Bradley S. Fletcher

Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates

The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with ∼100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35–50% stable gene transfer in human CD34+ cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis

Sleeping Beauty (SB) transposon is a natural nonviral gene transfer system that can mediate long‐term transgene expression. Its potential utility in treating organ transplantation‐associated long‐term complications has not yet been explored. In the present study we generated an improved SB transposon encoding the human gene indoleamine‐2,3‐dioxygenase (hIDO), an enzyme that possesses both T cell‐suppressive and antioxidant properties and selectively delivered the SB transposon in combination with a hyperactive transposase plasmid to donor lung using the cationic polymer polyethylenimine (PEI) as transfection reagent. This nonviral gene therapeutic approach led to persistent and uniform transgene expression in the rat lung tissue without noticeable toxicity and inflammation. Importantly, IDO activity produced by hIDO transgene showed a remarkable therapeutic response, as evident by near normal pulmonary function (peak airway pressure and oxygenation), histological appearance, and reduced collagen content in lung allografts. In addition, we established a hIDO‐overexpressing type II cell line using the SB‐based gene transfer system and found that hIDO‐overexpressing lung cells effectively inhibited transforming growth factor–stimulated fibroblast proliferation in vitro. In summary, the SB‐based gene therapy with hIDO represents a new strategy for treating lung transplantation‐associated chronic complications, e.g., obliterative bronchiolitis.—Liu, H., Liu, L., Fletcher, B. S., Visner, G. A. Sleeping Beauty‐based gene therapy with indoleamine 2,3‐dioxygenase inhibits lung allograft fibrosis. FASEB J. 20, E1694 –E1703 (2006)

Impaired proteasome activity and accumulation of ubiquitinated substrates in a hereditary neuropathy model

Accumulation of misfolded proteins and alterations in the ubiquitin–proteasome pathway are associated with various neurodegenerative conditions of the CNS and PNS. Aggregates containing ubiquitin and peripheral myelin protein 22 (PMP22) have been observed in the Trembler J mouse model of Charcot‐Marie‐Tooth disease type 1A demyelinating neuropathy. In these nerves, the turnover rate of the newly synthesized PMP22 is reduced, suggesting proteasome impairment. Here we show evidence of proteasome impairment in Trembler J neuropathy samples compared with wild‐type, as measured by reduced degradation of substrate reporters. Proteasome impairment correlates with increased levels of polyubiquitinated proteins, including PMP22, and the recruitment of E1, 20S and 11S to aggresomes formed either spontaneously due to the Trembler J mutation or upon proteasome inhibition. Furthermore, myelin basic protein, an endogenous Schwann cell proteasome substrate, associates with PMP22 aggregates in affected nerves. Together, our data show that in neuropathy nerves, reduced proteasome activity is coupled with the accumulation of ubiquitinated substrates, and the recruitment of proteasomal pathway constituents to aggregates. These results provide novel insights into the mechanism by which altered degradation of Schwann cell proteins may contribute to the pathogenesis of certain PMP22 neuropathies.

Sleeping Beauty-mediated eNOS gene therapy attenuates monocrotaline-induced pulmonary hypertension in rats

Pulmonary hypertension (PH) is a life‐threatening disorder with high mortality rates and limited treatment options. Gene therapy is an alternative treatment strategy, yet viral vectors have inherent disadvantages including immune activation. The Sleeping Beauty (SB) transposon is a nonviral method of gene delivery that overcomes some of these drawbacks. A SB‐based transposon harboring a constitutively active endothelial nitric oxide synthase (eNOS) gene was administered to Sprague‐Dawley rats via tail vein injection using the carrier polyethylenimine. Two days after transposon delivery, monocrotaline (MCT) was administered to induce PH. Hemodynamic, histological, and molecular measurements were performed four weeks later. Animals coinjected with transposase showed a significant reduction in pulmonary arterial pressure (PABP, 31.67±6.03 mmHg, P<0.01), an attenuation of right ventricle (RV) to whole heart (WH) wt ratios (0.227±0.0252, P<0.05) and a decrease in the pulmonary vessel wall thickness index (36.87%, P<0.001), compared with those animals receiving the eNOS transposon and a nonfunctional transposase (PABP 44.33±4.04 mmHg; RV/WH ratio 0.280±0.01; wall thickness index 62.14%) or control animals receiving MCT injection alone (PABP 49.67±3.22 mmHg; RV/WH ratio 0.290±0.0265; wall thickness index 71.99%). The physiological improvements correlated with therapeutic gene expression, suggesting that transposon‐based genetic approaches have utility in the treatment of PH.—Liu, L., Liu, H., Visner, G., Fletcher, B. S. Sleeping Beauty‐mediated eNOS gene therapy attenuates monocrotaline‐induced pulmonary hypertension in rats. FASEB J. 20, E2068–E2076 (2006)

