Transposon-based Vector Systems Research Articles

Generating and Validating Transgenic Mosquitoes with Transposon-Mediated Transgenesis.

Transposon-mediated transgenesis has revolutionized both basic and applied studies of mosquito vectors of disease. Currently, techniques such as enhancer traps and transposon tagging, which rely on remobilizable insertional mutagenesis, are only possible with transposon-based vector systems. Here, we provide general descriptions of methods and applications of transposon-based mosquito transgenesis. The exact procedures must be adapted to each mosquito species and comparisons of some differences among different mosquito species are outlined. A number of excellent publications showing detailed and specific protocols and methods are featured and referenced.

Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR.

DNA transposon-based gene vehicles - scenes from an evolutionary drive

DNA transposons are primitive genetic elements which have colonized living organisms from plants to bacteria and mammals. Through evolution such parasitic elements have shaped their host genomes by replicating and relocating between chromosomal loci in processes catalyzed by the transposase proteins encoded by the elements themselves. DNA transposable elements are constantly adapting to life in the genome, and self-suppressive regulation as well as defensive host mechanisms may assist in buffering ‘cut-and-paste’ DNA mobilization until accumulating mutations will eventually restrict events of transposition. With the reconstructed Sleeping Beauty DNA transposon as a powerful engine, a growing list of transposable elements with activity in human cells have moved into biomedical experimentation and preclinical therapy as versatile vehicles for delivery and genomic insertion of transgenes. In this review, we aim to link the mechanisms that drive transposon evolution with the realities and potential challenges we are facing when adapting DNA transposons for gene transfer. We argue that DNA transposon-derived vectors may carry inherent, and potentially limiting, traits of their mother elements. By understanding in detail the evolutionary journey of transposons, from host colonization to element multiplication and inactivation, we may better exploit the potential of distinct transposable elements. Hence, parallel efforts to investigate and develop distinct, but potent, transposon-based vector systems will benefit the broad applications of gene transfer. Insight and clever optimization have shaped new DNA transposon vectors, which recently debuted in the first DNA transposon-based clinical trial. Learning from an evolutionary drive may help us create gene vehicles that are safer, more efficient, and less prone for suppression and inactivation.

MDR1 Gene Expression Predicts Response and Progression-Free Survival Of Ph+ CML Patients On Second-Line Nilotinib Therapy After Imatinib Failure - 4-Year Follow-Up

