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...

References

D., and Perry, M. C. (1995). Chemotherapy. In Clinical Oncology (M. D. Abeloff, J. O. Armitage, A. S. Lichter, and J. E. Niederhuber, Eds.), pp. 201-218. Churchill Livingston, New York. 2. Scher, H. I., Isaacs, J. T., Fuks, Z., and Walsh, P. C. (1995). Prostate. In Clinical Oncology (M. D. Abeloff, J. O. Armitage, A. S. Lichter, and J. E. Niederhuber, Eds.), pp. 1439-1472. Churchill Livingston, New York. 3. Golomb, H. M., Fefer, A., Golde, D. W., Ozer, H.,...

Analytical Methods for Process Development and Process Tracking

Analytical methods are as important for purification as the process steps themselves. Analytical methods are essential for following the process and assessing the purity of adenovirus particles throughout the process. The methods must be rapid, reliable, and informative about the quantity and quality of adenovirus particles. They should be sensitive enough to detect subtle changes. As with the process techniques, characteristics of the virus are useful for selecting analytical techniques. In...

Safety Symposia in Conjunction with OBA

Following the death of the study participant discussed above, the OTC trial was immediately placed on clinical hold, and the FDA initiated a search of its database to identify all protocols involving adenoviral vectors used for therapeutic intent. A total of 12 protocols were identified which used adenovirus administered by either systemic or intrahepatic artery infusion, or by direct injection into the liver. The sponsors of these protocols were informed of the death of the patient in the OTC...

Pneumonia

Pneumonia and influenza infection remain the sixth leading cause of death in the United States 16 . Drug-resistant organisms are increasingly isolated from infected patients, presumably due to the broad use of antibiotics. As mentioned above, several biological response modifiers such as granulocyte colony stimulating factor (G-CSF) and IFNy have been investigated in patients as protein-based therapies. However, due to pharmacological advantages of adenovirus and other gene-based vector...

Flp

Figure 5 Ad genomic plasmids used for vector rescue by in vivo site-specific recombination. The plasmids pBHGloxE3Cre, pBHGloxAE3(Xl)Cre, and pBHGloxAEI ,3Cre are used to rescue vectors by Cre-mediated recombination bearing a wildtype E3 region, a 1864 bp deletion or a 2653 bp deletion of E3, respectively. Analogous plasmids pBHGfrtE3FLP, pBHGfrtAE3(Xl )FLP and pBHGfrtAEl,3FLP are used to rescue vectors by FLP-mediated recombination. The unique Pad restriction enzyme site in pBHGloxAEI ,3Cre,...

Animal Models of Lung Cancer

Lung cancer is the leading cause of cancer-associated mortality in both men and women. Although susceptibility to environmental carcinogens may be predetermined and follow a pattern of autosomal dominant Mendelian inheritance 2, 3 , lung cancer results from an accumulation of acquired genetic mutations 4-6 . In fact, it is suggested that 10-20 genetic mutations may be necessary for the development of lung cancer 7 , although the discrete steps for the progression of a hyperplastic bronchial...

E1B 55kDa Gene Deletion Mutant dl 1520

Dl1520 (Onyx-O15) was the first adenovirus described to mirror the gene deletion approach pioneered by Martuza with herpesvirus. McCormick et al. hypothesized that an adenovirus with deletion of a gene encoding a p53-binding protein, E1B 55-kDa, would be selective for tumors that already had inhibited or lost p53 function. p53 function is lost in the majority of human cancers through mechanisms including gene mutation, overexpression of p53-binding inhibitors (e.g., mdm2, human papillomavirus...

Standard Operating Procedures

Standard operating procedures are a mechanism to ensure that process controls and protocols for product manufacture and characterization are carried out in a reproducible and documented fashion for each stage of manufacturing and product testing. SOPs consist of detailed written documents describing each step of a process conducted in manufacturing. SOPs can also refer to many different types of processes that impact adenovirus production, such as required training of personnel, acquisition and...

The Airway Epithelium Cellular Targets for CF Gene Therapy

Airway epithelial cells are present throughout the conducting airways of the lung, including the nasal, tracheal, bronchial, and bronchiolar regions. In the upper airway, the surface epithelium lines these structures and is continuous with the tubulo-acinar submucosal mucus-secreting glands that invaginate from the airway surface. Airway epithelial cell-type composition is dependent both on the regional location and on the particular species studied and the reader is referred to comprehensive...

