Joel L. Frandsen

Gene insertion and long-term expression in lung mediated by the Sleeping Beauty transposon system.

Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, alpha1-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.

Lung-directed gene therapy in mice using the nonviral Sleeping Beauty transposon system

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.

338. Non-Viral Gene Transfer into Hematopoietic Cells of Artemis Deficient ScidA Mice Using the Sleeping BeautyTransposon System|[ast]|

The Sleeping Beauty (SB) transposon system has been shown to mediate gene insertion into mouse and human chromosomes, which is favored for cells where molecular therapies seek to provide durable gene expression. Although conditions have been established for non-viral delivery of DNA into cultured hematopoietic cells, no conclusive result has been published showing stable in vivo expression following non-viral delivery into mouse hematopoietic cells. Here we describe studies designed to test the ability of the SB system to mediate gene transfer into mouse total bone marrow (TBM) for the purpose of achieving stable gene expression in hematopoietic stem cells. Experiments were conducted in normal mice and in a model of T-B-NK+ SCID (ScidA) resulting from deficiency of Artemis for which selective engraftment of normal cells has been observed.

823. Use of a Cre Recombinase Silencing Strategy to Characterize Sleeping Beauty-Mediated Transposition In Vivo

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.

279. Methotrexate Chemotherapy of L1210 Leukemia in Mice Transplanted with Transgenic Marrow Expressing Drug-Resistant Dihydrofolate Reductase

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.

165. Long-term factor VIII expression and phenotypic correction in a murine model of hemophilia A achieved by immunosuppression and non-viral gene transfer using the sleeping beauty transposon

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.