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The Sleeping Beauty (SB) transposon is an integrative nonviral plasmid system. Here, we describe a protocol for SB-mediated transgene delivery using DNA/polyethyleneimine (PEI) complexes for long-term expression in mouse lungs. This protocol can be used for delivery of any plasmid-based vector system to mouse lungs, although long-term transgene expression will be obtained only when using the SB transposon or other integrating vector systems. The stages of this protocol are preparation of DNA-PEI complexes and injection of the complexes into the lateral tail vein of mice. We also provide protocols for assessing transgene expression using in vivo bioluminescence imaging and enzymatic assay of lung homogenates. The procedure can be completed within 24 h, starting from preparation of DNA-PEI complexes to analysis of transient transgene expression.

Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.

The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.

The Sleeping Beauty (SB) transposon system can integrate foreign sequences of DNA in the genome of mouse somatic cells eliciting long-term expression in vivo . This technology holds great promise for human gene therapy as a nonviral technology to deliver therapeutic genes. SB also provides a means to study the effects of defined genetic elements, such as oncogenes, on somatic cells in mice. Here, we test the ability of the SB transposon system to facilitate somatic integration of a transposon containing an activated NRAS oncogene in mouse hepatocytes to elicit tumor formation. NRAS oncogene-driven tumors developed when such vectors were delivered to the livers of p19 Arf -null or heterozygous mice. Delivery of the NRAS transposon cooperates with Arf loss to cause carcinomas of hepatocellular or biliary origin. These tumors allowed characterization of transposon integration and expression at the single-cell level, revealing robust NRAS expression and both transposase-mediated and random insertion of delivered vectors. Random integration and expression of the SB transposase plasmid was also observed in one instance. In addition, studies using effector loop mutants of activated NRAS provide evidence that mitogen-activated protein kinase activation alone cannot efficiently induce liver carcinomas. This system can be used to rapidly model tumors caused by defined genetic changes.

Hemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain–deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.

Murine models of lysosomal storage diseases provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease. To determine the potential for treatment of mucopolysaccharidosis type I using a gene delivery approach, a recombinant adeno-associated virus (AAV) vector, vTRCA1, transducing the human iduronidase (IDUA) gene was constructed and 1 x 10(10) particles were injected intravenously into 1-day-old Idua(-/-) mice. High levels of IDUA activity were present in the plasma of vTRCA1-treated animals that persisted for the 5-month duration of the study, with heart and lung of this group demonstrating the highest tissue levels of gene transfer and enzyme activity overall. vTRCA1-treated Idua(-/-) animals with measurable plasma IDUA activity exhibited histopathological evidence of reduced lysosomal storage in a number of tissues and were normalized with respect to urinary GAG excretion, craniofacial bony parameters, and body weight. In an open field test, vTRCA1-treated Idua(-/-) animals exhibited a significant reduction in total squares covered and a trend toward normalization in rearing events and grooming time compared to control-treated Idua(-/-) animals. We conclude that AAV-mediated transduction of the IDUA gene in newborn Idua(-/-) mice was sufficient to have a major curative impact on several of the most important parameters of the disease.

Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, α1-antitrypsin deficiency, emphysema, and cancer. DNA-mediated gene transfer to the lung has been previously demonstrated, but anticipated effectiveness has been limited by low gene transfer efficiencies and by transient expression of the transgene. Here, we combine plasmid-based gene transfer with the integrating capacity of the nonviral Sleeping Beauty (SB) transposon vector system to mediate gene insertion and long-term gene expression in mouse lung. We observed transgene expression after 24 h in lungs of all animals injected with the luciferase transposon (pT/L), but expression for up to 3 months required codelivery of a plasmid encoding the Sleeping Beauty transposase. We also observed long-term expression in pT/L-injected animals transgenic for SB transposase. Transgene expression was localized to the alveolar region of the lung, with transfection including mainly type II pneumocytes. We used a linker-mediated PCR technique to recover transposon flanking sequences, demonstrating transposition of pT/L into mouse chromosomal DNA of the lung.

