Showing papers in "Current Gene Therapy in 2013"
TL;DR: Current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells are covered.
Abstract: The unlimited proliferation capacity of embryonic stem cells (ESCs) combined with their pluripotent differentiation potential in various lineages raised great interest in both the scientific community and the public at large with hope for future prospects of regenerative medicine. However, since ESCs are derived from human embryos, their use is associated with significant ethical issues preventing broad studies and therapeutic applications. To get around this bottleneck, Takahashi and Yamanaka have recently achieved the conversion of adult somatic cells into ES-like cells via the forced expression of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. This first demonstration attracted public attention and opened a new field of stem cells research with both cognitive – such as disease modeling - and therapeutic prospects. This pioneer work just received the 2012 Nobel Prize in Physiology or Medicine. Many methods have been reported since 2006, for the generation of induced pluripotent stem (iPS) cells. Most strategies currently under use are based on gene delivery via gamma-retroviral or lentiviral vectors; some experiments have also been successful using plasmids or transposons-based systems and few with adenovirus. However, most experiments involve integration in the host cell genome with an identified risk for insertional mutagenesis and oncogenic transformation. To circumvent such risks which are deemed incompatible with therapeutic prospects, significant progress has been made with transgene-free reprogramming methods based on e.g.: sendai virus or direct mRNA or protein delivery to achieve conversion of adult cells into iPS. In this review we aim to cover current knowledge relating to both delivery systems and combinations of inducing factors including chemicals which are used to generate human iPS cells. Finally, genetic instability resulting from the reprogramming process is also being considered as a safety bottleneck for future clinical translation and stem cell-therapy prospects based on iPS.
105 citations
TL;DR: The present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery are reviewed.
Abstract: The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of “facultative” progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery.
60 citations
TL;DR: Recent studies that have propelled the field forward are presented and challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease are discussed.
Abstract: Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somatic cells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the way for retinal therapies with the ongoing development of safe animal cell transplantation studies and characterization of patient- specific and disease-specific human iPSc. The retina is an optimal model for investigation of neural regeneration; amongst other advantageous attributes, it is the most accessible part of the CNS for surgery and outcome monitoring. A recent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severely degenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitro increase the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. In this review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease.
53 citations
TL;DR: The current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages is discussed, focussing on studies which have demonstrated a disease phenotype in the tissue of interest.
Abstract: Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling, as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic.
51 citations
TL;DR: The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications and the main issues that such guidelines should address.
Abstract: Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address.
46 citations
TL;DR: An overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications, and how the rapid pace of development has led to a transition in the field of genome engineering.
Abstract: Over three years following the discovery of the TAL code, artificial TAL effector DNA binding domains have emerged as the premier platform for building site-specific DNA binding polypeptides for use in biological research. Here, we provide an overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications. We also discuss considerations for choosing TAL effector/mDBD or alternative nuclease technologies for genome engineering projects ranging from basic laboratory gene editing of cultured cell lines to therapeutics. Finally, we highlight how the rapid pace of development of mDBD-based, such as monomeric TALENs (I-TevI-TAL), and more recently RNA-guided nucleases (CRISPR-Cas9) has led to a transition in the field of genome engineering towards development of the next generation of technologies aimed at controlling events that occur after targeted DNA breaks are made.
46 citations
TL;DR: The origins of the understanding of IM are reviewed and applied to the clinical gene therapy trials conducted with retroviral vectors, with a view to provide a comprehensive account of this emerging field.
Abstract: Retroviral vectors derived from gammaretroviruses or lentiviruses have now been used extensively in clinical gene therapy trials for several diseases including primary immunodeficiencies, beta thalassaemia and adrenoleukodystrophy. Their utility in this setting has been readily demonstrated by the largely favourable outcomes in recent clinical trials, however this success has been marred by the emergence of malignancies in some trials. These malignancies were a consequence of perturbation of cellular proto-oncogene expression by the integrated retroviral vectors, the process of which is referred to as ‘insertional mutagenesis’ (IM). In this review, the origins of our understanding of IM are reviewed and applied to the clinical gene therapy trials conducted with retroviral vectors. Old and new methods for assessing this phenomenon are discussed with a view to provide a comprehensive account of this emerging field.
34 citations
TL;DR: UTMD has a number of advantages, such as target-specific, highly effective, non-invasivity, relatively low-cost and no radiation, and has broad application prospects, which is regarded as one hot spot in medical studies.
