GTMB 7 - Gene Therapy & Molecular Biology

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Stoll et al: The role of EBV and genomic sequences in gene expressionsequences from Epstein-Barr virus (EBV) that can aid inextrachromosomal plasmid maintenance and expression.Epstein-Barr virus (EBV) is a human herpes virusthat is capable of maintaining its genomeextrachromosomally in dividing primate cells.Maintenance is accomplished by the viral latent origin ofreplication, oriP, and the EBV nuclear antigen 1, EBNA1,which act together to replicate the viral genome and retainit in the nucleus (Yates et al, 1984, 1985; Reisman et al,1985). Plasmids containing EBNA1 and a truncated oriPcarrying only the tandem array of 21 EBNA1 binding sites(family of repeats) from oriP for retention, but lacking theoriP dyad symmetry element for replication, are retainedin the nucleus of the cells, but can replicate efficientlyonly if the plasmid also contains a functional mammalianorigin of replication, such as the 19 kb SERPINA1sequence ( Krysan et al, 1989; Heinzel et al, 1991; Stoll etal, 2001). These same EBV components that providereplication and retention functions are also associated withtranscriptional enhancer and anti-silencing activity(Reisman and Sugden, 1986; Kaneda et al, 2000).Furthermore, in addition to the replication function of thegenomic SERPINA1 sequence demonstrated in ourprevious experiments (Stoll et al, 2001), the full AAT genewas also able to provide more stable expression in vivothan its equivalent cDNA sequence, which may be subjectto silencing.Silencing of cDNA vectors may occur because thetransgenes are often driven by viral promoters. It has beenobserved that many common viral promoters, such asthose from cytomegalovirus (CMV), simian virus 40(SV40), and Rous sarcoma virus (RSV) often exhibitmarkedly decreased activity in mammalian cells in vivowithin a few weeks of transfection, a phenomenon that hasbeen attributed to inhibition by various cytokines (Paillard,1997). Gill (2001) recently demonstrated that the use ofthe cellular elongation factor 1α (EF1α) and ubiquitin C(UbC) mammalian promoters gave increased persistenceand ~10-fold higher expression levels of a luciferasereporter gene in lungs, compared to a control constructthat expressed luciferase from the CMV promoter.Quantitative PCR analysis of plasmid vector in the lungtissue revealed that there were no significant differences inplasmid copy number in the CMV versus EF1α or UbCpromoter vectors.In addition to the reduced transgene silencingobserved when mammalian promoters are used, genomicsequences may provide additional benefits that lead toincreased transgene expression. Studies in transgenic micehave indicated that introns are essential for stable, highlevels of transgene expression. In comparing transgenicmice generated with cDNA constructs versus full genomicsequences, the intronless constructs resulted in a lowerfrequency of transgenic mice expressing rat growthhormone (rGH), mouse metallothionein I (mMTI), orhuman β-globin (hBG) reporter genes, as well asdecreased expression levels in those mice that did haveobservable expression (Brinster et al, 1988). Similarresults have been observed for AAT and β-lactoglobulinexpression constructs in mammary cells of transgenic mice(Whitelaw et al, 1991). It is possible that genomic intronscontain transcriptional enhancer sequences that may act ontheir own to increase transgene expression or may act inconcert with upstream/promoter sequences. These intronicsequences may also act to help the transgene attain anopen chromatin configuration, making it more accessibleto transcription factors. This idea is supported byobservations that deletion of intronic sequences makestransgenes more susceptible to chromosomal positioneffects in vivo than their full genomic counterparts(Webster et al, 1997).Unfortunately, the large size of most full genes oftenprecludes their use in vectors. In order to obtain