Gene delivery into primary T cells

Technologies for transfer of exogenous genes into primary T cells have been limited until recently. The introduction of new approaches for gene transfer viadifferent viral vectors has expan ded the options for genetic manipulation of primary T cells and has provided powerful tools for studies of T cell activation and differentiation. We provide a brief overview of the systems currently available and contranst the advantages and disadvantages of each. We also describe a new transgenic model that enables highly efficient gene delivery into primary T cells by nonreplic ating adenoviral vectors.     

基因强化骨组织工程中的病毒载体

背景：不同的基因输送策略也被应用到骨组织工程中以修复破坏的骨组织,作为最有效率的基因转运载体,病毒载体在骨组织工程中的应用方兴未艾。 目的：系统回顾和讨论目前基因强化骨组织工程中常用的病毒载体相关应用。 方法：利用PubMed数据库对2002年1月至2015年1月的相关文献进行了检索,检索的文章主要聚焦在病毒载体基因转导方法和其在骨组织工程中的应用。对腺病毒、反转录病毒、腺相关病毒和嵌合病毒在骨组织工程的相关应用及不足进行了讨论。总共24篇相关文献被纳入此篇综述。 结果与结论：总结了近年来病毒载体联合基因治疗促进骨组织再生的研究工作。讨论了包括装载目的基因的病毒载体联合种子细胞例如间充质干细胞植入支架材料修复骨缺损。研究表明,基因强化的骨组织工程比传统组织工程具有更多的优点;病毒载体介导的基因转染效率比普通载体更高;病毒载体介导的基因强化骨组织工程用于人体的安全性仍需要漫长的临床观察研究。病毒载体系统仍然是最有效的将外源基因转入种子细胞的手段之一。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Non-viral delivery of RNA interference targeting cancer cells in cancer gene therapy

RNA interference (RNAi) is a collection of small RNA-directed mechanisms that result in sequence-specific inhibition of gene expression. RNAi delivery has demonstrated promising efficacy in the treatment of genetic disorders in cancer. Although viral vectors are currently the most efficient systems for gene therapy, potent immunogenicity, mutagenesis, and the biohazards of viral vectors remain their major risks. Various non-viral delivery vectors have been developed to provide a safer approach for gene delivery, including polymers, peptides, liposomes, and nanoparticles. However, some concerns and challenges of these non-viral gene delivery approaches remain to be overcome. In this review, we summarize the recent progress in the development of non-viral systems delivering RNAi and the currently available preclinical and clinical data, and discuss the challenges and future directions in cancer therapy.     

Light directed gene transfer by photochemical internalisation

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.     

From leading role to the backstage: mesenchymal stem cells as packaging cell lines for in situ production of viral vectors

Gene therapy is based on the genetic manipulation of target cells. The genetic information required to genetically engineer these cells can be delivered through non-viral or viral vectors that present different biologic properties. The production of viral vectors for gene therapy depends on the nature of the cells transfected with plasmids containing the genetic information for recombinant viral assemblage. These so-called packaging cell lines (PCL) can be injected into the target organ, for the in situ transduction of target cells. There have been recent reports about the capacity of mesenchymal stem cells (MSCs) to target tumor cells. Different research groups, including our own, have isolated these MSCs, but they have not yet been studied as potential PCL to produce viral vectors. We propose here that a MSC packaging cell line could be employed for in situ gene therapy of solid tumors. The tropism of MSCs for tumor cells may render this PCL more efficient in that microenvironment, producing viral vectors for longer periods of time, shifting MSCs from target cell to the backstage level of viral gene therapy.     

Transductional targeting with recombinant adenovirus vectors

Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed.     