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...

Adenoviral Vectors as Gene Transfer Vectors in the Lung

Animal Models for CF Airway Gene Transfer Studies The generation of CF mouse models was an important step for understanding the physiology of CF disease. There have now been over 10 different mouse models produced displaying a range of CF-associated genetic mutations 39 , Although most of the models reflect the most common human mutation, either a complete gene knockout or a AF508 mutation of the mouse CFTR, other models with less common human mutations (e.g., G551D) have also been reported....

Info

Figure 4 Postulated mechanisms of vector construction by (A) homologous recombination between a shuttle plasmid bearing a single ITR and an genomic plasmid and (B) Cre-mediated recombination between a shuttle plasmid bearing an ITR junction and an Ad genomic plasmid. method to utilize the yeast FLP-mediated site-specific recombination system 39 . With loxP sites in the Ad genomic and shuttle plasmids replaced with frt sites and the Cre-expression cassette replaced with a FLP-expression cassette...

Ad Vector Construction by in Vitro Ligation

The notion of separation of the molecular construction of the rAd vector from the virus production is also guiding the direct cloning procedures described below. Efficient and improved in vitro ligation techniques have recently been described 40-42 . Due to the paucity of unique restriction sites within the adenoviral genome, direct ligation of a transgene expression cassette into the viral vector backbone is facilitated by the use of intron-encoded endonucleases. The technique basically...

Master Virus Bank

A master virus bank (MVB) consists of a well-characterized stock of virus-based vector that serves as the inoculum for all subsequent large-scale vector production. It is sometimes referred to as a vector seed stock. Table III gives a summary of the types of characterization recommended by the FDA. Safety testing for a master virus bank is very similar to that done for a master cell bank. Thus a master virus bank is tested for sterility and mycoplasma, in vitro and in vivo adventitious virus,...

Purity Safety and Potency

When an adenovirus-based vector is used for the first time in humans, a major goal of FDA oversight is to ensure the safety of patients who receive the investigational product. A crucial component of safety and effectiveness is careful attention to the details of manufacturing and product characterization. The extent and quality of this information allows an assessment of the purity of the final vector preparation that will be administered to patients. Assessment of purity involves biological...

Hybrid Viral Vectors

The inadequacies of each viral vector system are illustrated in Table II. The negative attributes of one vector, however, generally emphasize the positive attributes of another. Thus most of the criteria defined for a hypothetical perfect gene therapy might actually be met by considering defined properties of the currently available vectors defined in Table II. Hence, although at present no individual virus system alone can meet all the criteria, current research is focusing on combining...

Replication Selective Oncolytic Adenovirus ElRegion Mutants Virotberapy for Cancer

Attributes of Replication-Selective Adenoviruses for Cancer Treatment 332 III. Biology of Human Adenovirus 332 IV. Mechanisms of Adenovirus-Mediated Cell Killing 333 V. Approaches to Optimizing Tumor-Selective Adenovirus Replication 333 VI. E1A-CR2 Region Deletion Mutants 334 VII. El B 55-kDa Gene Deletion Mutant d 1520 335 VIII. Clinical Trial Results with Replication-Competent Adenoviruses in Cancer Patients 337 A. Clinical Trial Results with Wild-Type Adenovirus 337 B. A Novel Staged...

Results from Clinical Trials with cf1520 OnyxOl 5 or CI1042

No maximally tolerated dose or dose-limiting toxicities were identified at doses up to 2xl012 particles administered by intratumoral injection. Flulike symptoms and injection-site pain were the most common associated toxicities 48 . This safety is remarkable given the daily or even twice-daily dosing that was repeated every 1-3 weeks in the head and neck region or pancreas 49 , Intraperitoneal, intraarterial, and intravenous administration were also remarkably well tolerated in general....

Homologous Recombination with Circular Ad Vector Genome Plasmids

Another way to circumvent the need for single cutting sites is obviously to use a circular instead of a linear template for the homologous recombination. However, in that case the selection process will necessarily involve a step of counterselection against the parental Ad plasmid, rendering the selection process more complex than the simple recircularization to be selected for in the systems described above. Two different approaches where the viral backbone is used as a supercoiled circular...