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). 32Dp210 tumor cells were found to be sensitive to methotrexate (MTX) in vitro, whereas cells expressing the tyrosine-22 DHFR gene were protected from MTX at up to micromolar concentrations. MTX administered at low dose (2 mg/kg/day) did not protect normal C3H-He/J mice from 32Dp210 tumor infused intravenously, with drug toxicity limiting the administration of higher doses. Animals engrafted with transgenic tyrosine-22 DHFR marrow were protected from greater MTX doses (up to 6 mg/kg/day). However, the increased doses of MTX afforded by drug-resistance gene expression surprisingly resulted in decreased survival of the transplanted tumor-bearing animals, with increased levels of tumor detected in peripheral blood. This apparent exacerbation of tumor progression by MTX was not observed in DHFR transgenic mice in which all cells and tissues contain the drug-resistance gene. This suggests that increased tumor progression in MTX-administered animals resulted from MTX sensitivity of a nonhematopoietic host component, thus allowing tumor expansion. We conclude that MTX exacerbates tumor progression in the 32Dp210 model of CML, and that based on this model alternate DHFR inhibitors combined with drug-resistant DHFR or other chemotherapeutic agent/drug-resistance gene combinations may be required for the application of drug-resistance gene expression to the treatment of CML. ispartof: Journal of Pharmacology and Experimental Therapeutics vol:300 issue:3 pages:1075-84 ispartof: location:United States status: published

Intercellular signaling by bone morphogenetic proteins (BMPs) regulates developmental decisions in virtually all animals. Here, we report that Decapentaplegic (Dpp; a Drosophila BMP family member) plays a role in blood cell homeostasis and immune responses by regulating a transcription factor cascade. The cascade begins with Dpp repression of Zfh1, continues with Zfh1 activation of Serpent (Srp; a GATA factor), and terminates with Srp activation of U-shaped (Ush) in hematopoietic cells. Hyperactivation of Zfh1, Srp, and Ush in dpp mutants leads to hyperplasia of plasmatocytes. Salmonella challenge revealed that in dpp mutants the misregulation of this cascade also prevents the generation of lamellocytes. These findings support the hypothesis that Ush participates in a switch between plasmatocyte and lamellocyte fate in a common precursor and further suggests a mechanism for how all blood cell types can arise from a single progenitor. These results also demonstrate that combining Drosophila and Salmonella genetics can provide novel opportunities for advancing our knowledge of hematopoiesis and innate immunity.

The Sleeping Beauty (SB) transposon system mediates chromosomal integration and stable gene expression when an engineered SB transposon is delivered along with transposase. One concern in the therapeutic application of the SB system is that persistent expression of transposase could result in transposon instability and genotoxicity. Here, we tested the use of transposase-encoding RNA plus transposon DNA for correction of murine fumarylacetoacetate hydrolase (FAH) deficiency. A bi-functional transposon containing both mouse FAH and firefly luciferase sequences was used to track the growth of genetically corrected liver tissue by in vivo bioluminescence imaging after delivery of DNA or RNA as a source of transposase. Supplying SB transposase in the form of RNA resulted in selective repopulation of corrected hepatocytes with stable expression of FAH and luciferase. Plasma succinylacetone and amino acid levels were normalized, suggesting normal liver metabolism of catabolized protein products. Secondary FAH-deficient animals transplanted with hepatocytes (250,000) isolated from primary treated animals survived 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) withdrawal, gained weight consistently, and demonstrated stable expression of luciferase. We conclude that transposase-encoding messenger RNA (mRNA) can be used to mediate stable non-viral gene therapy, resulting in complete phenotypic correction, and is thus an effective source of recombinase activity for use in human gene therapy.