theexpression advantages of intronic sequences, while stillminimizing transgene size, heterologous introns andgenomic minigenes have been developed. Palmiter (1991)found that including only select introns, specifically thefirst one, in the rGH gene resulted in transgenicfrequencies and expression levels comparable to thoseachieved when the full rGH was used. Heterologousintrons inserted between promoter and cDNA gave similarresults (Palmiter et al, 1991). This strategy has beenapplied to the construction of expression vectors fortherapeutically relevant genes. Miao (2000) constructed ahuman factor IX minigene, which included the ApoEhepatic locus control region (HCR), the hepatocytespecificAAT promoter, and the human factor IX cDNA,with its intron A and 3' untranslated region (UTR). This6.1 kb minigene was reported to provide therapeutic serumlevels of factor IX (0.5 - 2 µg/ml) that were sustained forat least 225 days, whereas a 2.0 kb AAT promoter-factorIX cDNA construct gave transient expression that fell to
Gene Therapy and Molecular Biology Vol 7, page 213heterologous gene, by inserting a human factor IXminigene (Miao et al, 2000) upstream of the first exon inSERPINA1. Here we report the successful reduction in sizeof the AAT gene, without loss of expression, as well as thesuccessful stabilization of factor IX expression in vivowhen expressed from within the AAT gene.II. Materials and methodsA. Vector constructionPlasmids pEF, pEF-AAT, pEF-cAAT, and pcAAT havebeen described previously (Stoll et al, 2001). Removal of theEBV components (EBNA1 and family of repeats) from plasmidpEF-AAT was achieved by digestion with ClaI and vectorreligation, creating the plasmid pAAT.Vectors pcAAT2 and pEFcAAT2 are identical to pcAATand pEF-cAAT, respectively, except that the RSV promoter hasbeen replaced with the endogenous, hepatocyte-specific AATpromoter. 471 bp containing the AAT promoter was PCRamplified from vector pF9 (Sclimenti et al, 2003) and cloned intothe vector pCR2.1 (Invitrogen, Carlsbad, CA), making the vectorpTA-AATpro. From there it was subcloned as an MluI-HinDIIIfragment into the MluI-HinDIII sites in the plasmid pcAAT,replacing the RSV promoter. This step created the vectorpcAAT2. From that plasmid, the 2.1 kb AATpro-cDNA fragmentwas liberated as an XhoI fragment and cloned into the SalI site inplasmid pEF, creating plasmid pEF-cAAT2.Minimization of the AAT genomic sequence began withremoval of some of the flanking sequences. From the 5' upstreamsequence, 3.7 kb was removed by digesting pAAT with ClaI andMfeI and religating the vector, creating the vector pAAT(-5').From the 3' flanking sequence in pAAT, 970 bp was removed bydeleting an AflII-SalI fragment, generating pAAT(-3'). Thisplasmid was further reduced by digestion with ClaI and MfeI andreligation, generating the double deletion plasmid pAATΔ, whichis reduced by a total of 4.7 kb, compared to pAAT.Cloning of the AAT minigene vector was performed asfollows. The AAT cDNA was removed from pcAAT as a 1.3 kbBamHI-PstI fragment, and cloned into the BamHI-PstI sites ofpBCSK+ (Stratagene, La Jolla, CA), creating the vectorpBcAAT. An AflII linker was cloned into its BglI-PstI sites,generating vector pBcAATii. A 1529 bp fragment of AAT 3'sequence was PCR amplified and cloned into vector pCR2.1,creating pTA-AAT3', from which the AAT3' sequence wassubcloned as an EcoRI fragment into the EcoRI sites ofpBcAAT, creating pBcAAT-AAT3'. From this plasmid, a 622 bpAvaI-PstI fragment was cloned into the AvaI-PstI sites ofpBcAATii, generating the vector pBcAATiii. Next, a 499 bp PstIfragment from pBcAAT-AAT3' was cloned into pBcAATiii,creating plasmid pBcAATiv. A 552 bp StuI-AflII fragment frompBcAAT-AAT3' was cloned into pBcAATiv, generatingpBcAATv. Next, a 771 bp BamHI fragment from pAAT wascloned into the BamHI site of pBcAATv, to create the plasmidpBcAATvi. The last step in creating the minigene was to clonethe 2.9 kb minigene from pBcAATvi, as an AflII-SnaBIfragment, into