IntroductionThe activity of the drug efflux transporter protein MDR1 reduces the intracellular concentration of nilotinib and thereby impairs its efficacy. Nilotinib has been shown to be efficacious in imatinib-resistant patients. In the face of competing second-generation tyrosine-kinase inhibitors (TKI), early identification of favorable responders is crucially important. We have reported the unexpected positive prognostic impact of high MDR1 gene expression at time of imatinib resistance for subsequent 2nd line nilotinib treatment. Here, we present (i) a 48-month follow-up of our clinical data and (ii) additional functional analyses studied in an established in vitro, transposon-based vector system with stable siRNA mediated knockdown of MDR1. AimWe sought to assess (i) whether high MDR1 expression remains associated with improved cumulative rates of major molecular remission (MMR), complete cytogenetic remission (CCyR) and patients' outcome (progression-free survival, PFS) after 48 months and (ii) the impact of MDR1 expression on nilotinib in vitro sensitivity. Methods(i) We analyzed 83 patients resistant to imatinib frontline treatment in chronic phase CML treated with nilotinib 400 mg bid within the CAMN107A2101 trial. MDR1 and BCR-ABL mRNA expression levels were determined by qRT-PCR using LightCycler™ technology, normalized against beta-glucuronidase (GUS) and standardized according to the international scale (IS). Log-rank tests were performed to compare PFS and the cumulative incidences (CI) of MMR and CCyR at 48 months. (ii) MDR1high overexpressing (K562-DoxH1) and MDR1low knockdown (K562-DoxMM) cell lines were used for in vitro testing (Rumpold et al., Exp Hematol 2005). Results(i) (a) At 24 and 48 months, patients with MDR1/GUS ratios ≥2.0 attained MMR in 39% and 41%, CCyR in 58% (at both time points), and PFS rates of 75% and 67%, whereas patients with initial MDR1/GUS ratios <2.0 had significantly worse response and PFS rates, i.e. MMR in 13% and 16% (p=0.014), CCyR in 35% and 39% (p=0.044), and PFS of 50% and 46% (p=0.032). (b) BCR-ABL tumor burden prior to nilotinib revealed a significant impact on molecular response rates. BCR-ABLIS <28% separated best concerning cumulative incidences of MMR by 24 and 48 months (41% vs 21% and 48% vs 21%, p=0.009). (c) Nilotinib in vitro sensitivity of BCR-ABL kinase domain mutations at time of imatinib resistance was associated with improved PFS under nilotinib therapy: patients without any mutation showed PFS rates of 71% and 63% at 24 and 48 months, whereas those with either sensitive mutations, intermediately sensitive mutations or mutations with unknown IC50attained PFS rates of 67% and 61%; patients with mutations resistant to nilotinib achieved PFS rates of 23% at both time points (p=0.01). (ii) Even though MDR1high K562 cells are less sensitive than MDR1low expressing cells, nilotinib (applying doses from 0.01 µM up to 0.5 µM) was still able to significantly impede proliferation of both MDR1high and MDR1low, whereas imatinib-mediated growth inhibition was only seen in MDR1low, but not in MDR1highcells. Conclusion(i) At the time of imatinib-resistance, a high MDR1 gene expression predicts favorable MMR, CCyR, and PFS on consecutive 2nd line nilotinib treatment. As shown earlier, single nucleotide polymorphisms (SNPs) within MDR1 (1236CT/TT and 2677GT/TT) were significantly associated with higher MDR1 expression. (Agrawal et al., ASH 2010) (ii) Our functional data support our clinical observation that nilotinib remains efficacious in MDR1 overexpressing cells, whereas even dose-escalated imatinib does not reverse resistance. High MDR1 gene expression might select patients whose mode of resistance is essentially determined by increased efflux activity of MDR1 and not by other pathways of resistance that cannot be overcome by nilotinib. Altogether, our data might be used for the clinical risk stratification in case of imatinib resistance before switching to nilotinib and are undergoing prospective validation within the ENEST1st study. Disclosures:Saussele:Novartis: Honoraria, Research Funding, Travel Other; BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria. Purkayasatha:Novartis: Employment. Woodman:Novartis: Employment. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Müller:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.

In vivo RNAi screening identified MKK4 as a new therapeutic target for the improvement of liver regeneration

We established a unique system for performing direct in vivo RNAi screens to genetically dissect cellular signaling networks that regulate proliferation control of hepatocytes during liver damage and regeneration. By combining the well characterized FAH-/- mouse model with a transposon based vector system, we generated chimeric mouse livers. In these mouse livers each hepatocyte expresses FAH, a marker gene and a microRNA based shRNA. To such mouse livers any form of liver damaging protocol can be applied and the regenerative process can be studied. We conducted a first in vivo RNAi screen, using a focused shRNAmir library consisting of 631 constructs targeting 362 genes. Mouse livers were stably repopulated with the shRNAmir library and after repopulation mice were subjected to chronic CCl4 treatment to induce chronic liver damage. The representation of each shRNAmir in the pool, the chimeric mouse liver and after chronic liver damage was determined by deep sequencing analysis. ShRNAs targeting MKK4 showed strong enrichment during liver damage and regeneration. MKK4 belongs to the Map kinase family and is part of two stress kinase signaling pathways, the JNK as well as the p38 pathway. Functional in vivo validation experiments showed that stable knock down of MKK4 by different shRNAs can significantly increase the repopulation efficiency of mouse hepatocytes and also increases the regenerative capacity of chronically damaged mouse livers. In addition we could show that MKK4 suppression also confers an increased robustness of hepatocytes in a model of acute liver failure, resulting in better survival of these animals. Beyond this, primary hepatocytes with stable MKK4 knockdown showed prolonged survival in cell culture and these cells could even be passaged and used to repopulate FAH-/- mouse livers. Protein array and western blot analyses of MKK4 shRNAs versus control-shRNA expressing livers revealed a complex activation pattern of JNK- and p38-signaling downstream factors. Interestingly, the observed pattern does not merely represent an increased JNK over p38 signaling but our data rather suggest that complex cross signaling events between both pathways are involved. Also hepatocytes with stable MKK4 knock down already show higher levels of CyclinD1, suggesting a priming of the cells to leave the G0 state and entering the G1 state of the cell cycle. We developed a new screening platform for pinpointing new regulators of hepatocyte proliferation and liver regeneration. A first in vivo RNAi screen identified MKK4 as an interesting new therapeutic target structure for improving the regenerative capacity under acute and chronic liver damaging conditions.

