rAd Vectors Research Articles

005 Cyclodextrin Polymer Based Biocompatible Matrix for Local Gene Delivery

Biocompatible matrices, such as bovine collagen, have demonstrated usefulness in delivering gene therapy vectors that express growth factors to local environments for tissue repair. Unlike animal derived matrices, we have developed a new synthetic matrix consisting of a linear cyclodextrin‐polyethyleneglycol co‐polymer that is non‐covalently cross‐linked with di‐adamantane‐polyethyleneglycol via inclusion complex formation between adamantane and cyclodextrin (CD‐Ada). We performed both in vitro and in vivo experiments for biocompatibility and localized transgene expression using a recombinant adenovirus (rAd) vector containing either the reporter gene, GFP, or the therapeutic gene, PDGF‐B. In vitro results demonstrated cell migration, adenoviral transduction, and gene expression with no visible signs of toxicity in human skin fibroblasts. Qualitative gene expression from the CD‐Ada containing rAd was delayed by approximately two days when compared to collagen, but the level of expression was greater over a longer period of time. In vivo experiments demonstrated gene expression after local delivery to mouse skin using rAd‐GFP in CD‐Ada. Again, the expression was slightly delayed but duration of expression was comparable to collagen. Expression studies using rAd‐PDGF‐B, were performed in the rat polyvinyl alcohol sponge model and showed comparable quantitative DNA and RNA levels between CD‐Ada and collagen (DNA: 4.1 × 1010 and 4.5 × 1010 MEQ of PDGF‐B/assayed sponge, respectively; RNA: 7.0 × 108 and 3.2 × 108 MEQ of PDGF‐B/assayed sponge, respectively). Additionally, we explored the use of plasmid DNA with the CD‐Ada matrix and observed PDGF‐B expression in vivo. Our results show that this new delivery system provides a safe, efficient, and adaptable medium for both viral and non‐viral gene delivery.

Transient overexpression of κ and μ opioid receptors using recombinant adenovirus vectors

In order to study the trafficking and signal transduction mechanisms of the multiple opioid receptors, these receptors are expressed either transiently or stably in cell lines. Often, it is difficult to express receptors at a sufficiently high density to obtain reproducible results. To achieve a high density of receptors, replication-defective adenovirus (rAd5) vectors encoding the μ (MOR) and κ (KOR) opioid receptors, both in their native form and as fusion proteins bearing the green fluorescent protein (GFP) at their C-terminus, were constructed. These vectors efficiently and reproducibly infected Chinese hamster ovary (CHO) cells that stably express the human coxsackie–adenovirus receptor (hCAR), with up to 90% of cells becoming infected at a low multiplicity of infection (MOI). Saturation receptor binding studies using μ- and κ-selective agonists, [ 3 H ][ d-Ala 2, N-Me-Phe 4, Gly 5-ol]enkephalin (DAMGO) and [ 3 H ](5α7α,8β)-(−)- N-methyl- N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69,593), respectively, and a nonselective antagonist, [ 3 H ]diprenorphine, revealed that rAd5-transduced cells expressed MOR and KOR for at least 3 days, at levels which exceeded those present on widely-used CHO sublines that stably express MOR or KOR. Expression levels were highest for the vectors encoding native MOR or KOR, and slightly reduced for the GFP fusion proteins. These findings demonstrate the feasibility of using rAd5 vectors to express opioid receptors at high densities, which may facilitate opioid receptor studies.

Developing a convenient large animal model for gene transfer to salivary glands in vivo.