Abstract: Ultrasound is a common used technique for clinical imaging. In recent years, with the advances in preparation technology of microbubbles and the innovations in ultrasound imaging, ultrasound is no longer confined to detection of tissue perfusion, but extends to specific ultrasound molecular imaging and target therapy gradually. With the development of research, ultrasound molecular imaging and target therapy have made great progresses. Targeted microbubbles for molecular imaging are achieved by binding target molecules, specific antibody or ligand to the surface of microbubbles to obtain specific imaging by attaching to target tissues. Meanwhile, it can also achieve targeting gene therapy or drug delivery by ultrasound targeted microbubble destruction (UTMD) mediating genes or drugs to specific target sites. UTMD has a number of advantages, such as target-specific, highly effective, non-invasivity, relatively low-cost and no radiation, and has broad application prospects, which is regarded as one hot spot in medical studies. We reviewed the new development and application of UTMD in gene therapy and drug delivery in this paper. With further development of technology and research, the gene or drug delivery system and related methods will be widely used in application and researches.
33 citations
TL;DR: The latest strategies for producing helper-dependent adenovirus (HDAd) vectors are summarized, the main areas of interest for which HDAd vectors are being used are described, and the future prospects forHDAd vectors in gene therapy are commented on.
Abstract: Sixteen years after Graham and coworkers described the most used system for generating helper-dependent adenovirus (HDAd) vectors, production systems have evolved considerably, and most resulting preparations have titres of 1 × 10(13) IU/ml (infection units/ml) and very low helper contamination levels (<0.1%). These advances in production, as well as the attractive characteristics of these vectors (large insert capacity and low cell immune response compared with first-generation Ad vectors) make them very interesting for many research purposes as they have become more accessible to the scientific community. In this review we summarise the latest strategies for producing HDAd vectors, describe the main areas of interest for which HDAd vectors are being used, and comment on the future prospects for HDAd vectors in gene therapy.
31 citations
TL;DR: This review addresses the various strategies employed to help the establishment of rAAV-based clinical gene therapy in the near future, namely exploring distinct serotype tropisms, the production of mosaic and chimeric capsids, the selection of vectors through directed evolution, the development of self-complementary vectors, the use of pharmacological adjuvants and the induction of specific capsid mutations.
Abstract: Recombinant adeno-associated viral vectors (rAAV) have now been used in several clinical trials to treat a variety of diseases, and are currently the preferred choice of many investigators in the field, due to both their low pathogenicity and immunogenicity compared with other viral vectors, as well as localized long-term gene expression, despite their limitations of DNA size packaging and speed of expression. Recently, a number of advances have led to new generations of rAAV vectors, with improved features. This review addresses the various strategies employed to such effect, namely exploring distinct serotype tropisms, the production of mosaic and chimeric capsids, the selection of vectors through directed evolution, the development of self-complementary vectors, the use of pharmacological adjuvants and the induction of specific capsid mutations. Such approaches are expected to help the establishment of rAAV-based clinical gene therapy in the near future.
21 citations
TL;DR: This field did not meet the expectation raised initially, mainly because of difficulties with obtaining sustained therapeutic plasma levels in animal models, but this situation is changing rapidly and the therapeutic potential of these approaches is getting closer to clinical applications.
Abstract: Monoclonal antibodies are successfully used in the clinic. However, repeated high-dose bolus injections imply high costs. As an alternative to recombinant protein administration, gene therapy may provide a novel mean for systemic delivery of monoclonal antibodies. This strategy has been used in preclinical studies of a wide variety of pathological conditions, including cancer, infectious diseases, drug addiction, retinal neovascularisation and Alzheimer's disease. The two main gene therapy approaches are based on direct gene delivery (using viral or non-viral vectors) or on inoculation of ex vivo genetically modified cells (autologous or allogenic). Viral vectors are highly efficient as gene delivery vehicles and have been tested in numerous clinical trials, but still raise concerns about safety and limitation of the therapeutic effect due to immune responses against viral antigens. On the other hand, use of standard non-viral vectors has been limited by their low transduction efficiency. Practical application of ex vivo genetically modified cells would imply the availability of stocks of "ready-to-use" gene-modified allogenic cells that should be protected from the host immune system. Actually, this field did not meet the expectation raised initially, mainly because of difficulties with obtaining sustained therapeutic plasma levels in animal models. However, this situation is changing rapidly and the therapeutic potential of these approaches is getting closer to clinical applications. This review focuses on the current achievements in preclinical studies, as well as the challenges and future prospects of antibody gene therapy.
