Salmonella pathogenesis reveals that BMP signaling regulates blood cell homeostasis and immune responses in Drosophila.

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.

Messenger RNA as a source of transposase for sleeping beauty transposon-mediated correction of hereditary tyrosinemia type I.

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.

RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues.

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.

Sleeping Beauty-mediated transposition and long-term expression in vivo: use of the LoxP/Cre recombinase system to distinguish transposition-specific expression.

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.

Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse.

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 p19Arf-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.

Dynamic gene expression after systemic delivery of plasmid DNA as determined by in vivo bioluminescence imaging.

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.

Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system.

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.

Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human alpha-L-iduronidase gene.

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 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.

Trimetrexate inhibits progression of the murine 32Dp210 model of chronic myeloid leukemia in animals expressing drug-resistant dihydrofolate reductase.

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.

Methotrexate exacerbates tumor progression in a murine model of chronic myeloid leukemia.

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.