FIGURES AND TABLES

Figure 1.

Comparison of serum hAAT expression profiles obtained with different promoters. Mice (n = 5 mice per group) were injected at day 0 with 25 g plasmid carrying the hAAT gene directed by the indicated promoters: CMV (cytomegalovirus), EF1 (elongation factor 1), RSV (Rous sarcoma virus LTR), MTH (metallothionein), PGK (phosphoglycerate kinase), TTR (transthyretin), MoU3 (Moloney murine leukemia virus U3 region), eG3xAlb (albumin with upstream enhancer), DHFR (dihydrofolate reductase), Alb (albumin), and No (no promoter). Expression from the MTH promoter was induced in one of two groups injected with pBS.MTH-hAAT by adding ZnSO₄ (indicated by +) to the drinking water (25 mM final concentration). For all time points, the serum hAAT level relative to that of CMV is shown. Serum hAAT levels for pBS.CMV-hAAT were 5.6 10⁵, 3.6 10⁵, and 9.9 10² ng/ml at day 1, 7, and 122, respectively. For all promoters, hAAT expression levels did not change significantly after day 32; data from day 122 are shown. Mean relative values standard deviation are shown. The relative strength of all promoters, determined by serum hAAT levels at day 7 after injection relative to levels obtained with pBS.CMV-hAAT, is shown in the gray box. Based on the distribution of relative serum hAAT levels at day 7, CMV, EF1, MTH, PGK, TTR, DHFR, and Alb promoters (indicated by black bars) were selected for further studies. Serum hAAT was detected for pBS.No-hAAT only at day 1 after injection. Further details on the tested promoters are given in Table 2. ND, not detectable.

Figure 2.

Sleeping Beauty transposon–transposase vectors induce persistent transgene expression in mice. (A) Schematic representation of injected SB-based HITT vectors. Plasmids carry the transposon-embedded hAAT and flanking transposase expression cassettes in the same orientation. Promoters indicated by black, gray, and white boxes (strong, weak, and intermediate promoters, respectively) were inserted into pT/hAAT.PIL-SB carrying the promoter insertion linker (PIL) upstream of the transposase coding region. In pT/hAAT.CMV-SB.ds, the transposase cassette was inserted downstream (ds) of the RSV-hAAT transposon. SB, transposase; IR, inverted repeat; pA, poly(A) sequence. (B) Patterns of long-term hAAT expression after administration of SB HITT vectors. Equal molar amounts of indicated vectors were administered to mouse livers (n = 4–5 mice per group) by hydrodynamic injection of 30 g HITT-vector plasmid or 24 g pT/hAAT into tail veins. The concentration of serum hAAT was followed for 79 days after injection. All HITT vectors, except those with CMV-directed transposase production, induced levels of persistent expression that were improved over the basic level obtained with pT/hAAT. Promoters support transposition and establishment of stable gene expression according to the following hierarchy: PGK > MTH(-Zn) > MTH(+Zn) > EF1 > TTR > Alb > DHFR > PIL > CMV > CMV(ds). For details on promoters, see legend to Fig. 1 and Table 2. Mean values standard deviation are shown. (C) Transposition from HITT vectors varies with promoter strength. By relating relative promoter strength (Fig. 1, bottom) with level of persistent hAAT expression after HITT-vector injection (B) an optimal window for transposase expression could be defined.

Figure 3.

Improved transgene transposition with SB HITT vectors. Serum hAAT levels in mice receiving 24 g pT/hAAT, 30 g pT/hAAT.PGK-SB, or 30 g pT/hAAT.MTH-SB (data from Fig. 2C) compared with 24 g pT/hAAT + 20 g pCMV-SB (1:1 ratio based on hAAT and SB transposase gene copy numbers) and 24 g pT/hAAT + 0.8 g pCMV-SB (25:1). When necessary, pUC19 "stuffer" DNA was included to ensure that each mouse was injected with the same total amount of DNA (44 g). Gene copy numbers determined amounts of DNA in the two-component injections; hAAT and SB transposase genes were injected in equal copy numbers for HITT vector and 1:1 two-component injections. Average values standard deviation are shown. n = 5 mice per group.

Figure 4.

Persistence of transgene expression is induced by transposase-dependent gene insertion. (A) Comparison of long-term stability of hAAT expression in mice (n = 4 mice per group) injected with SB HITT vectors harboring active (SB) or inactive (mSB) transposase. Animals received 30 g of HITT vectors indicated. Mean values standard deviation are shown. (B) Serum hAAT levels after induced liver cell division in HITT-vector-treated mice. Mice (n = 5 per group) treated with 24 g pT/hAAT, 30 g pT/hAAT.PGK-SB, or 30 g pT/hAAT.MTH-SB either were subjected to partial hepatectomy (PHx) at day 70 after DNA injection (n = 2, symbols marked with +) to facilitate loss of extrachromosomal DNA or did not undergo surgery (n = 3). The persistent high serum hAAT levels even after partial hepatectomy demonstrate that integrated copies of the hAAT gene and not putative episomal hAAT-encoding DNA account for the persistence of gene expression. In the absence of transposase (pT/hAAT), we could detect a significant reduction in serum hAAT, indicating loss of a significant portion of episomal pT/hAAT due to hepatocyte cell division. Mean values standard deviation are shown for groups that did not undergo surgery, mean values are shown for pairs of animals subjected to partial hepatectomy. Black arrow indicates time of partial hepatectomy.

Figure 5.

Effects of the orientation of expression cassettes on SB HITT-vector function. HITT vectors carrying the transposase expression cassette in orientation opposite to that of the transposon-embedded hAAT cDNA (constructs labeled with bid for "bidirectional") were compared to constructs with both expression cassettes oriented in the same direction. Plasmids (30 g) were injected into the tail vein of mice and the serum hAAT concentration was monitored over time. (A) Comparison of HITT vectors with CMV-, Alb-, and PIL-directed transposase expression (n = 5). The expression profiles of the "unidirectional" vectors (indicated with large symbols), presented previously in Fig. 2B, are included for direct comparison. (B) Comparison of HITT-vector-based PGK- and MTH-directed transposase production (n = 4–5). Profiles of pT/hAAT.PGK-SB and pT/hAAT.MTH-SB, shown earlier in Fig. 4A, are included for easy comparison. Mean values standard deviation are shown.

Figure 6.

Transposition from SB HITT vectors induces long-term expression of hFIX. Animals received equal copy numbers of the hFIX-tagged transposon: 35 g pT/EF1-hFIX + 7 g pUC19 or 42 g pT/EF1-hFIX-derived HITT vector plasmid harboring different transposase expression cassettes as indicated were injected into the mouse tail vein (n = 5 per group of mice). Serum hFIX concentration was monitored over time by serum ELISA. Mean values standard deviation are shown.

Table 1 - Partial phenotypic correction in hemophilia B mice treated once with HITT-vector plasmids.