TL;DR: It is demonstrated that bone marrow derived mesenchymal stem cells (MSCs) and overexpression of human manganese superoxide dismutase (MnSOD) could ameliorate radiation-induced intestinal syndrome and intravenously transplanted MnSOD-MSCs could colonize the irradiated intestine and repair injured sites.
Abstract: Radiation-induced intestinal injury is a common complication in radiotherapy for solid organ malignancies in abdomen or pelvis. However, currently there are no approved medical countermeasures for radiation-induced intestinal injury. Therefore, it is urgent to develop new treatments for radiation-induced intestinal injury. In the present study, we demonstrated that bone marrow derived mesenchymal stem cells (MSCs) and overexpression of human manganese superoxide dismutase (MnSOD) could ameliorate radiation-induced intestinal syndrome. NOD/SCID mice received abdominal irradiation at a selected dose of 5 Gy, and then infused intravenously with MnSOD-MSCs. Mice body weight, survival and diarrhea were monitored for 30-days. Colonization and differentiation of MnSOD-MSCs in the irradiated intestine were analyzed by histological and immunohistochemical methods. Consequently, our data demonstrated that intravenous administration of MnSOD-MSCs improved survival, decreased diarrhea occurrence and protected the small intestinal structural integrity of irradiated mice. Moreover, intravenously transplanted MnSOD-MSCs could colonize the irradiated intestine and repair injured sites. These findings suggested that MnSOD-MSCs may be an attractive and potential option for radiation-induced intestinal injury.
TL;DR: The data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.
Abstract: Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 μm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.
TL;DR: It is shown that p53, pRB, and p130 gene transfer mediated by US cavitation of microbubbles at the tumor site resulted in targeted gene transduction and increased reduction in tumor growth compared to DU-145 prostate cancer cell xenografts treated intratumorally with adenovirus or radiation alone.
Abstract: Combining radiation therapy and direct intratumoral (IT) injection of adenoviral vectors has been explored as a means to enhance the therapeutic potential of gene transfer. A major challenge for gene transfer is systemic delivery of nucleic acids directly into an affected tissue. Ultrasound (US) contrast agents (microbubbles) are viable candidates to enhance targeted delivery of systemically administered genes. Here we show that p53, pRB, and p130 gene transfer mediated by US cavitation of microbubbles at the tumor site resulted in targeted gene transduction and increased reduction in tumor growth compared to DU-145 prostate cancer cell xenografts treated intratumorally with adenovirus (Ad) or radiation alone. Microbubble-assisted/US-mediated Ad.p53 and Ad.RB treated tumors showed significant reduction in tumor volume compared to Ad.p130 treated tumors (p<0.05). Additionally, US mediated microbubble delivery of p53 and RB combined with external beam radiation resulted in the most profound tumor reduction in DU-145 xenografted nude mice (p<0.05) compared to radiation alone. These findings highlight the potential therapeutic applications of this novel image-guided gene transfer technology in combination with external beam radiation for prostate cancer patients with therapy resistant disease.
TL;DR: 3D cell culture models are more relevant concerning in vivo cell organization since cell-cell contacts are present as well as extracellular matrix and spheroid as a model to address and improve the electrotransfer processes.
Abstract: The use of electrotransfer to deliver therapeutic agents such as cytotoxic drugs and nucleic acids to cells and tissues has been successfully developed over the last decade. This strategy is promising for the systemic secretion of therapeutic proteins, vaccination and gene therapy. The safe and efficient use of this physical method for clinical purposes requires knowledge of the mechanisms underlying the DNA electrotransfer and expression phenomena. Despite the fact that the pioneering work on plasmid DNA electrotransfer to cells was initiated 30 years ago, many of the underlying mechanisms remain elucidated. While efficient in vitro, the method faces a lack of efficiency in packed tissues. Until now, the great majority of studies have been performed on cells in 2D cultures in Petri dishes or in suspension. However, these studies cannot get access to the tissue-specific architecture and organization present in 3D living tissues. In this context, 3D cell culture models are more relevant concerning in vivo cell organization since cell-cell contacts are present as well as extracellular matrix. The aim of this review is to describe the relevance of using spheroid as a model to address and improve the electrotransfer processes.
TL;DR: The ability of liposomes coated with specific ligands such as Apo-E derived RGD R9 and Tat peptide, to reverse the conceptualisation of drug resistance and cross the blood brain barrier, provides promising future for their use as an efficient drug delivery system.
