Hyperactive piggyBac transposons: A platform technology for sustained and robust liver targeted gene therapy and functional genomics in vivo

Given these encouragingresults, we subsequently validated our hepatotropic PB transposons as anemerging platform technology to dissect andstudying the role of oncogenes or tumor-suppressor gene and their consequenceon hepatocellular carcinomagenesis invivo. This strategy proved to be an efficient alternative to classicaltransgenesis for the generation ofgenetically defined mouse model of liver cancer and evaluation of in vivo relevant oncogenes crosstalk. Inparticular, we demonstrated that liver-directed gene transfer with hyperactivePB transposons encoding c-Myc in mice pretreated with a chemical carcinogen(DEN) resulted in the formation of hepatocellular carcinoma (HCC). Morever,cotransfection of mice with PB transposons encoding c-Myc and and H-RASG12Vresulted in an accelerated HCCdevelopment, obviating the need for chemical carcinogenesis induction with DEN.The hyperactive PB transposons were also well suited to over-express miRNA ormiRNA decoys (or sponge) inhepatocytes. This allowed us to performa semi-high throughput functional genomic screen in an attempt to establish a causalrelationship between a given miRNA and HCC. In particular, the geneticdissection of the miR-17-92 cluster provided evidence for a possible tumorsuppressor role of miR-20a in hepatocarcinogenesis. Consequently,liver-directed modulation of miRNA expression using the transposon technology isan attractive approach to exclude fortuitous associations between miRNAexpression levels and malignancy and hereby strengthen the diagnostic andtherapeutic relevance of a given miRNA.The piggyBac (PB) transposon system, originally derived fromthe cabbage looper moth Trichoplusia ni, is among the most promising transposons for gene therapy applications.To convert PB transposon into a gene delivery tool, a binary system is requiredthat is composed of an expression plasmid that encodes the PB transposase and adonor plasmid containing the gene of interest, which is flanked in cis by thetransposon terminal repeat sequences required for transposition. PB transposonscan efficiently transpose in mammalian cells and can deliver large transgenes,which cannot be readily accommodated into most viral vectors due to theirintrinsic packaging constraints. PB has been efficiently applied as a tool forfunctional genomic studies, ex vivo genetic modification of somatic andembryonic cells and generation of induced pluripotent stem cells. However,there are only few studies that focus on direct in vivo gene transfer with PBand most of these relied on reporter genes. The main objective of our presentstudy therefore consisted of establishing proof-of-concept that PB transposonsencoding FIX can be used for liver-directed gene delivery cure hemophilia B andto assess their overall efficacy and safety in appropriate mouse models. Maximizing the therapeutic index of a givengene transfer vector is a crucial step towards implementation of successfulclinical trials. Hence, we wanted to augment the efficiency oftransposon-mediated gene therapy using a multi-layered strategy by optimizingeach one of its components including the PB transposase and the transposon, theliver-specific promoter used to drive FIX and the FIX transgene itself. Ourresults demonstrated that PB transposons in conjunction with a mousecodon-optimized PB transposase (mPB) resulted in prolonged FIX expression andcure hemophilia B in FIX-deficient mice, which had not been shown previously.Moreover, we showed that the efficiency of PB-mediated gene therapy could beenhanced by using the latest generation hyperactive PB transposase (hyPB) andby modifying the transposon terminal repeats. In addition, the use of aliver-specific promoter coupled to in silico designed cis-regulatorymodules (CRMs) further increased FIXexpression levels. Finally, we demonstrated thatthe overall efficacy could befurther increased by using a codon-optimized FIX containing a hyper-functionalgain-of-function mutation (i.e. FIX Padua R338L). This combinatorial strategyresulted in robust FIX activity at supra-physiologic levels, substantiallyreducing the vector dose requirement for reaching therapeutic efficacy.Moreover, it enabled induction of immune tolerance to FIX and prevented thegeneration of anti-FIX antibodies upon immunization with recombinant FIX. Wealso validated the use of the hyperactive PB platform for delivery ofrelatively large transgenes, such as FVIII. Using a highly sensitivetumor-prone hepatocellular carcinoma mouse model, we did not observe anysignificant increase in oncogenic risk