the AflII-SnaBI sites of vector pAAT. The result isvector pAATmg, which lacks introns 2-4, resulting in a totalreduction of 3.5 kb in gene size.Vector pAAT-fIXmg was constructed as follows. A BglIIlinker was ligated into the SacI site of pF9, generating pfIXmgi.Next, a 2.6 kb fragment of the AAT first intron was PCRamplified, digested with BamHI and BglII, and cloned into theBglII site of pfIXmgi, creating pfIXmgii. The AAT promoter wasreleased from pTA-AATpro as a SapI-ClaI fragment and clonedinto the SapI-ClaI sites of pfIXmgii, generating the vectorpfIXmgiii. Finally, the 7.9 kb fIXmg was released from plasmidpfIXmgiii by digestion with BglII and cloned into the BglII sitesin plasmid pAAT, resulting in the final vector pAAT-fIXmg.B. In vivo delivery and analysis.C57BL/6 mice were injected over a period of 6 – 9 s with25 µg of DNA in 1.8 ml of 0.9% NaCl, by hydrodynamic tailveininjection (Liu et al, 1999; Zhang et al, 1999). Serumsamples were periodically obtained by retro-orbital bleed. Allanimal procedures were performed under the guidelines set forthby Stanford University and the National Institutes of Health. Thesamples were assayed by a polyclonal capture ELISA assay tomeasure serum AAT (Song et al, 1998; Yant et al, 2000) and/orserum fIX (Sclimenti et al, 2003). Some animals were subjectedto partial, surgical hepatectomy (PH) (Chen et al, 2001) and thenallowed to recover. Serum samples were obtained periodically byretro-orbital bleed and analyzed by ELISA. At 51 d post-PH,treated and control animals were sacrificed and the livers wereremoved for analysis of plasmid DNA. The liver tissue that wasremoved during PH surgery was also subjected to analysis. Livertissue was diced and total DNA was prepared using the Bloodand Cell Culture DNA Maxi kit (Qiagen, Valencia, CA).Southern blot analysis was performed as follows. DNA wasdigested with ScaI and separated on a 0.65% agarose gel. The gelwas depurinated in 0.25 M HCl, denatured in 0.5 M NaOH,neutralized in 0.5M Tris-HCl (pH 7.0) and transferred to an S&SNytran blotting membrane (Schleicher & Shuell, Keene, NH) in20X SSC transfer buffer. The membrane was probed with a 484bp fragment from the hygromycin resistance gene common toboth plasmids, labeled with alkaline phosphatase enzyme, usingthe AlkPhos Direct DNA labeling kit (Amersham PharmaciaBiotech). Hybridization occurred at 55°C for 16 h inhybridization buffer provided with the kit. Membranes werewashed according to protocol and incubated with CDP-StarDetection Reagent for 4 min. The membrane was then exposed toHyperfilm ECL (Amersham Pharmacia Biotech). Southern blotquantification was performed using Kodak 1D software.III. ResultsA. Role of EBV sequences inextrachromosomal plasmid expression in vivoOur previous research showed that the plasmid pEF-AAT, bearing the 19 kb SERPINA1 genomic locusencoding human α 1 -antitrypsin on a vector with the EBVEBNA1 and family of repeats retention sequences, wascapable of maintaining long-term high expression levelsafter transfection into mouse liver (Stoll et al, 2001). Wefirst wanted to determine what role the EBV componentson this plasmid played in sustaining these high expressionlevels. EBNA1 and the EBV family of repeats wereremoved, creating plasmid pAAT. Naked plasmid DNA ofpEF-AAT and pAAT, along with pEF-cAAT and pcAATcontrol cDNA plasmids with and without EBV retentionsequences (Figure 1A), were injected into the mouse livervia hydrodynamic tail-vein injection (Liu et al, 1999;Zhang et al, 2000). Serum AAT levels were determined byELISA. The results are shown in Figure 1B. Bothgenomic vectors were able to provide expression levels~1000-fold greater than the cDNA control vectors, whichfell to ≤ 100 ng/ml within 3 weeks post-injection. Whilethe non-EBV plasmid pAAT was still capable of providingstable, high-levels of expression of AAT (151 µg/ml at 6months), the levels provided by pEF-AAT were ~4-fold213
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