GTMB 7 - Gene Therapy & Molecular Biology

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Kren et al: Hepatocyte-targeted delivery of Sleeping BeautyAn ampicon of predicted size was obtained when bothprimers corresponded to the GFP coding region, but noamplification product was observed when the sense primerwas upstream to the direct repeat (data not shown). Theresult indicated that spontaneous integration of the plasmidwas rare or absent, strongly suggesting that the integrationhad occurred at the transposon DR.IV. DiscussionWe have shown that a single cis transposon plasmidthat expresses SB and carries a transgene can effectivelypromote long-term gene expression in the liver of miceand rats. Using hepatocyte-targeted in vivo delivery, thecis transposon system was almost 2-fold more efficient byPCR and western blot analysis than the SB transposasedelivered in trans. This finding is in contrast to a recentstudy using hydrodynamic delivery of a cis SB transposonin a murine model of tyrosinemia type I (Montini et al,2002). The authors reported reduced transposition usingthe cis construct, and concluded that this had resulted fromoverproduction of the transposase in mouse liver (Yant etal, 2000, 2002; Montini et al, 2002). Therefore, wecompared the efficiency of the cis and the trans systemsby using equal amounts of the transposase and transposonplasmid vectors when co-delivering the two plasmids intrans. In fact, both systems produced similar levels oftransposase protein immediately after transfection,suggesting that inhibition by transposase overproductionwould be equivalent. In contrast, other mouse studies(Yant et al, 2000, 2002; Montini et al, 2002) used a 1 to 25ratio of transposase construct to transposon. Interestingly,the optimal transposon/transposase ratio appears to beinfluenced by the amount of transposon (Geurts et al,2003). The lower doses were optimal at a 1:3 transposonto transposase construct ratio, while increasing thetransposon levels 5-fold decreased the optimal ratio to1:0.2. Thus, the reduced amounts of transposon in ourstudy may, in part, account for the observed increasedtransposition in cis. Additionally, the more rapid loss ofthe cis construct may have reduced levels of transposase,thereby increasing transposition. By targeted delivery, thecis plasmid showed greater efficacy for GFP transpositionin mice. A potential advantage of using the cis system isthat the transposition can result in self-destruction of thecis plasmid, eliminating the possibility of repeatedtransposition from the effect of any persisting SBexpression.In the present study, the DNA delivery systemprovides a potentially useful application to human trials. Inprevious studies, naked plasmids were delivered to theliver by rapid high volume intravenous administration thatcauses transient congestive heart failure and hepatic stasis,thereby enhancing DNA uptake by liver cells (Budker etal, 2000). This method, although useful in animal studies,is unlikely to find clinical application. In contrast, weachieved targeted delivery of DNA to the liver viahepatocyte-specific ASGR-mediated endocytosis (Wu andWu, 1998; Wu et al, 2002). The fate of plasmid DNAdelivered to the murine liver by the hydrodynamic methoddiffers dramatically from that with PEI (Oh et al, 2001).Using the branched 25 kDa polycation, 50% of theplasmid initially delivered to the liver was still present upto 10 days after transfer. In contrast, 50% of the nakedplasmid DNA delivered to the liver was lost within 15 minof administration, and there was no detectable plasmid at 3days. Although delivery of branched PEI to the lung hasbeen associated with a systemic immune response(Regnstrom et al, 2003), branched PEI-DNA complexesshowed no significant liver toxicity (Oh et al, 2001).Moreover, it efficiently transfects quiescent cells such ashepatocytes (Pollard et al, 1998), protects the DNA fromnuclease degradation (Boussif et al, 1995), and promotesefficient endosomal disruption (Behr, 1997). L-PEI mayalso enhance nuclear uptake of DNA by binding to alectin-like protein with galactose specificity in the nuclearpore complex (Klink et al, 2001). Finally, in contrast tocationic lipids, PEI does not appear to inhibit transgeneexpression (Pollard et al, 1998). Safety, efficacy andhepatocyte-specificity