Genome-wide target profiling of piggyBac and Tol2in HEK 293: pros and cons for gene discovery and gene therapy

BackgroundDNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery.ResultsWe have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2.ConclusionsThe non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting.

Genome-wide screen identifies PVT1 as a regulator of Gemcitabine sensitivity in human pancreatic cancer cells

Gemcitabine has been a first-line chemotherapy agent for advanced pancreatic cancer, which is associated with one of the lowest 5 years survival rates among human cancers. Due to our lack of understanding of the genetic determinants of Gemcitabine sensitivity in pancreatic cancer, the therapeutic effectiveness of Gemcitabine chemotherapy is typically unpredictable. Using a genome-wide and piggyBac transposon-based genetic screening platform, we identified the PVT1 gene as a regulator of Gemcitabine sensitivity and showed that functional inactivation of the PVT1 gene led to enhanced Gemcitabine sensitivity in human pancreatic cancer ASPC-1 cells. The integration of the piggyBac transposon-based vector system into intron 3 of PVT1 was within a common site of oncogenic retroviral insertions and chromosomal translocations. PVT1 is a non-protein encoding gene; the genomic arrangement of PVT1 and its co-amplification with MYC have been implicated in the tumorigenesis of a variety of cancers. The molecular mechanism of PVT1 transcripts in gene regulation remains a puzzle. We demonstrated that overexpression of a full length PVT1 cDNA in the antisense orientation reconstituted enhanced sensitivity to Gemcitabine in naïve ASPC-1 cells, whereas overexpression of a full length PVT1 cDNA in the sense orientation resulted in decreased sensitivity to Gemcitabine. Our results identified PVT1 as a regulator of Gemcitabine sensitivity in pancreatic cancer cells and validated the genome-wide genetic screening approach for the identification of genetic determinants as well as potential biomarkers for the rational design of Gemcitabine chemotherapies for pancreatic cancer.

Knockdown of PgP resensitizes leukemic cells to proteasome inhibitors

Overexpression of MDR-1 represents a critical mechanism of drug resistance in cancer. Proteasome inhibitors recently entered the clinic for treatment of multiple myeloma. We provide evidence that the proteasome-inhibitors Bortezomib and MLN273 are both substrates of MDR-1 by using knockdown of MDR-1 via a transposon-based vector system stably expressing siRNA against MDR-1 in MDR-1-overexpressing K562/Dox cells. Notably, the efficacy of MLN273 (EC50 from 253ng/ml in MDR-1+ to 9.7ng/ml in MDR-1− cells) was much more dependent on MDR-1 expression than Bortezomib (EC50 from 24.9ng/ml in MDR-1+ to 4.5ng/ml in MDR-1− cells). Growth inhibition in MDR-1 negative cells was in part due to increased rate of apoptosis. The enhanced inhibitory effect on the proteasome by loss of MDR-1 was corroborated by a reduced proteasomal activity. Our report provides evidence that MLN273 and, to a lesser degree, Bortezomib are both MDR-1-substrates, which might be relevant for drug-resistance in cancer.

A multipurpose transposon-based vector system mediates protein expression in Rhodococcus erythropolis