Many applications of gene therapy require long-lasting expression. In mice, hydrodynamic delivery directs DNA to the liver where when integrated expression can last the lifetime of the animal. Because this form of delivery will be invasive in larger animals, we have been seeking alternative methods of delivery, including condensing DNA with polyethelenimine (PEI). PEI has been reported to deliver genes to the lung with expression lasting for up to 3 months (Belur et al, Molecular Therapy 8: 501|[ndash]|507; 2003). However, using standard hydrodynamic delivery as a |[ldquo]|gold standard|[rdquo]| for achievable levels of gene expression in mice, we have found that simple PEI condensation results in expression of delivered genes at levels that are 10 to more than 100-fold lower than with hydrodynamic delivery. Following injection into the tail vein of PEI-complexed plasmids containing an SB transposon with a luciferase gene, DNA expression was most pronounced in the lung one day post-injection. By 7 days post-injection, expression was most evident in the liver rather than the lung as visualized with a Xenogen|[trade]| in vivo imaging system. JetPEI|[trade]| at N/P ratios of 7 and 9 led to expression levels that ranged from 1|[ndash]|22 |[times]| 106 photons/second (p/s) in the lung 1-day post-injection. However, by day-7 the ranges were from 2|[ndash]|80 |[times]| 104 to 2|[ndash]|9 |[times]| 104 p/s for N/P ratios of 7 and 9, respectively. Because particle sizes should be 100 nm or less for uptake by endocytosis, we sized our PEI-plasmid particles by dynamic laser light scattering. Particles ranged in size from 80|[ndash]|100 nm in 5% dextrose at an N/P of 7 with JetPEI|[trade]|. Because uncomplexed, free PEI is cytotoxic to cells, we hypothesize that the removal of free PEI will permit higher N/P ratios, which might provide increased levels of gene delivery and expression. Consequently, we are testing methods for removal of free PEI so that PEI transposon complexes prepared at higher N/P ratios can be tested for improved levels of long-term expression and animal survival after in vivo delivery.

Nonviral gene therapy approaches face the challenge of achieving stable gene expression in target organs and tissues. We used fluorescence microscopy and in vivo imaging after rapid, high-volume delivery of plasmid DNA (pDNA) encoding DsRed and luciferase as products of the same mRNA to detect localized gene expression in liver. Plasmids encoding the luciferase gene transcriptionally regulated by cellular and viral promoters were injected under similar conditions to test for potency and stability of gene expression in the liver of adult mice. Animals were imaged at 3, 6, 12, 24, and 48 hr and followed up at 1- to 2-week intervals over 2 months to identify maximum and persistent luciferase expression after injection of equimolar amounts of each plasmid. Emitted light representing luciferase expression was detected as early as 3 hr after pDNA infusion, reaching maximum levels at 12 hr for promoters containing viral sequences and at 24 hr for elements from human genes. Viral elements displayed 10- to 20-fold higher peak levels of expression but also yielded the most dramatic decline in expression over 8 weeks. In contrast, only a moderate decrease was observed for the cellular ubiquitin C promoter during that same period of time. Both promoter-deleted and phosphoglycerate kinase (PGK)-containing plasmids failed to maintain luciferase expression at levels above the limit of detection. These results demonstrate fine temporal analyses of reporter gene activity using promoters of various strength, suggesting an effective and reproducible method for studying gene therapy vectors in vivo.

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). Administration of the maximum tolerated dose of trimetrexate (TMTX) with the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine ribose-5'-monophosphate (NBMPR-P) inhibited 32Dp210 tumor progression in mice engrafted with transgenic tyrosine-22 DHFR marrow and improved survival of tumor-bearing animals as long as drug administration was continued. NBMPR-P coadministration was necessary for maximal tumor inhibition, as administration of TMTX alone delayed but did not prevent tumor progression. The chemoprotection afforded by engraftment with transgenic tyrosine-22 DHFR marrow was necessary for effective chemotherapy, as normal mice lacking transgenic marrow could not tolerate the higher TMTX dose (60 mg/kg/day) administered to mice with transgenic marrow, and the decreased dose of TMTX with NBMPR-P tolerated by normal tumor-bearing animals did not inhibit tumor progression or improve animal survival. We conclude that TMTX with NBMPR-P inhibits tumor progression in the 32Dp210 model of CML in animals engrafted with drug-resistant tyrosine-22 DHFR transgenic marrow, and that based on this model the introduction of a drug-resistant DHFR gene into marrow combined with TMTX and NBMPR-P administration may provide an effective treatment for CML.

Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the transposase component has been provided as a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to remobilization and reintegration of transposons. Such continued expression of transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of transposase, we have previously shown that in vitro transcribed transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of transposase- encoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time that transposase-encoding mRNA can be used to mediate non-viral gene therapy resulting in complete phenotypic correction that is stable and not associated with any incidents of cellular transformation.

Deficiency of fumaryl acetoacetate hydrolase (FAH) in hereditary tyrosinemia type I results in oxidative damage to hepatocytes. Treatment includes restriction of dietary intake of tyrosine and administration of 2-(2-nitro-4-trifluoromethylbenzyol)-1,3-cyclohexanedione (NTBC), which inhibits an enzyme upstream of FAH in tyrosine catabolism. FAH-/- knockout mice recapitulate the human disorder and require NTBC for survival. The mice have been used as a model system to study liver repopulation by wild-type hepatocytes or by FAH mutant hepatocytes that were corrected with wild-type FAH via viral or Sleeping Beauty (SB)-mediated gene transfer. Here we introduce a method to quantitatively track repopulating liver cells in vivo by bioluminescent imaging. We assembled a SB transposon plasmid with the strong CAGGS promoter regulating murine FAH expression. To the region 3' of the FAH coding sequence we attached an internal ribosome entry site (IRES) sequence and the luciferase transgene such that FAH and luciferase are translated from the same mRNA. Also, a SB transposase gene regulated by the PGK promoter was inserted into the plasmid outside of the transposon. For gene delivery to hepatocytes, we used rapid, high-volume injection into the tail vein of FAH-/- mice. Liver repopulation by SB-transposed cells was initiated by withdrawal of NTBC, which induces tyrosinemia. Six weeks after DNA injection the mice are steadily increasing in weight without NTBC, suggesting that the metabolic defect has been corrected. We also studied these mice for expression of the luciferase reporter gene contained on the same transposon sequence as the FAH gene. At various times after injection of tranposon DNA, the mice were injected i.p. with luciferin, and luciferase expression was visualized and quantified in vivo using the IVIS|[reg]| imaging system (Xenogen Corp.). We found that luciferase expression increased by 10-fold during two weeks of selective repopulation. We have engineered additional transposon constructs to link FAH expression with that of any transgene of interest, such as a mutant alpha1-antitrypsin gene. One potential application of this method is to generate liver-transgenic models without the necessity of germline transgenesis. In conclusion, we have developed a method to quantitatively track repopulating liver cells in vivo using luciferase as a reporter, and demonstrate that FAH-/- mice can be used for transgenesis of the adult mouse liver.

The Sleeping Beauty (SB) transposon system has been demonstrated to mediate gene insertion and long-term expression, suggesting the potential application of this plasmid based system for non-viral gene therapy. All reported studies of SB-mediated gene transfer to somatic cells have supplied the transposase by expression of co-injected plasmid DNA. Although successful for both in vitro and in vivo transposition, there is a lack of control with respect to the transient and long-term levels of transposase expressed in a target cell. There is also the potential for genomic integration of the transposase encoding DNA, resulting in sustained transposase expression and potential destabilizing effects on the integrated transgene or host genome integrity. We address these problems by supplying the transposase-encoding molecule in the form of mRNA. Rapid, high pressure injection of m7G-capped luciferase (Luc) mRNA through the tail vein of mice resulted in efficient luciferase expression with respect to a DNA element where the Caggs promoter controlled luciferase transcription. Using this system as a model for efficient gene transfer in the liver, we have taken steps to optimize delivery/expression of the mRNA by generating constructs to promote stability and translation. Real-time whole body in vivo imaging of Luc mRNA translation was conducted to measure the effect of an IRES and UTR sequences on luciferase expression. These studies determined that incorporation of 5' and 3' UTR elements from the Xenopus -globin gene were essential to achieve peak luciferase expression levels from an otherwise unprotected transcript. Co-delivery of these mRNAs with an inhibitor of or a substrate for endogenous RNases provided luciferase expression sufficient to mimic levels achieved using promoters known to provide optimum transposase expression for in vivo transposition. Using this genetic arrangement, we have co-delivered SB transposase mRNA with a transposon encoding the Luc gene (driven by the Caggs promoter) to the liver of transgenic mice in which transcription of Cre recombinase is under the control of an interferon response element. To distinguish between plasmid-mediated expression and expression mediated by transposition, we have engineered this transposon encoding plasmid with lox-P sites positioned such that Cre-mediated recombination excises a sequence which includes the Caggs promoter, unless it has been segregated away from the plasmid by the SB transposase (Score et al., Mol. Ther. 7: S9, 2003). We found that pIpC induction of Cre recombinase caused a 1600-fold reduction in luciferase expression in animals infused with the transposon alone. While co-delivery of transposase mRNA resulted in a moderate degree of inadvertent Cre induction, we observed only a 45-fold reduction in luciferase expression following analogous pIpC administration. This remaining luciferase expression is most likely attributable to SB mediated transposition. These results provide the first evidence for use of SB transposase mRNA to mediate transposition and long-term expression in an in vivo setting.

