Recent developments in transposon-mediated gene therapy

INTRODUCTION: The continuous improvement of gene transfer technologies has broad implications for stem cell biology, gene discovery, and gene therapy. Although viral vectors are efficient gene delivery vehicles, their safety, immunogenicity and manufacturing challenges hamper clinical progress. In contrast, non-viral gene delivery systems are less immunogenic and easier to manufacture. AREAS COVERED: In this review, we explore the emerging potential of transposons in gene and cell therapy. The safety, efficiency, and biology of novel hyperactive Sleeping Beauty (SB) and piggyBac (PB) transposon systems will be highlighted for ex vivo gene therapy in clinically relevant adult stem/progenitor cells, particularly hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), myoblasts, and induced pluripotent stem (iPS) cells. Moreover, efforts toward in vivo transposon-based gene therapy will be discussed. EXPERT OPINION: The latest generation SB and PB transposons currently represent some of the most attractive systems for stable non-viral genetic modification of primary cells, particularly adult stem cells. This paves the way toward the use of transposons as a non-viral gene therapy approach to correct hereditary disorders including those that affect the hematopoietic system. The development of targeted integration into "safe harbor" genetic loci may further improve their safety profile.     

Hyperactive piggyBac gene transfer in human cells and in vivo

We characterized a recently developed hyperactive piggyBac (pB) transposase enzyme [containing seven mutations (7pB)] for gene transfer in human cells in vitro and to somatic cells in mice in vivo. Despite a protein level expression similar to that of native pB, 7pB significantly increased the gene transfer efficiency of a neomycin resistance cassette transposon in both HEK293 and HeLa cultured human cells. Native pB and SB100X, the most active transposase of the Sleeping Beauty transposon system, exhibited similar transposition efficiency in cultured human cell lines. When delivered to primary human T cells ex vivo, 7pB increased gene delivery two- to threefold compared with piggyBac and SB100X. The activity of hyperactive 7pB transposase was not affected by the addition of a 24-kDa N-terminal tag, whereas SB100X manifested a 50% reduction in transposition. Hyperactive 7pB was compared with native pB and SB100X in vivo in mice using hydrodynamic tail-vein injection of a limiting dose of transposase DNA combined with luciferase reporter transposons. We followed transgene expression for up to 6 months and observed approximately 10-fold greater long-term gene expression in mice injected with a codon-optimized version of 7pB compared with mice injected with native pB or SB100X. We conclude that hyperactive piggyBac elements can increase gene transfer in human cells and in vivo and should enable improved gene delivery using the piggyBac transposon system in a variety of cell and gene-therapy applications.     

Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.

Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.     

Hyperactive transposase mutants of the Sleeping Beauty transposon.

Transposable elements have enormous potential to overcome one of the major hurdles in nonviral gene delivery, namely the lack of long-term gene expression. The Sleeping Beauty (SB) transposon is a promising vector system for nonviral gene therapy as it has the highest transposition activity of all known DNA transposons within mammalian cells. In an effort to generate a more efficient delivery vehicle, we conducted a systematic evaluation of several novel and previously identified SB transposase mutants. The results indicate that certain combinations of mutants do not enhance transposition, whereas others give a synergistic response. The most active mutant, designated HSB17, shows nearly 17-fold higher transposition activity compared to the original transposase SB10 when tested within the same expression cassette. In addition, synergistic activity is observed when this hyperactive mutant is combined with an improved transposon. Animal studies utilizing the hyperactive transposase show enhanced long-term reporter gene expression. These modifications further expand the utility of this transposon-based gene transfer system.     

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.     

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.     

Nonviral gene delivery with the sleeping beauty transposon system

Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine, and gene- and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors, and potential risks associated with random genomic insertion.     

Human satellite cells have regenerative capacity and are genetically manipulable

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7⁺ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7⁺ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7⁺ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7⁺ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7⁺ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7⁺ satellite cells have potential to restore gene function in muscular dystrophies.     

Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo

The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.     