In the current study we developed two transposon-based vectors; namely pTNR-KA and pTNR-TA and utilized them for expression of proteasome complex, derived from Streptomyces coelicolor, in Rhodococcus erythropolis. The two vectors can be transposed into Rhodococcus cells by means of electroporation, either individually in two consecutive processes or in combinations by a single step. During transposition, each of the two vectors liberates its transposable-marker gene, which integrated in a single copy into a random site in the Rhodococcus chromosomal DNA. Southern blot analysis indicated that the two transposable-marker genes of both vectors does not alter or knock out each other. To utilize these vectors for Streptomyces proteasome expression, two expression cassettes were constructed; each cassette comprised a constitutive promoter (P nit ), the DNA fragment, prcA or prcB that encodes α- or β-subunits of Streptomyces proteasome, and T thcA transcriptional terminator. The cassettes were then individually introduced into the multiple cloning sites that are located in the transposable-marker gene of the two vectors. The two cassettes-harboring vectors were subsequently co-transposed, in combinations, into the Rhodococcus genome by a single electroporation step and the Streptomyces proteasome was successfully expressed in the rodococcal host cell. The isolated proteasome was further characterized and the peptidase activity was confirmed and indicated that it was biologically active. The present study concluded that both pTNR-KA and pTNR-TA can be used as transposon-based protein expression systems in Rhodococcus species.

Sleeping Beauty Transposon-Mediated Nonviral Gene Therapy

Safe and effective delivery of genetic material to mammalian tissues would significantly expand the therapeutic possibilities for a large number of medical conditions. Unfortunately, the promise of gene therapy has been hampered by technical challenges, the induction of immune responses, and inadequate expression over time. Despite these setbacks, progress continues to be made and the anticipated benefits may come to fruition for certain disorders. In terms of delivery, nonviral vector systems are particularly attractive as they are simple to produce, can be stored for long periods of time, and induce no specific immune responses. A significant drawback to nonviral systems has been the lack of persistent expression, as plasmids are lost or degraded when delivered to living tissues. The recent application of integrating transposons to nonviral gene delivery has significantly helped to overcome this obstacle, because it allows for genomic integration and long-term expression. Recent advances in transposon-based vector systems hold promise as new technologies that may unlock the potential of gene therapy; however, technical and safety issues still need refinement.

RNAi-mediated knockdown of P-glycoprotein using a transposon-based vector system durably restores imatinib sensitivity in imatinib-resistant CML cell lines

Resistance to therapeutic drugs is a frequent phenomenon in hematologic malignancies, causing treatment failure in patients with leukemias and lymphomas. Overexpression of the multidrug-resistance gene (MDR-1) and its translational product P-glycoprotein (PgP) represents one mechanism of fatal drug resistance. We constructed a nonviral, transposon-based vector system for the stable knockdown of PgP in chronic myeloid leukemia cell lines resistant to imatinib and doxorubicin. Using this strategy, PgP expression was completely knocked down 72 hours after vector inoculation and lasted for several months. Cellular efflux of the PgP substates rhodamine and doxorubicin was abolished. Vector-treated cells were resensitized to imatinib- and doxorubicin-induced cell death. Using chronic myeloid leukemia as a model, we show that PgP-mediated resistance to imatinib and anthracyclines can be durably reversed by nonviral, transposon-based knockdown of PgP in malignant cells.

Stable RNAi-Mediated Knock-Down of P-Glycoprotein by a Transposon-Based Vector System Restores Imatinib Sensitivity.

Overexpression of P-glycoprotein (PgP) is critical for resistance of malignant cells against cytotoxic agents. However, the role of PgP overexpression for the sensitivity of chronic myeloid leukemia (CML) to imatinib remains controversial. We therefore constructed a transposon-based (Sleeping beauty) RNAi system for the stable down-regulation of PgP in imatinib/doxorubicin resistant K562/Dox cell lines. RT-PCR demonstrated the stable integration of the transposon into the genome. Real-time PCR revealed a rapid and almost complete degradation of MDR1 mRNA 72 h after nucleofection. PgP knockdown on protein level was much more efficient in the stable setting as compared to the transient approach. This observation points out to the requirement of stable knockdown approaches for siRNA-mediated downregulation of proteins with long half-life time. The functional impact of stable PgP knock-down was demonstrated by a reduced efflux of rhodamine and doxorubicin. In parallel, MDR1 knockdown cells were re-sensitized to doxorubicin as well as to imatinib-induced cell death. This observation supports the notion that up-regulation of MDR1 in CML might contribute to imatinib resistance in vivo.In summary we describe a very efficient tool for the stable knock-down of PgP by transposon-mediated siRNA delivery in CML cells and propose that the down-regulation of PgP in CML can restore sensitivity to imatinib.

Prospects for using genetic transformation for improved SIT and new biocontrol methods.

The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems. This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods. For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing. Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline. Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile. Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.