of bulky disease in most patients, Ad.RSVf/c will be most useful as an adjuvant to a surgical debulking 
procedure. 
V.B. Preclinical Data 
V.B.1. Production of Recombinant Adenovirus Containing HSV-Thymidine Kinase 
Recombinant adenoviruses were generated that express the HSVrKgene. Detailed descriptions of the 
methods used to construct the vectors and produce the viruses are provided in Section V.D. 
The recombinant virus used in this protocol, H5.010RSVTK', is based on replication deficient 
serotype 5 adenovirus. In this vector, sequences spanning the El region from 1 to 9.2 map units are 
deleted and replaced with a RSV promoter, HSVTK" cDNA, and SV40 late gene polyadenylation 
sequences. The recombinant virus is partially deleted of E3 sequences. 
V.B. 2. In Vitro Studies Using Human Mesothelioma Cell Lines 
Cell lines: (see Appendix A-Smythe et al. 1994a): We have obtained 5 well characterized cell lines 
derived from human mesothelioma from Dr. Joseph Testa (Fox Chase Cancer Institute) and have 
developed and characterized an additional, highly malignant line (REN) from a patient with an 
inflammatory mesothelioma (Smythe et al., 1994a). All of tumors exhibited appropriate 
immunoperoxidase staining patterns (negative for mucicarmine, CEA and Leu-Mi antibody 
staining). We have focused on two of these lines, the REN line from our lab and the 1-45 line from 
Dr. Testa. 
Replication-deficient adenovirus is an effective vector for transfer of recombinant genetic material 
into mesothelioma cells in culture (see Appendix-Smvthe et al. 1994a1 : To test the ability of 
adenovirus to infect human mesothelioma cell lines, the 1-45 and REN lines were exposed to an 
adenovirus vector containing a marker gene (H5.010RSV/acZ) in cell culture. This construct is a 
type 5 adenoviruses in which significant portions of the El and E3 genes have been deleted, thus 
rendering the virus replication incompetent. The marker gene, lacZ gene (encoding for E. Coli p- 
galactosidase (p— gal) is driven by a Rous Sarcoma virus (RSV) promoter. 
At a MOI of 10 2 viral particle per cell or greater, the H5.010RSV/acZ vector construct transfected 
75-90% of both cell lines with minimal cytotoxicity (cytotoxicity defined by loss of adherence to 
culture plate surface within 24 hours of infection) (see Fig. 1 and Table 1 in Smythe et al., 1994a) 
Expression of this construct was detected by addition of the p-gal substrate, X-gal (5-bromo-4- 
chloro-3-indolyl-b-D-galactosidase), which is easily identified in culture by light microscopy by 
virtue of a staining the infected cells an intense blue color (Fig. 1 in Smythe et al., 1994a). 
Adenovirus-mediated transfer of the HSVTK gene to mesothelioma cell lines sensitizes them to 
ganciclovir treatment in vitro (see Appendix B-Smvthe et al. 1994b): To determine the ability of 
H5.01 0RSV7X to confer sensitivity to ganciclovir (GCV) in a manner similar to that described for 
the retrovirally delivered HSVTK, the human malignant mesothelioma cell lines 1-45 and REN were 
infected by adenoviral vectors carrying either the HS VTK gene driven by an RSV promoter 
(H5.010RSVTK) or a marker gene (H5.010CMV/acZ) at a concentration of 100 particles per cell 
(see Smythe et al., 1994b). Cell lines were stained with a polyclonal antibody against HSVTK 
provided by Dr. William Summers of Yale University. Two days after infection, the HSV77C enzyme 
was expressed strongly in about 40% of cells and to lesser extent in many other cells (see Figure 1, 
in Appendix B). 
To test the functional ability of the viral TK protein, non-infected and infected cells were then plated 
at sparse density and exposed to varying concentrations of GCV in media for 4 days. Viable cell 
growth was then assessed by colorimetric assay measuring viable cell dehydrogenase activity. 
Transduced cells were 10 3 -10 4 times more senstive the toxic effects of GCV than non-transduced or 
H5.010CMV/acZ transduced cells (see Fig. 2 of Appendix B, Smythe et al., 1994b). These data 
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Recombinant DNA Research, Volume 20 