Localized gene transfer to salivary glands has great potential for the treatment of salivary gland, systemic, and oral diseases. The minipig parotid gland, given its volume and morphological similarities to the human parotid gland, may be useful as a large animal model for pre-clinical gene transfer experiments. The purpose of this study was to perform an initial assessment of the efficacy and safety of adenoviral-vector-mediated gene transfer to parotid glands of miniature pigs. AdCMVluc, a recombinant type 5 adenoviral (rAd5) vector containing a luciferase reporter gene, was administered to miniature pig parotid glands by intraductal cannulation. Five regions of gland tissue were obtained to measure the distribution of luciferase activity. The effects of time, viral dose, infusate buffer volume, and gland anatomical region on transgene expression were determined. Detailed serum chemistry and hematological analyses were performed. In addition, AdCMVlacZ, a similar rAd5 vector encoding beta-galactosidase, was also delivered to determine the parotid gland cell types transduced. Luciferase assays indicated that gene transfer to miniature pig salivary glands could be readily accomplished using rAd5 vectors. Highest transgene expression was found in the center of glands, which was > posterior > inferior > anterior > superior tissue regions. Expression was maximal on day 2 and declined to background by day 14, and observed in both acinar and ductal cells. Several serum chemistry and hematology parameters were transiently changed following rAd5 administration. Transgene expression by, and inflammatory response to, rAd5 vectors in minipig parotid glands are similar to results seen earlier in rodent studies. This suggests that results of salivary gland gene transfer from rodent studies can be extended to a larger animal model, and supports the value of using minipigs for pre-clinical applications of gene transfer to these tissues. Published in 2004 by John Wiley & Sons, Ltd.

Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines.

Heterologous "prime-boost" regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.

Adenovirus and adeno-associated virus vectors.

Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.

Infection of mouse bone marrow-derived dendritic cells with recombinant adenovirus vectors leads to presentation of encoded antigen by both MHC class I and class II molecules—potential benefits in vaccine design

Dendritic cells (DCs) are highly specialised antigen-presenting cells (APCs) that are essential for the initiation and modulation of T cell-mediated immune responses. In order to induce effective CTL responses against most infections and tumours, DCs must prime both CD4 + and CD8 + antigen-specific T cells. It is, therefore, important in vaccine design to produce antigen-delivery systems that lead to the simultaneous presentation of multiple histocompatibility complex (MHC) class I- and class II-restricted antigenic peptides by DCs. In this study, the infection of immature mouse bone marrow-derived DCs (BMDCs) with recombinant adenovirus (rAd) vectors led to a marked upregulation of surface costimulatory molecules, IL-12 p40 production and capacity to stimulate both allogeneic and antigen-specific T cells. Furthermore, infection of immature and mature BMDCs with a rAd encoding chicken ovalbumin (OVA) led to presentation of the antigen to TCR-transgenic OVA-specific CD4 + and CD8 + T cells. In addition, the activation state of responding CD8 + T cells was further amplified if they recognised antigen on rAd-transduced BMDCs in the presence of antigen-specific CD4 + T cells. The results suggest that rAd-encoded OVA protein is secreted by BMDCs, taken up by endocytosis and presented in association with MHC class II molecules for activation of OVA-specific CD4 + T cells. Consequently, rAd-transduced immature BMDCs become better stimulators of antigen-specific CD4 + T cells than rAd-infected mature BMDCs. Taken together, these data have important implications for vaccine design, and suggest that infection of immature DCs with rAd encoding MHC class I and class II-restricted T cell epitopes could be an efficient means of inducing effective immune responses.

Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig.

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-kappaB-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-kappaB. Herein, we describe the combined use of NF-kappaB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-kappaB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.

Adenovirus-mediated gene transfer to renal glomeruli in rodents

The expression of foreign genes into renal glomerular cells holds enormous potential to modulate the outcome of renal diseases. Recombinant adenoviruses (rAds) are promising gene transfer vectors because they have the ability to infect a wide range of nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, adenovirus-mediated gene transfer into rodent glomeruli has been problematic. Here, we described our experience using rAd vectors to express foreign genes in rodent renal glomeruli in vivo and in cultured human renal glomerular cells. We developed two techniques--the "portal clamping" and "prolonged renal infusion"--to infect mouse and rat renal glomeruli in vivo, respectively. We used E-1-deleted rAd vectors carrying the lacZ gene encoding beta-galactosidase (Ad. CBlacZ) under the control of the cytomegalovirus enhancer and chicken beta-actin promoter. Cultured human renal glomerular podocytes, endothelial and mesangial cells were grown following standard techniques. Transgene expression was evaluated by doing beta-galactosidase staining and reverse transcription-polymerase chain reaction studies. We found that both a prolonged exposure and a high concentration of circulating adenoviral vectors were required to achieve efficient gene transfer to renal glomerular cells in rodents. The virus-mediated transgene expression in renal glomeruli lasted for at least 42 days in mice and 21 days in rats without causing significant renal injury. These data demonstrate the feasibility of using rAd vectors as a tool to express foreign genes in rodent renal glomerular cells and suggest that all types of human renal glomerular cells are equally susceptible to rAd infection.

