization. By labeling brainstem axons with EGFP before injury, numerous axon remnants undergoing Wallerian degeneration may be identified distal to the complete transection up to 6 weeks after injury. Serotype 2 AAV-EGFP enabled easy visualization of brainstem axon regeneration. Rigorous models of axonal injury (i.e. complete transection and cell implantation) should be used in combination with AAV-EGFP to directly assess AAV-mediated expression of therapeutic transgenes as intrinsic treatments to improve axonal regeneration.