and post gene therapy for this purpose. The interpretation of lymphocyte testing may be confounded by a 
clinical requirement for the use of corticosteroids. These tests may provide evidence for the 
development of an immune response to following adenovirus transduction and will play an important 
role in the design of future adenovirus vector clinical trials. The immune response to the vector may 
limit the efficacy of serial treatments, may lessen the duration of transgene expression, or may 
contribute to local, dose limiting, inflammation [52]. These problems may be overcome by the use of 
newer adenoviral vectors that are currently under development [53]. 
III.F Ethics Advisory Board 
An Ethics Advisory Board within the Institute for Human Gene Therapy will undertake the development 
of guidelines and recommendations with respect to all areas of patient participation in gene therapy 
research. This is described in detail in Appendix VI, F, page 33. 
IV. Isolation and Production of H5.01 ORSVT/C Adenovirus 
IV. A Construction of recombinant adenoviral vector-H5.010RSVT/C 
The strategy for constructing and producing this recombinant adenovirus is summarized as follows. 
Step 1. Construction of pAdBglll. The purpose of this step was to construct a plasmid containing the 5' 
portion of Ad 5 with deletion of El sequences and a unique cloning site. The plasmid pEHX-L3 contains 
sequences from Ad5 spanning map units 1 to 16.1. pEHX-L3 was digested with EcoRI and Bglll and a 5.2 
kb fragment isolated, which contains the adenoviral sequences from map units 9.2-16.1 and the plasmid 
backbone (derived from pAT 153). The adenoviral sequences from map unit 0-1 which contains the 5’ 
inverted terminal repeat, origin of replication and encapsidation signal were amplified from the 
original pEHX-L3 using PCR to insert a Nhe I site immediately downstream of the EcoR I site, and a Bgl 
II site at the 3’ end. This PCR fragment and the EcoR l/Bgl II 5.2 kb fragment were ligated to produce 
the plasmid pAdBglll. 
Step 2. Construction of pAd.RS V7X. The RSV promoter, the HSV TK gene and the SV40 polyadenylation 
signal were cloned into the Bglll site of AdBglll. The source of the RSV promoter and the SV40 
polyadenylation signal was the commercially available plasmid pREP8 (InVitrogen). pREP8 contains 
the RSV promoter and SV40 polyadenylation signal flanking a small multiple cloning site. pREP8 was 
cut with Not I and Kpn I to remove the Nhel site. The ends were blunted, ligated to Not I linkers and 
excess linkers cleaved. The resultant plasmid was ligated (closed). The RSV-MCS-SV40 poly A was 
removed from this plasmid by digesting with Nru I and Xba I. This MCS in this cassette contains a 
unique Not I, Bam HI and Xho I site. Ends were blunted and Bgl II linkers added. This fragment was then 
cloned into pAdBglll and designated pAdRSV4. The HSVf/r gene was isolated from the plasmid 
pL(X)RNL.HSV//r (gift of XO Breakfield). pL(X)RNL.HSVf/c was cut with Bgl II and Bam HI to remove 
the HSV77C fragment The Bgl II digestion results in removal of a large portion of the 5' untranslated 
region. The fragment was gel isolated and cloned into Bam HI cut pAdRSV4. Recombinant plasmids in 
which the HSVf/rgene was correctly inserted were amplified and designated pAdRS Vtk (this adenovirus 
has been renamed, H5.010RSVTK). The plasmid DNA was linearized with Nhe I prior to generation of 
adenoviral recombinants. 
IV. B Generation of Recombinant H5.010RSV77C Virus 
The recombinant tk adenovirus was constructed from the plasmid pAdRS V//c and modified adenovirus 
type 5 (Ad 5), sub 360, in which a small portion of the E3 gene is deleted. pAdRS \/tk was linearized by 
Nhe I and co-transfected with the large fragment of Xbal/CIa I cut sub 360 into 293 cells (a human 
kidney cell line containing a functional Ela gene that provides a trans-acting Ela protein) to allow 
homologous recombination to occur, followed by replication and encapsidation of recombinant 
adenoviral DNA into infectious virions and formation of plaques. Individual plaques were isolated and 
amplified in 293 cells, viral DNA was isolated, and recombinant adenoviral plaques containing the 
human tk cDNA were identified by restriction cleavage and Southern blot analysis. Three of the positive 
Recombinant DNA Research, Volume 20 
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