Abstract: Understanding the cellular target structure and thereby proposing the best delivery system to achieve sustained release of drugs has always been a significant area of focus in biomedical research for translational benefits. Specific targeting of the receptors expressed on the target cell represents an effective strategy for increasing the pharmacological efficacy of the administered drug. Liposomes offer enhanced conveyance as a potential carrier of biomacromolecules such as anti-cancer proteins, drugs and siRNA for targeting tumour cell death. Commonly used liposomal constructs for various therapies are Doxil, Myocet, DepoCyt and Abraxanes. However, recent strategy of using multifunctional liposomes for the sustained release of drugs with increased plasma residence time and monoclonal antibody-based targeting of tumours coupled with imaging modalities have attracted enormous scientific attention. The ability of liposomes coated with specific ligands such as Apo-E derived RGD R9 and Tat peptide, to reverse the conceptualisation of drug resistance and cross the blood brain barrier, provides promising future for their use as an efficient drug delivery system. By outlining the recent advancements and innovations in the established concept of liposomal drug delivery, this review will focus on the multifunctional liposomes as an emerging novel lipid based drug delivery system.
TL;DR: Artificial pri-miRNAs targeting non-coding cis-active modules and/or conserved sequences of the Emx2 locus were delivered to embryonic cortico-cerebral precursors, by lentiviral vectors, resulting in an appreciable expansion of the neuronogenic precursor pool.
Abstract: Emx2 encodes for a transcription factor controlling several aspects of cerebral cortex development. Its overexpression promotes self-renewal of young cortico-cerebral precursors, it promotes neuronal rather than gliogenic fates and it protects neuronal progenitors from cell death. These are all key activities for purposes of gene-promoted brain repair. Artificial pri-miRNAs targeting non-coding cis-active modules and/or conserved sequences of the Emx2 locus were delivered to embryonic cortico-cerebral precursors, by lentiviral vectors. A subset of these pri-miRNAs upregulated Emx2, possibly stimulating its transcription. That led to enhanced self-renewal, delayed differentiation and reduced death of neuronally committed precursors, resulting in an appreciable expansion of the neuronogenic precursors pool. This method makes Emx2 overexpression for purposes of brain repair a more feasible goal, avoiding the drawbacks of exogenous gene copies introduction. Interestingly, the two genomic enhancers targeted by these pri-miRNAs were discovered to be naturally transcribed. Their expression profile suggests their possible involvement in regulation of Emx2 transcription.
TL;DR: The results for the first time showed in Chinese population, NF against AAV2 was present in serum of all the patients with AMD or PCV and controls, and there were no significant differences among these groups, demonstrating there was no correlations between A AV2 NF titer and these diseases.
Abstract: Adeno-associated virus type 2 (AAV2) mediated gene therapy providing a potential treatment in the eye. However, immune responses can limit virally mediated gene transfer and therapy. To assess preexisting AAV2 neutralizing factors (NF) titers in peripheral blood and the vitreous in patients with age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). 130 subjects were enrolled: 50 with neovascular AMD, 30 with PCV, and 50 controls. The serum and the vitreous were obtained for AAV2 NF assay. We found AAV2 NF are present in all of AMD, PCV patients and controls we tested. There were no significant differences in prevalence of NAb in serum between AMD, PCV and controls (P =0.999). There was no correlation between NF in serum and in vitreous (P>0.05), and NF in vitreous was significantly less than in serum. Our results for the first time showed in Chinese population, NF against AAV2 was present in serum of all the patients with AMD or PCV and controls, and there were no significant differences among these groups. Therefore, it demonstrated there were no correlations between AAV2 NF titer and these diseases. We found NF in vitreous was considerably less than in serum in all groups. We also found no direct correlation between NF in vitreous and in serum suggesting serum antibody levels may not be used to predict their counterparts in the vitreous. Our results will provide crucial information for future clinical studies in the development of new therapies based on AAV2 mediated gene delivery in the eye.
TL;DR: The recent demonstration that human somatic cells can be genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methods to differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a large number of human pathologies.