成体干细胞及其分化机理

成体干细胞是存在于胎儿和成人组织器官中具有自我更新，高度增殖和多向分化潜能的细胞，在适当的诱导条件下。可变成不同类型的细胞。成体干细胞的分化是指成体干细胞具有可在体内、体外分化成不同类型细胞的能力，将其分化物植入体内后在各种情况下都能稳定地存活。目前认为分化的可能机制是：多种成体干细胞可能在不同的器官中存在，包括出生后体内仍存在的多能性干细胞。美国Minnesota大学干细胞研究所一系列的研究证明成体骨髓中确实存在具有多分化潜能的成体干细胞，命名为MAPC，能在体外由单细胞分化为具有中胚层系、外胚层系或内胚层系特征的细胞；将其注入胚泡，能分化成各种组织细胞。多能的成体干细胞在今后能用于多个不同器官的变性或遗传性疾病的治疗。     

Integration and long-term expression in xenografted human glioblastoma cells using a plasmid-based transposon system.

Gene therapy has the potential to become an effective component of cancer treatment by transferring genes that cause immunomodulation or tumor cell death or that inhibit angiogenesis into tumor cells or tumor-associated stroma. Viral vectors have been the primary gene transfer vehicles used for intratumoral gene transfer to date. Plasmid-based vectors may be safer and more scalable than viral vectors. However, attempts at plasmid-based intratumoral gene transfer have been met with transient expression and poor gene transfer efficiency. Here we report integration and long-term expression of reporter genes in human glial tumors, growing in nude mice, using the Sleeping Beauty (SB) transposon system. A two-plasmid system was used, in which linear polyethylenimine was complexed with a GFP, NEO, or luciferase transposon plasmid and a SB transposase-expressing plasmid. SB-mediated transposition led to chromosomal integration of the NEO transgene in roughly 8% of tumor cells. SB-mediated insertions were cloned from the genomes of glial tumor cells to provide molecular proof of transposase-mediated integration. Luciferase studies showed that SB facilitated long-term expression of the transgene in glial tumors. SB-mediated intratumoral gene transfer is a novel, nonviral technique that could be used to augment conventional therapy for glioblastoma or other cancers.     

活体生物发光成像追踪大鼠跟腱内移植干细胞

背景：移植脂肪源干细胞在活体内的归巢、迁移、增殖和分化的机制仍未得到充分阐明。活体生物发光活体成像技术是近来发展起来的一种可以直接检测活细胞在动物体内生物学行为的新的技术方法。目的：探讨用活体生物发光成像技术检测大鼠跟腱内移植的经荧光基因修饰的脂肪源干细胞可行性。方法：分离培养大鼠腹腔来源的脂肪源干细胞,用浓度为3×10^10L^-1携带虫荧光素酶的腺病毒载体对其进行转染,观察转染对脂肪源干细胞的影响;将转染的脂肪源干细胞移植到大鼠跟腱缺损处,移植后1,4,7,14d用活体生物发光成像技术检测移植脂肪源干细胞荧光素酶的表达,移植后28d跟腱标本冰冻切片在荧光显微镜下观察。结果与结论：在体外,腺病毒转染对脂肪源干细胞的生长和增殖无明显影响（P〉0．05）。细胞移植后1,4,7,14d,活体生物发光成像技术在实验侧修复段跟腱检测到的荧光表达强度分别为（1．22±0．43）×10^6、（1．81±0．76）×10^6、（1．88±0．69）×10^6和（0．89±0．26）×10^5光子／s（n=6）。而对照侧跟腱修复段未检测到荧光表达;移植后28d,实验侧跟腱冰冻切片在荧光显微镜下见到大量表达荧光的细胞。表明活体生物发光成像技术可成功追踪大鼠跟腱内移植的经荧光基因修饰的脂肪源干细胞。脂肪源干细胞有望成为肌腱组织工程的种子细胞。     

Sustained FVIII expression and phenotypic correction of hemophilia A in neonatal mice using an endothelial-targeted sleeping beauty transposon.

