Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous ...transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.
Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and ...adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically.
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder ...with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients' clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.).
Gene therapy is a technique that aims at the delivery of nucleic acids to cells, to obtain a therapeutic effect. In situ gene therapy consists of the administration of the gene product to a specific ...site. It possesses several advantages, such as the reduction in potential side effects, the need for a lower vector dose, and, as a consequence, reduced costs, compared to intravenous administration. Different vectors, administration routes and doses involving in situ gene transfer have been tested both in animal models and humans, with in situ gene therapy drugs already approved in the market. In this review, we present applications of in situ gene therapy for different diseases, ranging from monogenic to multifactorial diseases, focusing mainly on therapies designed for the intra-articular and intraocular compartments, as well as gene therapies for the central nervous system (CNS) and for tumors. Gene therapy finally seems to blossom as a viable therapeutic approach. The growth in the number of clinical protocols shown here is evident, and the positive outcomes observed in several clinical trials indicate that more products based on in situ gene therapy should reach the market in the next years.
The use of a “size-tunable” polyurethane resistive pulse sensor for quantitative sizing of nano- and microparticles is presented. A linear relationship, as first suggested by Maxwell, between ...particle volume and change in electric resistance across the pore was observed. Particle sizes were quantified for a given size-tunable membrane, by first creating a linear calibration curve to a series of monodisperse carboxylated polystyrene particles of various diameters and then applying this curve to calculate the size of “unknown” nanoparticles. The diameters of a selection of synthetic and biological particles, being PMMA and nonfunctionalized polystyrene particles, along with biological nanoparticles (adenovirus) were calculated using this methodology. Calculated particle diameters and coefficients of variation were shown to be in good agreement with both transmission electron microscopy and dynamic light scattering results.
Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the ...wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.
Upstream production of AAV therapies is often driven by transient transfection of suspension HEK cells with multiple plasmid DNA constructs encoding essential virus proteins. AAV therapies require ...high-quality and high titers of viral genomes per dosage, prompting the need for a more efficient AAV manufacturing method to reduce overall costs. Improving AAV titers and quality hold significant implications for the economical manufacturing of high-dose assets and mainstream utilization of AAV gene therapy.
The RevIT™ AAV Enhancer is compatible with TransIT-VirusGEN® Transfection Reagent or conventional polymeric transfection reagents. RevIT™ AAV Enhancer has the potential to increase higher genome titers 2-4X in a range of suspension HEK 293 subtypes and cell culture media. When combined with the TransIT-VirusGEN® Transfection Reagent, this enhancer not only yields the highest titers but also allows for the use of lower amounts of plasmid DNA which represents a key cost-saving opportunity.
Additional features of RevIT™ include flexibility to produce multiple serotypes and transfection complex stability. RevIT™ AAV Enhancer produces high quality titers across a range of AAV serotypes, including AAV2, AAV5, AAV8, and AAV9. Complex stability is also a hallmark of TransIT-VirusGEN and RevIT with extended complex formation times required in large-scale AAV manufacture that do not negatively impact cell viability. We also implemented Design of Experiments (DOE) to optimize and determine the interactions of DNA dosage, the TransIT-VirusGEN:DNA ratio, and cell density. Optimized conditions were then tested in combination with a range of the RevIT™ AAV Enhancer to further increase genome titers by >2-fold. Productions with TransIT-VirusGEN® and RevIT™ AAV Enhancer are linearly scalable; small-scale shake flask model can be employed for DOE screening and be representative of results of stirred tank bioreactors. Through increasing recombinant AAV titers, RevIT™ AAV Enhancer paves the way for driving down upstream manufacturing costs, resulting in a lower cost per therapeutic dose.
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