Replication-deficient adenovirus is an effective vector for transfer of recombinant genetic material 
into mesothelioma tumor cells in vivo (see Appendix A-Smythe et al. 1994a) : To test the ability of 
adenovirus to transfer genetic material to tumor cells in an in vivo setting, SCID mice were injected 
intraperitoneally with H5. 01 OCMV/acZ following establishment of intraperitoneal REN tumor. At 
necropsy, all animals had nodular growth of REN xenograft tumor at scattered sites throughout the 
abdominal cavity. Microscopic examination of X-gal-stained frozen sections of tumor and organs in 
animals administered 1.5 X 10 11 particles in 0.2 cc showed uniform and extensive transfer of the 
lacZ gene with expression of p-gal in all areas of the normal mouse mesothelium covering the 
surfaces of abdominal organs (See Fig. 2, large arrows-Smythe et al., 1994a). In addition, 
extensive transfer of the lacZ gene was noted in all tumor nodules sectioned, as well as in smaller 
areas of tumor found incidentally adherent to abdominal organs as these were examined. As might be 
expected, surfaces of the tumor nodules exhibited extensive gene transfer, however, much deeper 
regions of the nodules also exhibited transfer of the lacZ gene as evidenced by positively staining 
areas (Fig. 2, arrowheads-Smythe et al., 1994a). No positive staining of within the abdomen, other 
than mesothelium, were noted, except for some scattered cells within the liver. Organs removed 
from a sham-infected animal exhibited neither intrinsic tissue p-gal activity nor evidence of false- 
positive X-gal staining artifact. 
Subcutaneo us xenoorafted human mesothelioma tumors generated from cells transfected with 
H5.01 ORSV 7~/< are completely eliminated with ganciclovir treatment in vivo (see Appendix C- 
Smvthe et al. 1994c1 : To determine if GCV could induce cell death in a tumor growing in an 
experimental animal, 20 million 1-45 cells infected in vitro with either H5.010RSVTK' or 
H5. 01 OCMV/acZ at an MOI of 100, were injected into subcutaneous flank tissues (R flank=/acZ, L 
flank=HSV77<) of three Severe Combined Immune Deficient mice. By day three, the animals had 
developed bilateral tumors of 6-8 mm. diameter and began treatment with 5 mg of intraperitoneal 
GCV/day. By day ten, left flank tumors in all animals (HS V77< side) were undetectable, with 
continued growth of the tumors derived from H5. 01 OCMV/acZ infected cells (Fig. 1-Smythe et al. 
1994c- Appendix C). Animals followed an additional two weeks after the cessation of GCV treatment 
showed no recurrence of tumor. 
Established Intraperitoneal tumors were erradicated in SCID mice using the H5.010RSVT/<'/GCV 
System (see Appendix C-Smvthe et al. 1994c1 : Based on these results, we designed experiments to 
evaluate in situ treatment of pre-existing mesothelioma in our model that approximated human 
disease. Forty million REN cells were injected into the peritoneal cavity of 30 SCID mice. Five days 
later, when macroscopic tumor nodules of 1-2 mm were present, we injected a control vector 
(H5. 01 OCMV/acZ ) into 6 mice, our therapeutic gene construct (H5.010RSV7K) into 15 mice, and 
diluent (sham-infected) into 9 mice via the intraperitoneal (IP) route. The dose of virus was 6 x 
10 9 pfu. Animals were treated two days later with IP GCV at a dose of 5 mg/day (200 mg/kg) or 
saline for seven days. One month after initial tumor inoculation, animals were euthanized and 
carefully necropsied. There was no grossly detectable disease in the 10 mice of the 
H5.010RSV7K7GCV group (Fig. 1, panels B,D, and F-previous page or Fig. 3B,D,F in Smythe et al., 
1993c) with the exception of one animal who had a solitary 3 mm macroscopic tumor nodule. In 
contrast, all but one of the 20 animals in the other groups had an extensive burden of 
intraperitoneal tumor (Table 1 and Figs. 2 & 3 in Smythe et al., 1993c). Scattered tumor nodules 
of up to 1.5 cm in diameter were noted in these animals, many of which were partially obstructing 
the bowel or biliary tree (Fig. 1 -previous page or Fig. 3 A,C,E in Smythe et al., 1993c). In 
addition to macroscopic disease (found primarily in the upper abdomen; pancreatic mesentery, 
region of the porta hepatis and diaphragm, all control animals exhibited microscopic tumor 
uniformly throughout the upper abdomen (Fig. 2D,E,F-Smythe et al., 1993c). Microscopic 
examination of intraabdominal organs and tissues removed from animals in the HSVTK/GCV group by 
conventional H&E staining of paraffin sections or a more sensitive staining technique utilizing an 
antibody against the human HLA-Class 1 shared determinant (Table 1 and Fig. 2D,E,F-Smythe et al., 
1993c) showed evidence of tumor in only two of the ten mice. In one animal (who also had the 
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