Duncan L. Mcvey

Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12

Direct intratumoral (i.t.) injection of adenoviruses (Ads) expressing specific immunostimulatory cytokines represents an attractive strategy for the clinical implementation of cytokine gene therapy of cancer. Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells and promotes a T helper 1-like immune response. We have constructed an Ad vector (AdCMV-mIL-12) containing both chains of the murine IL-12 (mIL-12) gene linked by an internal ribosomal entry site sequence under the transcriptional control of the cytomegalovirus immediate-early gene promoter, which is able to mediate the transient expression of very high levels of biologically active mIL-12 both in vitro and in vivo. An i.t. injection of 4 × 108 plaque-forming units of AdCMV-mIL-12 resulted in a complete regression of day 7 established subcutaneous MC38 murine adenocarcinomas and MCA205 murine fibrosarcomas. Treated animals rejected a subsequent rechallenge with MC38 and MCA205, respectively, demonstrating the induction of long-lasting antitumor immunity. Specific antitumor cytotoxic T lymphocyte reactivity was detected in splenocytes harvested from treated animals. A significant increase in the numbers of both CD4+ and CD8+ T cells in the AdCMV-mIL-12-infected tumors was observed. Ad-mediated IL-12 gene therapy was also associated with measurable serum levels of mIL-12 and profound changes in the composition of splenic lymphocytes. Taken together, these results demonstrate the feasibility and efficacy of delivering IL-12 directly i.t. using a recombinant adenoviral vector.

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.

Rapid Construction of Adenoviral Vectors by Lambda Phage Genetics

ABSTRACT Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.

Characterization of human adenovirus 35 and derivation of complex vectors

BackgroundReplication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.ResultsSequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.ConclusionsThe results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

Identification of a Suppressor Mutation that Improves the Yields of Hexon-Modified Adenovirus Vectors

ABSTRACT We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.

Repeat Administration of Proteins to the Eye With a Single Intraocular Injection of an Adenovirus Vector

Delivery of therapeutic proteins, such as antiangiogenic proteins, to the eye is a demonstrated method for the control of age-related macular degeneration (AMD). However, one of the key limitations is the requirement for frequent and repeated intraocular injections. In this article, we demonstrate that repeated protein production in the eye can be stimulated from the cytomegalovirus (CMV) promoter without repeat intraocular injections using a small molecule, all-trans retinoic acid (ATRA). ATRA by systemic delivery can stimulate protein production multiple times in the eye. Administration of ATRA resulted in stimulation of gene expression to relevant levels that block abnormal blood vessel growth in an experimental animal model for AMD. These data support the principles of this technological discovery to therapeutic applications for chronic ocular diseases.

875. Adenoviral Vector Targeting |[ndash]| New Targets for Old Capsids

Top of pageAbstract The goal of our adenoviral vector-targeting program is to identify strategies that enhance delivery of therapeutic proteins to the site of disease as well as reduce the impact of the therapy on nontarget cells and tissue. Therapeutic areas under consideration are cancer, ocular disease, vaccine, hearing loss and balance disorders. To accomplish this we have created vectors that are ablated for binding to native CAR and integrin receptors, have introduced specific binding ligands for selective entry into the cells of choice, and have evaluated vector performance in these target therapeutic areas. We have evaluated the pharmacokinetics and biodistribution of tropism-modified vectors in regional (intra-peritoneal) and disseminated cancer disease models and have shown the importance of selective delivery to tumors after regional and systemic administration. Delivery to the eye and ear of tropism-modified vectors results in transduction of new target cells and the modifications may also impact the kinetics of transgene expression. We have demonstrated low-level expression of the normal entry receptors for adenovirus in the inner ear of mice, which is inconsistent with the amount of gene delivery observed by native tropism vectors. Adenovirus capsids ablated of normal receptor interactions (CAR and integrin) are still capable of gene delivery, suggesting that vector entry may occur through alternative pathways. The efficiency of gene delivery in the inner ear by capsid-modified vectors is dependent on target volume and local vector concentration. While efficient delivery within the inner ear is retained by the ablated vector, it has a restricted tropism for gene delivery to tissue outside of the inner ear. These results collectively lend support to the design of targeted adenovirus vectors that could enhance the delivery of the therapeutic protein and thereby be used for multiple clinical applications.

989. Toward Multivalent Adeno-Based Vaccine for Malaria: Effects of Promoter Strength on Immunogenicity and Vaccine Efficacy

Malaria is the most devastating parasitic disease affecting humans. Each year 300-500 million people become infected with the parasite and more than 1.5 million people, primarily children, die of the disease. The feasibility of a malaria vaccine is supported by the demonstration of protective immunity following exposure to the intact Plasmodium parasite.

993. Activation of Gene Expression from Silenced Adenovector Genomes

Top of pageAbstract Transgene expression driven by the human cytomegalovirus (hCMV) promoter from adenovectors following intravitreal administration into the eye is characterized by an initial high-level burst of expression that drops quickly and is followed by eventual loss of transgene expression. The decrease in expression could arise from elimination of vector genomes or by loss of promoter activity. In the current studies we investigate the cause for the diminution of activity, evaluate quantitatively vector genomes and expression levels, describe a means in which to follow transgene expression in the eye in the same animal over time, and discover means in which silenced adenovector genomes can be reactivated to express transgenes. In these studies, replication-deficient adenovectors deleted of E1, E3 and E4 that express nothing, GFP, or luciferase from the hCMV promoter were delivered to the eyes of mice by direct intravitreal injection. The amount of vector genome was quantitated by qPCR, the amount of luciferase transgene expression quantitated by luminesence, and the level of GFP transgene expression monitored by fluorescence. From these analyses the loss of transgene expression over time from adenovectors can be attributed to loss of promoter activity. Adenovector genomes from which there was no detectable transgene expression still retained the ability to express their protein product. This was shown by the ability to completely restore expression from a silenced hCMV promoter by multiple means. The administration of non-expressing adenovector particles to the vitreous activated expression from the silenced adenovector genome to levels equal to or greater than that originally observed on day 1. These experiments also showed that quiescent adenovector genomes retain the ability to be reactivated for transgene expression even after being silenced for more than a year. A screen of potential molecules capable of activating the hCMV promoter found retinoic acid (RA) to be a potent activator of expression and systemic administration of RA was found to activate expression from silenced adenovector genomes to day 1 levels. Furthermore, we found that RA could activate expression from silenced adenovector genomes even after 2 months following initial administration into the eye, which further demonstrates that adenovector genomes remain expression competent. These data suggest that adenovectors may provide a means to deliver proteins to the eye requiring repeated expression for the treatment of ocular disease and expands the utility of adenovectors in ocular gene delivery.