PiggyBac Transposon-based Inducible Gene Expression In Vivo After Somatic Cell Gene Transfer

Somatic cell gene transfer has permitted inducible gene expression in vivo through coinfection of multiple viruses. We hypothesized that the highly efficient plasmid-based piggyBac transposon system would enable long-term inducible gene expression in mice in vivo. We used a multiple-transposon delivery strategy to create a tetracycline-inducible expression system in vitro in human cells by delivering the two genes on separate transposons for inducible reporter gene expression along with a separate selectable transposon marker. Evaluation of stable cell lines revealed 100% of selected clones exhibited inducible expression via stable expression from three separate transposons simultaneously. We next tested and found that piggyBac-mediated gene transfer to liver or lung could achieve stable reporter gene expression in mice in vivo in either immunocompetent or immune deficient animals. A single injection of piggyBac transposons could achieve long-term inducible gene expression in the livers of mice in vivo, confirming our multiple-transposon strategy used in cultured cells. The plasmid-based piggyBac transposon system enables constitutive or inducible gene expression in vivo for potential therapeutic and biological applications without using viral vectors.

Targeting tumor endothelial marker 8 in the tumor vasculature of colorectal carcinomas in mice.

Tumor endothelial marker 8 (TEM8) is a recently described protein that is preferentially expressed within tumor endothelium. We have developed a fusion protein that targets TEM8 and disrupts tumor vasculature by promoting localized thrombosis. Fusion protein specificity and function were evaluated using Western blot analysis, ELISA, and enzymatic assays. A xenograft model of colorectal carcinoma was used to test the efficacy of targeted and control fusion proteins. Mice treated with the gene encoding anti-TEM8/truncated tissue factor exhibited a 53% reduction in tumor volume when compared with the untreated animals (P < 0.0001; n = 10) and achieved a 49% increase in tumor growth delay by Kaplan-Meier analysis (P = 0.0367; n = 6). Immunohistochemistry confirmed tumor endothelial expression of TEM8, fusion protein homing to tumor vasculature, decrease in vessel density, and localized areas of thrombosis. These data support the hypothesis that targeting TEM8 can be an effective approach to influence tumor development by disrupting tumor vasculature.

The promise of nanotechnology for solving clinical problems in breast cancer

Approaches for breast cancer treatment are invasive, disfiguring, have significant side‐effects, and are not always curative. Nanotechnology is an emerging area which is focused on engineering of materials <100 × 10−9 m. There is significant promise for advancing nanotechnology to improve breast cancer diagnosis and treatment including non‐invasive therapy, monitoring response to therapy, advanced imaging, treatment of metastatic disease, and improved nodal staging. Current approaches and important future directions are discussed. J. Surg. Oncol. 2011; 103:317–325.

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.

Molecular alterations resulting from frameshift mutations in peripheral myelin protein 22: implications for neuropathy severity.

Alterations in peripheral myelin protein 22 (PMP22) expression are associated with a heterogeneous group of hereditary demyelinating peripheral neuropathies. Two mutations at glycine 94, a single guanine insertion or deletion in PMP22, result in different reading frameshifts and, consequently, an extended G94fsX222 or a truncated G94fsX110 protein, respectively. Both of these autosomal dominant mutations alter the second half of PMP22 and yet are linked to clinical phenotypes with distinct severities. The G94fsX222 is associated with hereditary neuropathy with liability to pressure palsies, whereas G94fsX110 causes severe neuropathy diagnosed as Dejerine‐Sottas disease or Charcot‐Marie‐Tooth disease type IA. To investigate the subcellular changes associated with the G94 frameshift mutations, we expressed epitope‐tagged forms in primary rat Schwann cells. Biochemical and immunolabeling studies indicate that, unlike the wild‐type protein, which is targeted for the plasma membrane, frameshift PMP22s are retained in the cell, prior to reaching the medial Golgi compartment. Similar to Wt‐PMP22, both frameshift mutants are targeted for proteasomal degradation and accumulate in detergent‐insoluble, ubiquitin‐containing aggregates upon inhibition of this pathway. The extended frameshift PMP22 shows the ability to form spontaneous aggregates in the absence of proteasome inhibition. On the other hand, Schwann cells expressing the truncated protein proliferate at a significantly higher rate than Schwann cells expressing the wild‐type or the extended PMP22. In summary, these results suggest that a greater potential for PMP22 aggregation is associated with a less severe phenotype, whereas dysregulation of Schwann cell proliferation is linked to severe neuropathy.