An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene - PubMed (original) (raw)

Figure 1

HD-leptin construct. (A) The DNA composite fragments of pΔSTK120-HCMV-mOb-BGHpA (≈19.6 kb total size) are from left to right: the left end terminus of Ad5, composed of the ITR sequences and the packaging signal ψ (nucleotides 1–440, solid arrow); the 5,072-bp fragment of hypoxanthine guanine phosphoribosyltransferase (HPRT) (nucleotides 12,373–17,853 in gb:humhprtb, ░⃞); the leptin expression cassette (1,835 bp), composed of the HCMV promoter, the murine leptin cDNA (500 bp) and the bovine growth hormone poly(A) tail (□) (inserted in the complementary orientation); the _Hin_dIII 9063-bp fragment of C346 cosmid (nucleotides 12,421–21,484 in gb:L31948, ░⃞); and the right end terminus of Ad5, composed of the ITR sequence (nucleotides 35,818–35,935). The ITRs are flanked by unique_Pme_I restriction sites used to liberate the vector fragment from the plasmid backbone before the initial transfection into 293-cre4 cells for viral rescue and propagation (released fragment is 16.7 kb). To the right of the vector structures is a representative cesium chloride banded HD-leptin vector stock (see Materials and Methods), at the final stage of band collection. The band is single, compact, and thick. (B) The structure and similarity of HD-leptin vectors (33 kb), which are all tail-to-tail concatamerizations (junction is at the 3′ ITR ends of ΔSTK120-HCMV-mOb-BGHpA), is verified by the restriction enzyme pattern of the three independently rescued viruses. The gel, labeled Vector DNA, shows 0.5 μg of DNA extracted from the HD-leptin viral stock (lane A), Ad-leptin stock (lane B) and the Pme_I cut pΔSTK120-HCMV-mOb-BGHpA (lane C) compared on a 0.5% agarose gel for sizing. Both HD-leptin (33 kb) and Ad-leptin (34 kb) extracted DNA migrate, as expected, between 38.5–29.9 kb, and the cut ΔSTK120-HCMV-mOb-BGHpA (16.7 kb) migrates between 17.1 and 15.0 kb, the smaller band corresponds to the plasmid backbone (2.9 kb), and the faint band in lane A represents the trace amount of the propagated 16.7-kb linearized vector. Structures of ΔSTK120-HCMV-mOb-BGHpA (lane 1) (gel extracted after separation of plasmid backbone by_Pme_I digestion) and the three HD-leptin vectors (lanes 2–4) are compared by restriction analysis, as described in_Materials and Methods. The expected fragment sizes for HD-leptin are: for Asp-718: 15,391-single band (s), 6,296-double band (d), and 2,501-d; _Eag_I: 20,445-s and 6,270/6,266-d;_Fse_I: 16,523/16,458-d; _Hin_dIII: 10,207/10,174-d, 5,845-d, and 454/450-d; _Pac_I: 16,516/16,465-d; _Sma_I: 6,701-d, 5,163-d, 2,180-d, 1,715-s, and 1,589-d, and _Xho_I: 11,833-d, 2,964/2,953-d; and 1,701/1,697-d bp. The expected fragment sizes for ΔSTK120-HCMV-mOb-BGHpA are: for Asp-718: 7,837-s, 6,296-s, and 2,501-s; _Eag_I: 10,364-s and 6,266-s;_Fse_I: 16,458-s and 172-s; _Hin_dIII: 10,174-s, 5848-s, 450-s, and 158-s; _Pac_I: 16,465-s and 165-s; _Sma_I: 6,701-s, 5,163-s, 2,180-s, 1,589-s and 997-s, and _Xho_I: 11833-s, 2964-s, 1697-s and 136-s bp. M1 and M2 are DNA markers (8–48 kb, Bio-Rad, and 1-kb DNA ladder, GIBCO/Life Technologies, Gaithersburg, MD, respectively).