Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.

The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice.

PiggyBac toolbox.

The PiggyBac (PB) transposon system was originally derived from the cabbage looper moth Trichoplusia ni and represents one of the most promising transposon systems to date. Engineering of the PB transposase enzyme (PBase) and its cognate transposon DNA elements resulted in a substantial increase in transposition activities. Consequently, this has greatly enhanced the versatility of the PB toolbox.

Transposon-mediated gene transfer into adult and induced pluripotent stem cells.

Transposon technology is a particularly attractive non-viral gene delivery paradigm that allows for efficient genomic integration into a variety of different cell types. In particular, transposon-mediated gene transfer is a promising tool for stem cell research, by virtue of its ability to efficiently and stably transfer genes into adult and induced pluripotent stem (iPS) cells. Moreover, transposons open up new perspectives for non-viral-mediated stem cell-based gene therapy.

Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.

Unlabelled: Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V).

Intrinsic cell memory reinforces myogenic commitment of pericyte-derived iPSCs.

Mesoangioblasts (MABs) are a subset of muscle-derived pericytes able to restore dystrophic phenotype in mice and dogs. However, their lifespan is limited and they undergo senescence after 25-30 population doublings. Recently, induced pluripotent stem cells (iPSCs) generated from reprogrammed fibroblasts have been demonstrated to have in vitro and in vivo myogenic potential when sorted for the SM/C-2.

Codon optimization of human factor VIII cDNAs leads to high-level expression.

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII.

Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation.

Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust nonviral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We have previously generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm.

Preclinical and clinical progress in hemophilia gene therapy.

Purpose Of Review: Hemophilia A and B are attractive target diseases for gene therapy, as stable expression of coagulation factor VIII and IX may correct the bleeding diathesis. This review focuses on the recent progress in preclinical and clinical studies in gene therapy for hemophilia A and B.

Recent Findings: Hepatic gene delivery using vectors derived from adeno-associated virus (AAV) resulted in therapeutic but transient functional clotting factor IX (FIX) expression levels in severe hemophilia B patients.

Comparative analysis of transposable element vector systems in human cells.

Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells.

Recent advances in lentiviral vector development and applications.

Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution.

Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors.

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells.

Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells.

Effective gene therapy requires robust delivery of the desired genes into the relevant target cells, long-term gene expression, and minimal risks of secondary effects. The development of efficient and safe nonviral vectors would greatly facilitate clinical gene therapy studies. However, nonviral gene transfer approaches typically result in only limited stable gene transfer efficiencies in most primary cells.

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 approximately 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.

Restoration of plasma von Willebrand factor deficiency is sufficient to correct thrombus formation after gene therapy for severe von Willebrand disease.

Objective: Gene therapy for severe von Willebrand disease (vWD) seems an interesting treatment alternative with long-term therapeutic potential. We investigated the feasibility of targeting the liver for ectopic expression of physiologically active von Willebrand factor (vWF).

Methods And Results: The capacity of transgene-encoded murine vWF to restore vWF function was studied in a mouse model of severe vWD after liver-specific gene transfer by hydrodynamic injection.

Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells.

Human bone marrow (BM) mesenchymal stem/progenitor cells are potentially attractive targets for ex vivo gene therapy. The potential of lentiviral vectors for transducing BM mesenchymal cells was examined using a self-inactivating vector that expressed the green fluorescent protein (GFP) from an internal cytomegalovirus (CMV) promoter. This vector was compared with oncoretroviral vectors expressing GFP from the CMV promoter or a modified long-terminal repeat that had been optimized for long-term expression in stem cells.

Cutting through the obstacles and resurrecting the promise of gene therapy.

Gene therapy is a promising biomedical discipline that could potentially lead to new treatments and perhaps long-term curative effects for a plethora of diseases including hereditary disorders, cardiovascular and neurological diseases, cancer, diabetes and even infectious or autoimmune diseases. These diseases affect millions of people worldwide and the development of effective and safe gene-based drugs obviously represent a tremendous market potential. Convincing evidence continues to emerge from clinical trials demonstrating that gene therapy can be effective in patients suffering from a limited number of different diseases.

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies.

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome.

Biosafety of adenoviral vectors.

Adenoviral vectors can efficiently transduce a broad variety of different cell types and have been used extensively in preclinical and clinical studies. However, early generation of adenoviral vectors retained residual adenoviral genes that contribute to inflammatory immune responses and toxicity. In addition, these vectors often result in transient expression of the potentially therapeutic transgene.

Biosafety of onco-retroviral vectors.

Extensive gene therapy studies in preclinical models and in clinical trials underscore the relative safety of onco-retroviral vectors. Up until recently, no adverse effects have been reported in nearly 2000 patients that were enrolled in gene therapy clinical trials involving onco-retroviral vectors. However, the main safety concern of using onco-retroviral vectors is related to the risk of malignant transformation following oncogene activation due to random onco-retroviral genomic integration.

Nanoparticles for the delivery of genes and drugs to human hepatocytes.

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues.

Efficiency of onco-retroviral and lentiviral gene transfer into primary mouse and human B-lymphocytes is pseudotype dependent.

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.

Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors.

High-capacity adenoviral (HC-Ad) vectors expressing B-domain-deleted human or canine factor VIII from different liver-specific promoters were evaluated for gene therapy of hemophilia A. Intravenous administration of these vectors into hemophilic FVIII-deficient immunodeficient SCID mice (FVIIIKO-SCID) at a dose of 5 x 10(9) infectious units (IU) resulted in efficient hepatic gene delivery and long-term expression of supraphysiologic FVIII levels (exceeding 15 000 mU/mL), correcting the bleeding diathesis. Injection of only 5 x 10(7) IU still resulted in therapeutic FVIII levels.

Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo.

High-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)-based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.