upon PB transposition, suggesting thatthis improved hyperactive PB platform has a favorable safety profile. Given the optimal results obtained in a hemophilia genetherapy context, and the growing interest in exploiting this new non-viral toolfor functional genomics studies, especially for cancer gene discovery, we thenvalidate our potent hepatotropic PB transposons as new genomic tools fordissecting and studying the role of oncogenes, tumor suppressor genes and theirconsequence on liver cancer in vivo. This strategy proved to be an efficientalternative to classical transgenesis for the generation of genetically definedmouse model of liver cancer and evaluation of in vivo relevant oncogenescrosstalk (i.e. c-Myc/H-RASG12V). Moreover, this allowed as to performsemi-high throughput functional genomic screenings using hepatocellularcarcinoma (HCC) as a model, given its poor prognosis and high prevalence. Inparticular, since recent evidence indicates that small non-protein-coding RNAmolecules, called microRNAs (miRNAs), can influence tumorigenesis and functionas either tumor suppressors or oncogenes, we focused our attention inestablishing a causal relationship between a given miRNA and malignancyfollowing hepatic over/under-expression of this miRNA in order to excludefortuitous associations between miRNA expression levels and the cancerousphenotype and hereby strengthen its intrinsic diagnostic and therapeuticrelevance. In particular, the genetic dissection of the miR-17-92 clusterprovided evidence for a tumor suppressor role of miR-20a inhepatocarcinogenesis. The piggyBac (PB) transposon system,originally derived from the cabbage looper moth Trichoplusia ni, is among the mostpromising transposons for gene therapy applications. To convert PB transposon into a gene deliverytool, a binary system is required that is composed of an expression plasmidthat encodes the PB transposase and a donor plasmid containing the gene ofinterest, which isflanked in cis bythe transposon terminal repeat sequences required for transposition. PBtransposons can efficiently transpose in mammalian cells and can deliver largetransgenes, which cannot be readily accommodated into most viral vectors due totheir intrinsic packaging constraints. PB has been efficiently applied as atool for functional genomic studies, exvivo genetic modification of somatic and embryonic cells and generation ofinduced pluripotent stem cells. However, there are only few studies that focuson direct in vivo gene transfer withPB and most of these relied on reporter genes. The main objective of our present studytherefore consisted of establishing proof-of-concept that PB transposonsencoding coagulation factor FIX (FIX) can be used for liver-directed genedelivery to cure hemophilia B and to assess their overall efficacy and safetyin appropriate mouse models. Maximizingthe therapeutic index of a given gene transfer vector is a crucial step towardsimplementation of successful clinical trials. Hence, we wanted to augment theefficiency of transposon-mediated gene therapy using a multi-layered strategyby optimizing each one of its components including the PB transposase and thetransposon, the liver-specific promoter used to drive FIX and the FIX transgeneitself. Our results demonstrated that PB transposons in conjunction with amouse codon-optimized PB transposase (mPB) resulted in prolonged FIX expressionand cure hemophilia B in FIX-deficient mice, which had not been shownpreviously. Moreover, we showed that the efficiency of PB-mediated gene therapycould be enhanced by using the latest generation hyperactive PB transposase(hyPB) and by modifying the transposon terminal repeats. In addition, the useof a liver-specific promoter coupled to insilico designed cis-regulatorymodules (CRMs) further increased FIX expression levels. Finally, wedemonstrated that the overall efficacy could be further increased by using acodon-optimized FIX containing a hyper-functional gain-of-function mutation(i.e. FIX Padua R338L). This combinatorial strategy resulted in robust FIXactivity at supra-physiologic levels, substantially reducing the vector doserequirement for reaching therapeutic efficacy. Moreover, it enabledinduction of immune tolerance toFIX and prevented the generation of anti-FIX antibodies upon immunization withrecombinant FIX. We also validated the use of the hyperactive PB platform fordelivery of relatively large transgenes, such as FVIII. Using a highlysensitive tumor-prone hepatocellular carcinoma mouse model, we did not observeany significant increase in oncogenic risk upon PB transposition, suggestingthat this improved hyperactive PB platform has a favorable safety profile.