Top of pageAbstract We have developed a series of anti-cancer Sleeping Beauty (SB) transposons designed for transfer of anti-angiogenic genes or Herpes Simplex Virus Thymidine Kinase (HSVTK) fusion genes into tumor cells, tumor-associated stroma, or organs undergoing metastatic attack. All genes were placed in SB T2 transposon vectors driven by the chicken beta-actin/CMV immediate early promoter/rabbit globin intron (CAGGS) promoter construct. Glioblastoma multiforme (GBM) and at least 6 other cancers overexpress IL-13 receptors. In order to take advantage of this, the human IL-13 ligand was fused to the N-terminus of HSVTK to make a secreted, tumor-targeted, suicide gene. Cell culture media collected from cells transfected with IL13-HSVTK is able to specifically sensitize IL-13 receptor positive GBM-derived cells to ganciclovir. Furthermore, we have delivered the IL13-HSVTK transposon intratumorally as plasmid DNA vector in polyethylenimine (PEI) to abolish glioma xenografts in immunodeficient nu/nu mice. We have also used angiostatin-endostatin fusion and dominant-negative VEGF transposons, delivered intratumorally as DNA/PEI complexes, to reduce glioma xenograft size and halt tumor growth. GBM generally causes death quickly as a primary tumor, however metastatic disease such as occurs in late-stage melanoma also remains difficult to treat. Therefore, we have investigated delivering anti-angiogenic transposons into the mouse lung as DNA/PEI complexes to prevent metastatic engraftment of syngenic B16F10 melanoma cells. Both the quantity and volume of lung metastases was reduced in a transposase-dependent fashion in mice treated 2 months prior to melanoma challenge. SB anti-cancer transposons represent a novel, non-viral vector system that could be used to augment conventional therapy for localized and metastatic cancers.

Methotrexate (MTX) is an effective chemotherapeutic agent in the treatment of several proliferative tumors, most notably acute lymphocytic leukemia, Ewings sarcoma and osteosarcoma. However, methotrexate also has considerable toxicity for normal proliferative tissues, including hematopoietic cells and cells of the gastrointestinal tract. The toxicity of methotrexate thus limits its chemotherapeutic effectiveness. MTX acts by binding to dihydrofolate reductase (DHFR) as a competitive inhibitor of the enzyme. We are investigating the possibility of expressing drug-resistant forms of DHFR in hematopoietic cells as a means of protecting the recipient from the toxicity associated with MTX chemotherapy. We previously demonstrated substantial protection of recipient mice from lethal toxicity of MTX by transplantation of transgenic marrow expressing Arg22 or Tyr22 forms of drug-resistant DHFR. Here we report that the increased MTX dose tolerance afforded by transplantation with marrow expressing the tyr22 form of drug-resistant DHFR allows for improved chemotherapy of murine L1210 leukemia. In our experimental model, animals were administered a subcutaneous dose of 10e6 L1210 tumor cells. Three days later, the animals were given a sublethal (650 cGy) dose of Cs irradiation. Our preliminary studies demonstrated that this course of treatments left a consistent amount of residual disease in the animals. One day after irradiation, animals were transplanted with 107 bone marrow cells flushed either from normal animals or from Tyr22 DHFR transgenic animals, and on the following day MTX administration was initiated. Control mice administered PBS developed tumor at a median of 12 days after subcutaneous injection of L1210 cells. Administration of 4 mg/kg MTX resulted in a considerable delay in the emergence of tumor for groups given either normal BMT or DHFR transgenic BMT. However, animals transplanted with normal marrow suffered from MTX toxicity, evidenced by reduced hematocrit, while there was no anemia observed in animals transplanted with DHFR transgenic marrow. A second experiment was conducted in the same way except that MTX was administered at a higher dose of 10 mg/kg/day. In this experiment, all control animals transplanted with normal marrow succumbed to toxicity associated with drug administration, while animals transplanted with DHFR transgenic marrow were substantially protected from drug toxicity. Appearance of tumor was delayed to a median of 28 days in comparison to 12 days for PBS administered controls. We conclude that expression of drug-resistant DHFR in hematopoietic cells confers substantial resistance to MTX, which can be used for more effective chemotherapy against L1210 leukemia in mice. These results support the application of DHFR gene transfer as strategy for improved cancer chemotherapy using MTX and other antifolates.

