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Comparative Study
. 2000 Feb 1;97(3):1002-7.
doi: 10.1073/pnas.97.3.1002.

Optimization of the helper-dependent adenovirus system for production and potency in vivo

V Sandig  1 , R Youil, A J Bett, L L Franlin, M Oshima, D Maione, F Wang, M L Metzker, R Savino, C T Caskey
Affiliations
Free PMC article
Comparative Study

Optimization of the helper-dependent adenovirus system for production and potency in vivo

V Sandig et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Helper-dependent (HD) adenoviral vectors devoid of all viral coding sequences provide for safe and highly efficient gene transfer with long-lasting transgene expression. High titer stocks of HD vectors can be generated by using the cre-recombinase system. However, we have encountered difficulties with this system, including rearranged HD vectors and variable efficiency of HD vector rescue. These problems represent a major hindrance, particularly with regard to large-scale production. To overcome these limitations, we have modified the system in two ways: We constructed a new helper virus with a modified packaging signal and enhanced growth characteristics. We also redesigned the vector backbones by including noncoding adenovirus sequences adjacent to the right inverted terminal repeat and by incorporated a number of different segments of noncoding DNA of human origin as "stuffer." Comparison of these vectors showed that the nature of the stuffer sequence affects replication of the HD vector. Optimization of the system resulted in a more robust and consistent production of HD vectors with low helper contamination and high in vivo potency.

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Figures

Figure 1
Figure 1
Rearrangement of HD vectors as a result of homologous recombination. Structure of the helper AdC8cluc, the HD vector STK120vegf145, and a mutant HD vector generated during amplification. ░⃞, adenovirus-derived sequences; formula image, hvegf145-cDNA and bGH poly A; ◂, loxP-site; arrow, CMV promoter; —, CMV intron A.
Figure 2
Figure 2
Competition between HD vectors containing different fragments from the right end of Ad5. (A) Structure of STK120 gfp (STK) and its derivatives containing 400 bp STKgfpE4p (STKE4p) or 3,135 bp STKgfpE4 (STKE4) of the Ad5 3′ end. Fragment sizes for BamHI and BglII restriction digests are shown. Discriminating fragments are marked in bold. (B) P33-labeled restriction fragments of purified HD vectors resulting from cotransfections of two HD vectors in equimolar ratios. Five or three preparations were generated by independent transfection, amplification to passage 6, and single-step gradient banding. The respective plasmids digested with PmeI and BamHI or PmeI and BglII are shown for comparison. *, plasmid backbone.
Figure 3
Figure 3
Effect of Ad5 3′ end sequences on replication or packaging. Cells were infected with helper 14 and either STKgfp or STKgfpE4p and were harvested at the indicated time points. Genomic DNA was extracted and analyzed for helper and HD vector sequences by TaqMan PCR. Changes in the ratio of HD vector and helper DNA over time indicate the replication of HD vectors relative to that of the helper. After a single-step gradient, the ratio between purified HD vector and helper was 14:1 (STKgfp) and 50:1(STKgfpE4p)
Figure 4
Figure 4
Competition of HD vectors with altered stuffer sequences. (A) Structure of new HD vectors. To build HD vectors of group A, a 8.4-kb bp fragment between the unique Swa and Eag sites in STK120 was deleted. The following new sequences were introduced together with 400 bp of the right Ad5 terminus: fragment AFO (11, 3 kb, locus AF011889) and HSU (9, 3 kb, locus HSU 71148) located on Xq28 and ER [5, 4 + 5, 8 kb, estrogen receptor β region, Merck sequencing group (M.L.M., unpublished work)] located on 14q21. Vectors of group B contain 19 kb of sequence from the genomic region HUMDXS455A (cosmid C346), part of which is also present in pSTK120. This sequence was interrupted into four segments of ≈4.5 kb each. Segments were cloned in reversed orientation compared with their original position in the genome. Unique restriction sites (as shown) were created at the junction between fragments to allow for insertion of therapeutic genes. (B) P33-labeled HindIII restriction fragments of purified HD vectors. Vectors were generated individually or as mixtures by cotansfection of equimolar ratios of the respective plasmids. All amplifications are carried out with helper H14. Vectors carrying the gfp expression cassette were compared within and between groups A and B (MixA, B, A/B). One preferred vector of group B (C4HSU) was compared with the STK120-based vector (STK), both carrying the EPO expression cassette (MixC). Representative bands of each vector in group A are marked in the individual preparations and in the Mix.
Figure 5
Figure 5
Effect of stuffer sequences on replication. 293cre415 cells were infected with helper 14 and purified HD vectors at a ratio of 3:1 and were harvested at the indicated time points. Genomic DNA was extracted and analyzed for helper and HD vector sequences by real time PCR.
Figure 6
Figure 6
In vivo comparison between HD vectors C4HSU-mEPO, C4AFO-mEPO, and STK120-mEPO. Groups of female BALB/c mice (n = 5) were infused in the tail vein with 1 × 106 infectious units of different HD-mEPO vectors. Blood samples were taken at the time points indicated and were analyzed for hematocrit levels. The results obtained for three independent stocks of STK120-mEPO prepared by different researchers are shown.
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