Abstract: The fundamental inaccessibility of the human neural cell types affected by neurological disorders prevents their isolation for in vitro studies of disease mechanisms or for drug screening efforts. Pluripotent stem cells represent a new interesting way to generate models of human neurological disorders, explore the physiopathological mechanisms and develop new therapeutic strategies. Disease-specific human embryonic stem cells were the first source of material to be used to study certain disease states. The recent demonstration that human somatic cells, such as fibroblasts or blood cells, can be genetically converted to induced pluripotent stem cells (hiPSCs) together with the continuous improvement of methods to differentiate these cells into disease-affected neuronal subtypes opens new perspectives to model and understand a large number of human pathologies. This review focuses on the opportunities concerning the use disease-specific human pluripotent stem cells as well as the different challenges that still need to be overcome. We also discuss the recent improvements in the genetic manipulation of human pluripotent stem cells and the consequences of these on disease modeling and drug screening for neurological diseases.
TL;DR: This review covers the most important, promising and successful approaches to improve siRNA delivery, including cell-specific delivery coupling ligands or antibodies with nanodevices to improveSiRNA efficiency and specificity.
Abstract: The delivery of small interfering RNAs (siRNAs) is a promising approach to silencing gene expression aimed at treating infections, cancer, neurodegenerative diseases and various other disorders. Recent progress in this area has been achieved with nanodevices possessing multiple properties and assembled with new, biodegradable, synthetic polymers and polysaccharides. Different synthetic routes and multiple strategies, such as multilayer systems and stimuliresponsive polymers, have been developed to attain high efficiencies. This review covers the most important, promising and successful approaches to improve siRNA delivery. It is a concise report on multiple strategies employed, including cell-specific delivery coupling ligands or antibodies with nanodevices to improve siRNA efficiency and specificity.
TL;DR: The results suggest that the genetic excision of the pathogenicity island ROD21 in S. Enteritidis modulates the capacity of this bacterium to cause disease in mice due to a change in the expression of virulence genes.
Abstract: Although the excision of unstable pathogenicity islands is a phenomenon that has been described for several virulent bacteria, whether this process directly affects the capacity of these microorganisms to cause disease in their hosts remains unknown. Salmonella enterica serovar Enteritidis (S. Enteritidis) is an enterobacterium that harbors several unstable pathogenicity islands that can excise from the main bacterial chromosome. Here we have evaluated whether excision of one of these pathogenicity islands, denominated as Region of Difference 21 (ROD21), is required for S. Enteritidis to cause disease in the host. By means of genetic targeting of the integrase encoded by the ROD21 we have generated S. Enteritidis strains unable to excise ROD21. The failure to excise ROD21 significantly reduced the capacity to cause a lethal disease and to colonize the spleen and liver of mice, as compared to wild type S. Enteritidis. On the contrary, S. Enteritidis strains overexpressing an excisionase protein increased the frequency of ROD21 excision and showed an improved capacity to cause lethal disease in mice. Accordingly, strains unable to excise ROD21 showed an altered expression of genes located in this pathogenicity island. Our results suggest that the genetic excision of the pathogenicity island ROD21 in S. Enteritidis modulates the capacity of this bacterium to cause disease in mice due to a change in the expression of virulence genes.
TL;DR: It has been demonstrated that PLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunity to interfere with different hallmarks of cancer, in tumors with diverse histological origin.
Abstract: The main goal of this work was to assess in vitro the potential of Polo-like kinase gene (PLK-1) as a molecular target within the tumor microenvironment, namely in both cancer cells of tumors of different histological origin and endothelial cells from angiogenic blood vessels, upon silencing with anti-PLK-1 siRNA. In addition, the effect of Plk-1 downregulation on the cancer cells chemosensitization to paclitaxel was further assessed. Downregulation of Plk-1 reduced cancer cells viability from 40 to 85% and up to 59% in endothelial cells. Regarding the latter, it compromised their ability to form new tube-like structures, decreasing the formation of network projections up to 46%. This suggested for the first time, PLK-1 as a valuable angiogenic molecular target. In combination with paclitaxel, anti-PLK-1 siRNA chemosensitized non-small cell lung cancer (NSCLC) and prostate carcinoma cell lines, leading up to a 2-fold increase in the drug cytotoxic effect. Moreover, the sequential incubation of anti-PLK-1 siRNA and paclitaxel led to a decrease in the IC50 of the latter up to 2.7- and 4.1-fold, in A-549 and PC-3 cells, respectively. The combination of anti-PLK-1 siRNA with paclitaxel led to cell cycle arrest, increasing the number of cells at the G2/M and S phases to 1.5 and 1.3-fold in PC-3 cells, and to 1.6 and 1.4-fold in A-549 cells, respectively. Overall, it has been demonstrated that PLK-1 silencing with siRNA can impact multiple cellular players of tumor aggressiveness, thus enabling the opportunity to interfere with different hallmarks of cancer, in tumors with diverse histological origin.