Bone marrow stem cells as a vehicle for delivery of heme oxygenase-1 gene

Bone marrow-derived mesenchymal stem cells (MSCs) are readily accessible adult stem cells that are capable of self-renewal and multilineage differentiation. Human MSCs have been well described and used in xenogenic models for investigation, but rodent MSCs, if available, would eliminate problems associated with transplantation across a species barrier. Here we describe an effective method to generate rat MSCs and use these cells to target gene delivery in vivo. MSCs that were capable of retaining their differentiation potential after several population doublings in culture were generated from rat bone marrow. Marrow-derived MSCs were enriched and infected with an adenoviral vector carrying the heme oxygenase gene (Ad5/HO-1). Transfected rodent MSCs retained their differentiation potential, even after 10 passages, as determined by their ability to differentiate into adipocytes. Western analyses clearly indicated that Ad5/HO-1-transfected rodent MSCs exhibited increased HO-1 expression. Trafficking of fluorescent rat MSCs was evaluated 24 and 48 h after MSC infusion. Most of the infused cells accumulated in the lungs of recipients where they expressed HO-1. Thus, bone marrow-derived MSCs are useful for gene delivery replacement of the products of deficient genes. These cells may be useful for potentiation of wound healing because they retain their pluripotential differentiation ability.     

Viral vectors in cancer immunotherapy: which vector for which strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.     

Tissue-specific targeting for cardiovascular gene transfer. Potential vectors and future challenges

The introduction of genes to cardiovascular cells in vivo remains the major challenge for current gene therapy modalities. However, recent developments in retargeting adenoviral vectors are promising to improve transduction efficiency in the cardiovascular cells. After systemic application, most adenoviral vectors are trapped by the liver, hampering delivery to target cardiovascular tissues. Furthermore, a majority of vectors for vascular gene transfer utilizes strong heterologous viral promoters, such as CMV. A potential side effect related to the use of such vectors is the systemic organ toxicity resulting from unrestricted transgene expression. These vectors have the additional problem of being frequently shut-down in vivo. Therefore, both retargeting adenoviral vectors and the use of tissue-specific promoter-driven vectors offer an enhanced safety profile by reducing ectopic expression in vital organs including the liver and lung. However, the limiting factor for the use of tissue-specific promoters is the low-level of expression compared with their viral counterparts. Both the development of efficient and strong vectors using cell-specific regulatory elements and the production of therapeutic proteins at sufficient levels is urgently needed to inhibit vasculoproliferative disorders. This review will focus on some of the recent achievements in vector development relevant to the delivery of vascular gene therapies targeted to the vascular endothelium, smooth muscle cells and macrophages during arterial remodelling.     

Development of retroviral vectors as safe, targeted gene delivery systems

The transfer of genes of potential therapeutic benefit is presently being attempted in the clinic to treat a number of genetic and virally induced diseases. Many of these protocols use retroviral vectors derived from murine leukemia retroviruses as gene delivery systems. Although these viral delivery systems are well suited for this purpose, a number of their characteristics, some of which are discussed here, are still troublesome. Future retroviral vectors will incorporate nonretroviral features and will be tailored to desired needs for specific uses. These vectors will be safer, more efficient, and targeted in their delivery. Further, expression of the therapeutic genes carried will be limited to the specific target cell type. Some of the recent advances that have been made towards this goal are reviewed here.     

Electrotransfer as a non viral method of gene delivery

Over the last few decades, various vectors have been developed in the field of gene therapy. There still exist a number of important unresolved problems associated with the use of viral as well as non viral vectors. These techniques can suffer from secondary toxicity or low gene transfer efficiency. Therefore an efficient and safe method of DNA delivery still needs to be found for medical applications. DNA electrotransfer is a physical method that consists of the local application of electric pulses after the introduction of DNA into the extra cellular medium. As electrotransfer has proven to be one of the most efficient and simple non viral methods of delivery, it may provide an important alternative technique in the field of gene therapy. The present review focuses on questions related to the mechanism of DNA electrotransfer, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses the current limitations of the method as applied to DNA transfer, in particular its efficiency in achieving in vitro gene expression in cells and also its potential use for in vivo gene delivery.     