Hybrid Adenoviral Vector Systems

A number of hybrid adenoviral vector systems have been reported in the literature, combining the properties of RV, AAV, and EBV vectors, as well as elements of other Ad serotypes, to enhance the therapeutic efficacy of Ad vectors in vivo. The principal aim of these new hybrid vectors is to overcome the limitations of transient Ad vector retention in infected cells. In addition to the well-documented limitations of Ad vectors Table II , some initially perceived advantageous properties of Ad...

Master Cell Bank

Testing of the cell banks used in adenovirus vector production is of two general types safety testing and characterization. Table I is an overview of the recommended characterizations. The safety testing is intended to demonstrate that the cell bank is free of any detectable microbial contamination including bacterial, fungal, and viral. Sterility testing is a universal requirement for biologies and is set forth in 21 CFR 610.12. Alternative sterility assays validated to be of equal sensitivity...

Formulation and Stability

Process development must determine product stability over a wide range of conditions. The time course of the purification must be scrutinized for excessive run times and delays because rapid processing favors high yields. A well-designed process includes holding points selected such that the viral Figure 7 The most useful method for analyzing both crude and pure samples of adenovirus is anion-exchange HPLC. Pure adenovirus elutes in a nearly symmetrical peak. The adenoviral peak from lysate...

Propagation of Adenoviral Vectors Use of PERC6 Cells

Ledwith, K. Brouwer, S. Manam, R. Vogels, D. Kaslow, D. Zuidgeest, A. J. Bett, L Chen, M. van der Kaaden, S. M. Galloway, R. B. Hill, S. V. Machotka, C. A. Anderson, J. lewis, D. Martinez, J. Lebron, C. Russo, D. Valerio, and A. Bout II. Cells Expressing El of Adenovirus 134 A. Transformation of Cells by El of Adenovirus 134 B. El-Expressing Cell Lines for Adenoviral Vector Production 135 III. PER.C6 Prevents RCA during Vector Production 136 B. PF.R.C6...

Va Rna Genes

Adenovirus encodes one or two VA virus-associated RNAs, depending on serotype, of about 160 nucleotides that are transcribed by host cell RNA polymerase III. VA-RNAi targets the protein kinase named PKR reviewed in 91 . PKR is activated by the presence of low levels of double-stranded RNA, a likely product from symmetrical transcription of the Ad genome. Upon activation, PKR phosphorylates and inactivates eukaryotic initiation factor 2 alpha eIF-2a , thus inhibiting protein synthesis in...

Homologous Recombination with Linear Ad Vector Genome Plasmids

The work by Chartier et al. 60 showed that stable maintenance of plasmids containing the entire Ad viral genome is achieved through the separation of the viral ITRs by the bacterial plasmid backbone, confirming the observations made earlier by Hanahan and Gluzman 64 . This was accomplished by the insertion of the left and right end of the Ad genome in their normal orientation into a colEI-derived bacterial plasmid ppolyll 65 , using conventional cloning techniques. Such a plasmid linearized...

Conclusion

The establishment of hybrid Ad vectors incorporating the advantageous properties of other viruses greatly expands their therapeutic potential. In the early 1990s, after the initial decade of proof-of-concept for Ad-mediated gene therapy, the main focus was on limiting the immunogenicity of the vectors to enhance transgene expression. Further restricting the expression of the highly immunogenic late viral transcripts by E4 deletions or by complete deletion of all viral genes in the gutless...

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...

Clinical Development of CV706 and CV787

CV706 and CV787 are novel therapeutic agents with a novel mechanism of action. As of this writing, a phase I II clinical trial of CV706 for recurrent local prostate cancer has been completed and CV787 has entered a multicenter Phase I II clinical trial for metastatic, hormonal refractory prostate cancer. A brief summary for CV706 trial result and factors impacting clinical efficacy and safety are discussed. A. CV706 Phase I II Trial for Locally Recurrent Prostate Cancer A Phase I trial of CV706...

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...

Testing of Adenoviral Vector Gene Transfer Products FDA Expectations

Pilaro, and Karen D. Weiss II. Manufacturing Control and Product Characterization 616 A. Purity, Safety, and Potcncy 616 B. Regulation of Process as Well as Product 617 C. Current Good Manufacturing Practices 617 III. Development of Recommendations for the Manufacture and Characterization of Adenoviral Vectors 618 IV. Considerations in Manufacturing Adenoviral Vectors 620 A. Components and Characterization 620 A. Standard Operating Procedures 621 B. Quality Assurance...