TL;DR: Results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy, and this treatment reduced glucose levels in diabetic mice.
Abstract: Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.
TL;DR: The history, recent advances, challenges and plausibility of applying cardiac reprogramming on patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field of cardiac regeneration.
Abstract: Cardiac diseases are the major causes of morbidity and mortality in the world. Cardiomyocyte death is a common consequence of many types of heart diseases and is usually irreversible. Scar tissues formed by cardiac fibroblasts serve compensatory roles for the injured heart but eventually weaken cardiac function and result in life-threatening heart failures. Unfortunately, adult human hearts have limited regenerative capacities. In the past decades, many interventional approaches have been taken in an attempt to restore functional cardiomyocytes in an injured heart. Promising advances have been made in directly reprogramming mouse fibroblasts into cardiomyocyte-like cells both in vitro and in vivo. Recently, several different methods have been reported, including the use of transcription factors and microRNAs. In addition, two in vivo studies showed heart function improvements with delivery of reprogramming factors in mouse infarcted hearts. Although many of these studies are at early preliminary stages, the plausibility of applying cardiac reprogramming on patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field. This review will discuss the history, recent advances and challenges of cellular reprogramming, specifically in the field of cardiac regeneration.
TL;DR: Preclinical studies with non-replicating herpes simplex virus-based vectors injected into the skin to transduce neurons in the dorsal root ganglion have demonstrated efficacy in preventing progression of sensory neuropathy without any possible systemic side effects.
Abstract: Dysfunction of peripheral nerves due to metabolic, toxic, infectious, or genetic causes is a common and debilitating syndrome resulting in sensory loss. Peripheral neuropathies are one of the most widespread neurological disorders, affecting nearly 20 million people in the United States alone. Pharmacologic treatment for peripheral neuropathies is one of the most challenging fields in the clinical research. Sensory neurons are widely distributed and relatively inaccessible to direct drug delivery. Targeted delivery of neurotrophic factors to the primary sensory afferent for treatment of polyneuropathy by gene transfer approach offers the possibility of a highly selective targeted release of bioactive molecules within the nervous system. Preclinical studies with non-replicating herpes simplex virus (HSV)-based vectors injected into the skin to transduce neurons in the dorsal root ganglion (DRG) have demonstrated efficacy in preventing progression of sensory neuropathy without any possible systemic side effects.
TL;DR: Anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells, which provides a promising approach for clinical treatment of leukemia in future.
Abstract: Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.
TL;DR: A system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation is generated and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.
Abstract: Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.
TL;DR: This less invasive catheter-based US therapeutic procedure offers observable gene delivery with higher therapeutic efficiency, enhanced angiogenesis, and improved myocardial perfusion and ventricular function following MI.
Abstract: To enhance the safety of transendocardial delivery and the efficacy of intramyocardial angiogenic gene expression, a visible, less invasive, targeted, high-efficiency gene delivery strategy was tested. Progress toward clinical approval of systemic administration of genes and microbubbles (MBs) has been limited. The feasibility of transendocardially delivering MBs as extracellular markers and gene carriers in conjunction with intracardiac ultrasound (US) treatment remains unknown. In a canine acute myocardial infarction (MI) model, a naked plasmid encoding 500 μg human hepatocyte growth factor (HGF) was delivered transendocardially to the myocardium via US/MB (HGF-US/MB), insonation (HGFUS), or alone (HGF alone). Control MI dogs received saline without US/MB (control group). During US/MB, intracardiac insonation was performed for 30 s with a 10-s pause, at 4.3-MHz, 1-W/cm(2), for 60 s at each site. Gene and MB distribution in the myocardium was visualized. Compared to the HGF alone group at 28 days, the HGF-US/MB group had an average 7.1-fold enhancement in gene expression (P < 0.01). Compared to the control group, there were 16% decreases in the ratio of left ventricle (LV) weight/body weight in the HGF-US/MB group and decreases in collagen volume fraction (CVF) of type I (33%) and type III (23%) collagen. Capillary density increased from 22.8 ± 6.3/mm(2) in the control group to 154.3 ± 42.9/mm(2) in the HGF-US/MB group (P < 0.01). This less invasive catheter-based US therapeutic procedure offers observable gene delivery with higher therapeutic efficiency, enhanced angiogenesis, and improved myocardial perfusion and ventricular function following MI.