of this endocytosis-based DNAdelivery system makes it attractive for potential use ingene therapy.This study is the first report of transposon-mediatedgene transfer in a mammal other than the mouse. In the ratliver, a single dose of the cis transposon, or pT/GFP aloneresulted in stable transgene expression, althoughsignificantly less homogeneous than in mice. For the cisplasmid, the expression observed in the intact liver wassimilar to that after regeneration post-70% PH. In contrastto the results reported with transposon delivery to themouse liver using adenovectors, PH in the rats markedlyreduced the transgene content and expression in theanimals that received pT/GFP alone (Yant et al, 2002).One explanation for this finding is the differentregenerative response of hepatocytes post-PH in the twospecies (Fausto, 2000; Higgins and Anderson, 1931). Also,there is increased persistence of the adenoviral vectorsrelative to plasmids during cell cycling in vivo (Ehrhardt etal, 2003). Finally, the method of DNA delivery may havecontributed to the observed differences.The finding of small clusters of cells expressing GFPafter PH in the cis transposon rats suggested that theintegration event preceded hepatocyte replication.Integration of the transgene was confirmed by Southernblot analysis and like the mouse studies (Dupuy et al,2001; Fischer et al, 2001; Horie et al, 2001; Yant et al,2000, 2002) SB-mediated gene transfer in rats alsooccurred randomly within the genome. Interestingly, onaverage a single copy of the transposon was observed perdiploid liver genome when clonal selection for transgeneexpression occurs in vivo (Montini et al, 2002), yet singlecopies of a transposon were not associated with transgeneexpression in other in vivo mouse systems (Dupuy et al,2002; Horie et al, 2001). Thus, the observed transgeneexpression may underestimate the overall transpositionfrequency. Expression of randomly inserted transgenes canbe variable because of the known positional effects (Ivicset al, 1997; Izsvák et al, 2000; Yant et al, 2000, 2002;Dupuy et al, 2001, 2002; Horie et al, 2001; Montini et al,2002). Insertion of insulator sequences flanking thetransgene carried by transposons might abrogate thepositional variation of transgene expression and time-236
Gene Therapy and Molecular Biology Vol 7, page 237related gene silencing (Pikaart et al, 1998). The efficiencyof transposition by the cis transposon will most likely beincreased using different promoters to regulate transposaseexpression (Mikkelsen et al, 2003).In summary, our data indicate that by using areceptor-mediated DNA delivery system and equivalentinitial levels of transposase expression, the cis delivery oftransposons is more efficient than trans for transgeneintegration into the liver. Furthermore, we havedemonstrated that the combination of a cis construct and anonviral DNA delivery system could achieve stabletransgene expression at levels required to potentially treatmany inherited metabolic disorders of the liver. SBpromises to play an important role in the gene therapy ofhuman genetic diseases.AcknowledgmentsWe thank Phillip Y.P. Wong, L. Xiaoming Ma, JoelFrandsen and Stefan Kren for excellent technicalassistance. This work was supported by National Institutesof Health grants P01 HD32652 to B.T.K. and P.B.H.,RO1-DK46057 to J.RC., and P01-HL65578 and P01-HL55552 to C.J.S.ReferencesBandyopadhyay P, Kren BT, Ma X and Steer CJ (1998)Enhanced gene transfer into HuH-7 cells and primary rathepatocytes using targeted liposomes and polyethylenimine.BioTechniques 25, 282-292.Behr JP (1997) The proton sponge: a trick to enter cells theviruses did not exploit. Chimia 51, 34-36.Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D,Demeneix B and Behr JP (1995) A versatile vector for geneand oligonucleotide transfer into cells in culture and in vivo:polyethylenimine. Proc Natl Acad Sci USA 92, 7297-7301.Budker V, Budker T, Zhang G, Subbotin V, Loomis A and WolffJA (2000) Hypothesis: naked plasmid DNA is taken up bycells in vivo by a receptor-mediated process. J Gene Med 2,76-88.Chen ZY, Yant SR, He CY, Meuse L, Shen S and Kay MA(2001) Linear DNAs concatemerize in vivo and result insustained transgene expression in mouse liver. Mol Ther 3,403-410.Cui Z, Geurts AM, Liu G, Kaufman CD and Hackett PB (2002)Structure-function analysis of the inverted terminal repeats ofthe Sleeping Beauty transposon. J Mol Biol 318, 1221-1235.Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, MarkleyKM, Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn Sand Largaespada DA (2002) Mammalian germ-linetransgenesis by transposition. Proc Natl Acad Sci U S A 99,4495-4499.Dupuy AJ, Fritz S and Largaespada DA (2001) Transpositionand gene disruption in the male germline of the mouse.Genesis 30, 82-88.Ehrhardt A and Kay MA (2002) A new adenoviral helperdependentvector results in long-term therapeutic levels ofhuman coagulation factor IX at low doses in vivo. Blood 99,3923-3930.Ehrhardt A, Xu H and Kay MA (2003) Episomal persistence ofrecombinant adenoviral vector genomes during the cell cyclein vivo. J Virol 77, 7689-7695.Fausto N (2000) Liver regeneration. J Hepatol 32, 19-31.Fischer SEJ, Wienholds E and Plasterk RHA (2001) Regulatedtransposition of a fish transposon in the mouse germ line.Proc Natl Acad Sci U S A 98, 6759-6764.Follenzi A, Sabatino G, Lombardo A, Boccaccio C and Naldini L(2002) Efficient gene delivery and targeted expression tohepatocytes in vivo by improved lentiviral vectors. HumGene Ther 13, 243-260.Geurts AM, Yang Y, Clark KJ, Liu G, Cui Z, Dupuy AJ, Bell JB,Largaespada DA and Hackett PB (2003) Gene transfer intogenomes of human cells by the sleeping beauty transposonsystem. Mol Ther 8, 108-117.Hara T, Tan Y and Huang L (1997) In vivo gene delivery to theliver using reconstituted chylomicron remnants as a novelnonviral vector. Proc Natl Acad Sci USA 94, 14547-14552.Harui A, Suzuki S, Kochanek S and Mitani K (1999) Frequencyand stability of chromosomal integration of adenovirusvectors. J Virol 73, 6141-6146.Higgins GM and Anderson RM (1931) Experimental pathologyof the liver. I. Restoration of the liver of the white ratfollowing partial surgical removal. Arch Pathol 12, 186-202.Hillgenberg M, Schnieders F, Loser P and Strauss M (2001)System for efficient helper-dependent minimal adenovirusconstruction and rescue. Hum Gene Ther 12, 643-657.Horie K, Kuroiwa A, Ikawa M, Okabe M, Kondoh G, Matsuda Yand Takeda J (2001) Efficient chromosomal transposition ofa Tc1/mariner- like transposon Sleeping Beauty in mice.Proc Natl Acad Sci U S A 98, 9191-9196.Ivics Z, Hackett PB, Plasterk RH and Izsvák Z (1997) Molecularreconstruction of Sleeping Beauty, a Tc1-like transposonfrom fish, and its transposition in human cells. Cell 91, 501-510.Izsvák Z, Ivics Z and Plasterk RH (2000) Sleeping Beauty, awide host-range transposon vector for genetic transformationin vertebrates. J Mol Biol 302, 93-102.Johnson AD and Krieg PA (1994) pXeX, a vector for efficientexpression of cloned sequences in Xenopus embryos. Gene147, 223-226.Kalpana GV (1999) Retroviral vectors for liver directed genetherapy. Semin Liver Dis 19, 27-37.Kay MA, Glorioso JC and Naldini L (2001) Viral vectors forgene therapy: the art of turning infectious agents intovehicles of therapeutics. Nat Med 7, 33-40.Klink DT, Chao S, Glick MC and Scanlin TF (2001) Nucleartranslocation of lactosylated poly-L-lysine/cDNA complex incystic fibrosis airway epithelial cells. Mol Ther 3, 831-841.Kren BT, Bandyopadhyay P, Roy Chowdhury N, RoyChowdhury J and Steer CJ (2002) Oligonucleotide-mediatedsite-directed gene repair. Meth Enzymol 346, 14-31.Kren BT, Parashar B, Bandyopadhyay P, Roy Chowdhury N,Roy Chowdhury J and Steer CJ (1999) Correction of theUDP-glucuronosyltransferase gene defect in the Gunn ratmodel of Crigler-Najjar syndrome type I with a chimericoligonucleotide. Proc Natl Acad Sci USA 96, 10349-10354.Maruyama H, Higuchi N, Nishikawa Y, Kameda S, Iino N,Kazama JJ, Takahashi N, Sugawa M, Hanawa H, Tada N,Miyazaki J and Gejyo F (2002) High-level expression ofnaked DNA delivered to rat liver via tail vein injection. JGene Med 4, 333-341.Mikkelsen JG, Yant SR, Meuse L, Huang Z, Xu H and Kay MA(2003) Helper-independent Sleeping Beauty transposontransposasevectors for efficient nonviral gene delivery andpersistent gene expression in vivo. Mol Ther 8, 654-665.Montini E, Held PK, Noll M, Morcinek N, Al-Dhalimy M,Finegold M, Yant SR, Kay MA and Grompe M (2002) Invivo correction of murine tyrosinemia type I by DNAmediatedtransposition. 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