The Sleeping Beauty transposon system has been shown to reproducibly mediate gene integration resulting in long-term expression of various marker genes and even therapeutic benefits. We previously reported a method to isolate expression originating from integrated transposons. Briefly this procedure involves use of the Cre recombinase system and placement of LoxP sites within the transposon-encoding plasmid such that transposition segregates the recombinase sites from each other. In this system, transposition prevents Cre-mediated excision of the transposons promoter region, thus allowing maintenance of expression from transposed elements while greatly reducing plasmid mediated expression (Score et al., Mol. Ther. 7: S9, 2003). Expression from untransposed plasmids can be reduced in this way by nearly 99%, allowing the assessment of expression resulting from transposition in as little as three weeks. Approximately 90% of plasmids recovered from the livers of animals injected with floxed transposons were found to have been silenced in this way. Southern blots also confirmed the recombination and subsequent silencing of episomal plasmids. The system is being further characterized through PCR analysis for transposon excision as well as sequencing of transposon flanking genomic DNA. We are currently using this system to quantitatively compare transposition efficiencies in vivo, comparing the relative activity of transposase and transposon variants, and also establishing a time course of transposition based on the expression of different levels of transposase. Using this model we are thus optimizing performance of the SB transposon system for its application to therapeutic models.

Hemophilia A is an X-linked recessive genetic disorder that is characterized by sustained bleeding after trauma or injury. Currently, patients are treated by self-administration of recombinant factor VIII protein (FVIII), but this treatment is expensive and cumbersome. Gene therapy using viral vectors has been successfully used to correct the bleeding disorder in FVIII deficient mice and dogs. However, anti-viral immune responses, problems with scale-up, and insertional mutagenesis, due to a predilection for most integrating viruses to insert near genes, all raise safety and practicality questions for viral vectors. Therefore we used the Sleeping Beauty (SB) transposon system, which is delivered as plasmid DNA, to integrate a B-domain deleted FVIII genes into the chromosomes of C57BL/6J-F8−/− mice and correct the phenotype. The transposon plasmid was co-delivered with a Ubiquitin 3 promoter-regulated SB 10 transposase expression plasmid using the “hydrodynamic” method. Plasma FVIII levels were assayed in treated mice by ELISA, COAT test, and APTT clotting assay. Long-term FVIII expression (>9 weeks at present) required delivery of 280 micrograms of plasmid DNA, co-delivery of Ub-SB10 plasmid, and immunosuppression by bi-monthly 150 mg/kg cyclophosphamide injections or immunotolerization by FVIII (RefactoTM) injection in C57BL/6J-F8−/− neonates. Without immunosupression or tolerization, anti-FVIII antibody was detected in treated mice using the Bethesda assay. Transposon excision products were detected in DNA extracted from the liver of mice treated with transposon and transposase, providing evidence for transposition. Transposon insertion site analyses in hepatocytes from treated mice are ongoing. The SB transposon system thus provides non-viral means for obtaining stable FVIII gene insertion and expression for the treatment of hemophilia A.