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

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Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 103–104-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (~15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion. © 2001 Cancer Research Campaign http://www.bjcancer.com

Combined Effect of Cyclosporine and Sirolimus on Improving the Longevity of Recombinant Adenovirus–Mediated Transgene Expression in the Retina

To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy.

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.

602 - Intratumor (IT) gene transfer with recombinant adenovlral (rAd) vectors in lung cancer (LC) patients (PTS): The institut Gustave Roussy (IGR) experience

602 - Intratumor (IT) gene transfer with recombinant adenovlral (rAd) vectors in lung cancer (LC) patients (PTS): The institut Gustave Roussy (IGR) experience

Immunological approaches in the gene therapy of cancer

Background: despite vigorous efforts at curbing tobacco consumption and aggressive combined modality treatment programmes, both incidence and mortality from lung cancer have remained virtually unchanged in the last 10 years and there is a clear need for more effective innovative therapies. The direct transfer of tumour suppressor genes or toxic gene products that specifically promote tumour cell death and spare non-malignant cells is a potentially novel anticancer treatment approach that should be investigated. Purpose: based on compelling preclinical data, we conducted a phase I study in humans to evaluate the feasibility, tolerance and clinical, biological and immunological effects of the intratumoral administration of a recombinant, replication-deficient adenovirus (Ad RSVB-gal), using the RSV promoter to drive transcription of the Escherichia coli lacZ marker gene that encodes for the bacterial enzyme beta-galactosidase (B-gal), in patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy. The first single dose level was 10 7 (3 patients), the second was 10 8 (3 patients) and the third was 10 9 plaque forming unit (pfu) (2 patients). Biopsies of tumour and surrounding mucosa, performed at days 8, and at 1, 2 and 3 months following injection, were analysed using PCR and X-gal staining. Patients and medical staff were monitored for wildtype and rAd infections by PCR, culture and serologic analyses. Patients remained in TL2 isolation until negative results of PCR and cultures of biologic fluid specimens were confirmed. All patients received concomitant chemotherapy. Results: seven patients with NSCLC and one with neuroendocrine tumour entered this ongoing study. Expression of B-gal was observed in five out of eight tumour biopsies (one at 10 7, two at 10 8 and two at 10 9 dose level). Bronchoalveolar lavage collected immediately after injection were positive for recombinant adenovirus by culture and PCR. All biological fluids were negative by PCR after day 12. The three and two patients treated at 10 8 and 10 9 pfu respectively had PCR positive blood samples on day 1. Patients were in isolation for a median of 17 days. Moderate bleeding (two patients) during bronchoscopy and fever (four patients) were the most common toxicities. Endoscopic and clinical objective tumour responses were seen in five patients. The three patients treated at the second dose level, all with stage IIIB disease, were deemed resectable after chemotherapy. They all had a complete surgical resection of their tumour 112–152 days after viral injection. Hospital staff remained negative for recombinant adenovirus infection throughout the study. Conclusion: this ongoing study demonstrates that a marker gene can be safely introduced into humans using an rAd vector and that the gene product is expressed by host lung tumour cells. We will continue our programme by using this methodology to study the antitumour effects mediated by transfer of tumour suppressor or cytokine genes into endobronchial lesions in lung cancer patients.

98 Direct intratumor (IT) gene transfer using recombinant adenoviral (rAd) vectors: a phase I study in lung cancer (LC) patients (pts)

98 Direct intratumor (IT) gene transfer using recombinant adenoviral (rAd) vectors: a phase I study in lung cancer (LC) patients (pts)