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. Several transposon systems, especially the Sleeping Beauty (SB), the piggyBac (PB) and Tol2, have been optimized for gene transfer into mammalian cells. In particular, SB resulted in stable gene transfer into various adult stem cells including human CD34(+) hematopoietic stem cells (HSCs), myoblasts and mesenchymal stem cells (MSCs). This has been confirmed with PB, yielding stable gene transfer in human CD34(+) HSCs. Recently, PB transposons were used to deliver the genes encoding the reprogramming factors into somatic cells making it an attractive technology for generating iPS cells. Subsequent de novo expression of the PB transposase resulted in traceless excision of the reprogramming cassette. This prevented inadvertent re-expression of the reprogramming factors obviating some of the concerns associated with the use of integrating vectors. Transposons have also been used as a novel non-viral paradigm to coax differentiation of iPS cells into their desired target cells by forced expression of specific differentiation factors. This review focuses on the emerging potential of transposons for gene transfer into stem cells and its implications for gene therapy and regenerative medicine.     

Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor

MSCs have received attention for their therapeutic potential in a number of disease states, including bone formation, diabetes, stem cell engraftment after marrow transplantation, graft-versus-host disease, and heart failure. Despite this diverse interest, the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors, we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts, which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs, cardiac function improved in MCP-3-expressing hearts (88.7%, p < .001) but not in control hearts (8.6%, p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather, MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration.     

Biosafety of herpesvirus vectors

Herpesviruses are large DNA viruses, which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the viral genome exists as a stable episome in the nucleus. For gene therapy to become a potential future treatment option, biosafe therapeutically efficient gene transfer is a central, but more and more stringent requirement. This review highlights the progress in development of herpesvirus based vectors, describes their properties as wall as discusses the biosafety concerns that are associated with their use in gene therapy. Thought was also given to biosafety issues pertaining to design and production of herpesvirus vector systems in therapeutic gene delivery.     

Genomic context vectors and artificial chromosomes for cystic fibrosis gene therapy

Cystic fibrosis (CF) is caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP dependent chloride channel whose expression is finely tuned in space and time. Gene therapy approaches to CF lung disease have demonstrated partial efficacy and short-lived CFTR expression in the airways. Drawbacks in the use of classical gene transfer vectors include immune response to viral proteins or to unmethylated CpG motifs contained in bacterially-derived vector DNA, and shut-off of viral promoters. These limitations could be overcome by providing stable maintenance and expression of the CFTR gene inside the defective cells. This strategy makes use of large fragments of DNA of various sizes containing the CFTR transgene and its relevant regulatory regions, (genomic context vectors [GCVs], reaching ultimate complexity in the form of an artificial chromosome [AC]) as vector for the transgene. Appropriate regulation in space and time would be achieved by the presence of the endogenous promoter and other control elements, while retention in daughter cells could be ensured by the presence of sequences which guarantee episomal replication. In this review, we describe recent advances in GCVs and ACs and the technology underlying their construction. These vectors have been shown to be suitable for delivery and expression of therapeutically relevant genes, including CFTR. The major issue which now limits their routine use is delivery inefficiency. Once this issue is resolved, we will be closer to achieving the goal of regulated gene therapy for CF.     

Polycation-based gene therapy: current knowledge and new perspectives

At present, gene transfection insufficient efficiency is a major drawback of non-viral gene therapy. The 2 main types of delivery systems deployed in gene therapy are based on viral or non-viral gene carriers. Several non-viral modalities can transfer foreign genetic material into the human body. To do so, polycation-based gene delivery methods must achieve sufficient efficiency in the transportation of therapeutic genes across various extracellular and intracellular barriers. These barriers include interactions with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is a potential obstacle for functional delivery to target cells. Cationic polymers constitute one of the most promising approaches to the use of viral vectors for gene therapy. A better understanding of the mechanisms by which DNA can escape from endosomes and traffic to enter the nucleus has triggered new strategies of synthesis and has revitalized research into new polycation-based systems. The objective of this review is to address the state of the art in gene therapy with synthetic and natural polycations and the latest advances to improve gene transfer efficiency in cells.     