Hemophilia A, deficiency of coagulation factor VIII (FVIII), is an attractive candidate for gene therapy as expression of modest amounts of FVIII can provide therapeutic benefit. Most gene transfer approaches for hemophilia have focused on the liver, as this is the major source of endogenous FVIII; however, increasing evidence suggests that endothelial cells are capable of synthesis and release of FVIII. Here the Sleeping Beauty (SB) transposon is employed to target long-term expression of the human B-domain-depleted FVIII gene (approved gene symbol F8) within lung endothelia of hemophilic mice. As the formation of inhibitory antibodies to FVIII has been a significant impediment toward achieving therapeutic plasma levels after gene or protein therapy, we chose to perform gene transfer in neonatal mice, which are more likely to be immune tolerant. Using this approach, low therapeutic levels of FVIII ( approximately 10%), as well as phenotypic correction of the bleeding disorder, were achieved in all animals that received the FVIII transposon and functional transposase throughout the duration of the study (24 weeks). Rechallenge of these animals with additional gene transfer did not result in significant increases in FVIII levels, due mainly to increases in inhibitory antibodies. These studies demonstrate the feasibility of using endothelial-targeted SB transposons for the treatment of hemophilia A.     

Adipose tissue-derived therapeutics

Human adipose tissue provides a uniquely abundant and accessible source of adult stem cells for applications in tissue engineering and regenerative medicine. The adult stem cells are isolated by collagenase digestion, differential centrifugation and subsequent adherence to a plasticware surface. Based on their immunophenotype, the cells are relatively homogeneous, with shared expression of integrin beta(1), the hyaluronate receptor, and the tetraspan protein CD9, among other markers. In response to chemical, hormonal or structural stimuli, the adipose-derived adult stem (ADAS) cells can differentiate along multiple lineage pathways, including adipocytes, chondrocytes, myocytes, neurons and osteoblasts. The cells can be transduced with viral vectors and have potential utility as gene delivery vehicles. Further studies will facilitate the clinical and commercial development of ADAS cells. First, it will be necessary to develop closed system bioreactors for the large-scale manufacture of ADAS cells. Second, methods that improve the vascularisation of in vivo implants will allow transplantation of larger engineered tissues. Finally, experiments must investigate the feasibility of transplanting allogeneic, as compared to autologous, ADAS cells for therapeutic applications. Based on the promising findings from adipose-derived and other adult stem cells to date, it is likely that future studies will address these challenges.     

Adipose tissue-derived therapeutics

Human adipose tissue provides a uniquely abundant and accessible source of adult stem cells for applications in tissue engineering and regenerative medicine. The adult stem cells are isolated by collagenase digestion, differential centrifugation and subsequent adherence to a plasticware surface. Based on their immunophenotype, the cells are relatively homogeneous, with shared expression of integrin β₁, the hyaluronate receptor, and the tetraspan protein CD9, among other markers. In response to chemical, hormonal or structural stimuli, the adipose-derived adult stem (ADAS) cells can differentiate along multiple lineage pathways, including adipocytes, chondrocytes, myocytes, neurons and osteoblasts. The cells can be transduced with viral vectors and have potential utility as gene delivery vehicles. Further studies will facilitate the clinical and commercial development of ADAS cells. First, it will be necessary to develop closed system bioreactors for the large-scale manufacture of ADAS cells. Second, methods that improve the vascularisation of in vivo implants will allow transplantation of larger engineered tissues. Finally, experiments must investigate the feasibility of transplanting allogeneic, as compared to autologous, ADAS cells for therapeutic applications. Based on the promising findings from adipose-derived and other adult stem cells to date, it is likely that future studies will address these challenges.     

Duration of Expression and Activity of Sleeping Beauty Transposase in mouse liver following hydrodynamic DNA delivery

The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that “cuts” the transposon from a plasmid and “pastes” it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts.     

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.