Protocols for the Two Plasmid Rescue System

The remainder of this chapter provides detailed protocols for each of the steps involved in the rescue and propagation of recombinant Ad vectors. A flow chart of these steps is presented in Fig. 7. Briefly, 293 cells are cotransfected with the Ad genomic plasmid and the shuttle plasmid. The recombinant vector is generated by in vivo site-specific recombination between the two plasmids and forms a plaque in the cell monolayer. The plaques are isolated, the virus expanded, and the vector DNA is...

Inflammatory Bowel Disease

The gut has been proposed as a target for gene delivery for a variety of diseases including both metabolic diseases and primary diseases affecting the intestine, including the inflammatory bowel diseases, Chron's disease and ulcerative colitis 42, 43 , Toward this end Hogaboam and colleagues have shown that intraperitoneal delivery of adenovirus encoding interleukin-4 (AdIL-4), a prototypic TH2 cytokine, attenuates colitis induced by trinitroben-zene sulfonic acid (TNB) 44 , TNB-induced colitis...

Production of Adenoviral Vectors

When constructing El-deleted adenoviral vectors, a number of choices must be made regarding the structure of the vector backbone and the composition of the transgene. One must determine if the size of the El deletion will be adequate to accommodate the size of the transgene or if additional deletions, such as in the E3 region, will be needed. One must also decide on the placement of the transgene within the genome (El vs E3) and the orientation of the transgene (El parallel vs El antiparallel)....

Approaches of Transcriptional Regulation

Prior Rationale Universal Promoters Several universal promoters have been utilized to attempt to maximize gene expression. The LTR, CMV, and RSV promoters were isolated from Maloney retrovirus, cytomegalovirus, and Rous sarcoma virus, respectively. These promoter elements were used because of the universal transcriptional activation over a broad host range. This universal transcription allowed for excellent but nondiscriminatory gene transcription and subsequent transgene expression. Because...

Strategies to Overcome the Humoral Immune Response

The humoral and cellular immune response to recombinant adenoviral vectors, as described in several animal models, result in extinction of transgene expression, severe local inflammation, and production of neutralizing antibodies that prevent readministration 59, 63, 64 . A direct correlation between neutralizing antibody and the block to readministration of vector has been established by passive transfer of immunity by sera from treated to naive animals 59 , One approach to enhance...

Distribution and Clearance of Adenovirus from the Respiratory Tract

Clinical Aspects of Natural Adenoviral Infection in Humans Adenovirus is an important respiratory pathogen affecting individuals of all ages with an annual incidence of between 5 to 10 million in the United States. Infections can occur sporadically, epidemically and nosocomially but most individuals are infected at a young age adenovirus accounts for 7 to 10 of all respiratory illnesses in infants and children 8, 9 . Although adenovirus frequently causes a mild, acute upper respiratory...

Synergy of ARCA and Conventional Therapy

Although conventional cancer therapies (surgery, chemotherapy, and radiation) are effective at curing early-stage disease, few human cancers are curable with a single modality. The utilization of a replication-competent cytolytic adenovirus as a therapeutic modality shows much promise. In our laboratory and clinical trials, CV706 and CV787 have been shown to be effective against human prostate tumors 21-23 . However, single-dose total eradication of human LNCaP xenografts in our animal model...

The Two Plasmid Rescue System

One of the first methods that was developed to overcome the limitations of the earlier approaches was the two-plasmid rescue system (Fig. 3A). In this method, recombinant Ad vectors are generated by in vivo homologous recombination between two noninfectious plasmids cotransfected into 293 cells. Since its development, the two-plasmid rescue method has been widely used due to its simplicity. Other methods of constructing Ad vectors have Figure 3 Construction of Ad vectors by (A) in vivo...

Gamma Ray Imaging

Gamma-ray imaging has been applied in two different ways to evaluate gene-therapy vectors in animal models. First, the in vivo location of the administered vector can be imaged following dosing. Second, the vector-dependent location of transgene expression can be imaged. This is accomplished by detection of expressed genetic reporters encoded in the vector. Both methods yield different information and may, or may not be correlated. For example, following intravenous injection an Ad vector might...

Safety Considerations of PERC6

QC Testing of PER.C6 Cells for Use in the Manufacture of Biologicals and Vaccines The safety of vaccines and biologicals manufactured in continuous cell lines of animal or human origin is of paramount importance and must be ensured by the manufacturer through a program of quality control (QC) testing applied to the product before release for human administration. This QC testing is intended to (i) ensure the identity of the product, (ii) ensure the safety and sterility of the product by...

ARCAs for Prostate Cancer CV706 and CV787

Adenovirus Gene Expression and Regulation Members of the human Adenoviridae family were first cultured from the tonsils and adenoids of children in 1953 29 . They represent 51 different serotypes which are distinguishable by antibody reactivity to epitopes on the virion surface. Each serotype is assigned to one of five subgroups (A-E). Adenovirus type 5 (Ad5), a member of Subgroup C, is associated with a self-limiting, febrile respiratory illness and ocular disease in humans infectious virus...