Enhanced in vitro chondrogenesis of primary mesenchymal stem cells by combined gene transfer

Because articular cartilage has a poor regeneration capacity, numerous cell-based approaches to therapy are currently being explored. The present study involved the use of gene transfer as a means to provide sustained delivery of chondrogenic proteins to primary mesenchymal stem cells (MSCs). In previous work, we found that adenoviral-mediated gene transfer of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2), but not insulin-like growth factor 1 (IGF-1), could be used to induce chondrogenic differentiation of MSCs in an aggregate culture system. In the present study, we examined the effects on chondrogenesis of these transgenes when delivered in combination. Cultures of bone marrow-derived MSCs were infected with 2.5 x 10(2) or 2.5 x 10(3) viral particles/cell of each adenoviral vector individually, or in combination, seeded into aggregates, and cultured for 3 weeks in a defined serum-free medium. Levels of transgene product in the medium were initially high, approximately 100 ng/mL TGF-beta1, 120 ng/mL BMP-2, and 80 ng/mL IGF-1 at day 3, and declined thereafter. We found that co-expression of IGF-1 and TGF-beta1, BMP-2, or both at low doses resulted in larger aggregates, higher levels of glycosaminoglycan synthesis, stronger staining for proteoglycans and collagen type II and X, and greater expression of cartilage-specific marker genes than with either transgene alone. Gene-induced chondrogenesis of MSCs using multiple genes that act synergistically may enable the administration of reduced viral doses in vivo and could be of considerable benefit for the development of cell-based therapies for cartilage repair.     

Gene therapy of muscular dystrophy

Development of gene therapy for the muscular dystrophies represents a daunting challenge requiring significant advances in our knowledge of the defective genes, muscle promoters, viral vectors, immune system surveillance and methods for systemic delivery of vectors. However, tremendous progress has been made in developing improved viral vectors and avoiding immune reactions against gene transfer. This review summarizes recent progress and highlights problems that must be solved before an effective treatment is available.     

Mesenchymal stem cells in the aging and osteoporotic population

Because of their ability to self-renew and differentiate, mesenchymal stem cells (MSCs) are the in vivo source for replacing lost cells in high-turnover tissues during the life of an organism. MSCs have osteogenic potential and can be eligible for the repair and maintenance of the skeleton, thus they are very attractive for tissue engineering and regenerative medicine approaches. However, many changes in their behavior, caused by aging and bone disease, have been reported in the literature. These changes, which affect MSC self-renewal ability and differentiation potentiality, are related to cell proliferation, differentiation, cell cycle phases (depending on gene modification), and cytokine and growth factor production. This review summarizes the literature related to intrinsic and extrinsic characteristics of human bone marrow or adipose tissue MSCs during aging and osteoporosis. Although some studies reveal contrasting results, the results of this review suggest that the cellular modifications due to aging and osteoporosis should be carefully considered in relation to the use of MSCs for therapeutic application.     

Immune responses to lentiviral vectors

Efficient delivery and sustained expression of a therapeutic gene into human tissues are the requisite to accomplish the high expectations of gene therapy. A major challenge has concerned development of gene transfer systems capable of efficient cell transduction and transgene expression without harm to the recipient. A lot of work has been done to demonstrate the efficacy of gene therapy in animal models that mimic situations in humans. Use of lentiviral vectors (LVs) offers multiple advantages for gene replacement therapy, because they combine efficient delivery, ability to transduce proliferating and resting cells, capacity to integrate into the host chromatin to provide stable long-term expression of the transgene, absence of any viral genes in the vector and absence of interference from preexisting viral immunity. However, one of the major barriers to stable gene transfer by LVs and other viral vectors is the development of innate and adaptive immune responses to the delivery vector and the transferred therapeutic transgene. Since this greatly hinders the therapeutical benefits of gene therapy by LVs, developing strategies to overcome the host immune response to the transfer vector and the transgene is a matter of current investigation.