pRNA:一种安全、稳定、大容量的基因治疗分子载体

疾病的基因治疗需要安全、高效、靶向性的分子载体。通过对pRNA分子的结构和作用机理的研究,发现pRNA分子极易穿过细胞膜,有多个结合位点,能有效转导多个外源分子进入靶细胞,在体内几乎不引起免疫反应,而且可能通过设计多功能靶向复合物将疾病的检测和治疗有机的结合在一起,成为一种理想的基因治疗型载体。     

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome^1,2 or hyperoxic injuries of the neonatal lung³. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)⁴, genetic variants of surfactant deficiencies⁵ and α1-antitrypsin deficiency⁶.Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual''s immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies⁷.In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep⁸, and even in a clinical setting⁹, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection^10-12, (ultrasound-guided) intrapulmonary injection^13,14 and intravenous administration into the yolk sac vessels^15,16 or umbilical vein¹⁷. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques¹⁸.     

Gene therapy and retinitis pigmentosa: advances and future challenges.

It may be possible, one day, to use gene therapy to treat diseases whose genetic defects have been discerned. Because many genes responsible for inherited eye disorders within the retina have been identified, diseases of the eye are prime candidates for this form of therapy. The eye also has the advantage of being highly accessible with altered immunological properties, important considerations for easy delivery of virus and avoidance of systemic immune responses. Currently, adenovirus, adeno-associated virus and lentivirus have been used to successfully transfer genetic material to retinal pigment epithelium and photoreceptor cells. By harnessing therapeutic genes to these viruses, researchers have been able to demonstrate rescue in rodent models of retinitis pigmentosa, providing evidence that this form of therapy can be effective in delaying photoreceptor cell death. Future challenges include confirming therapeutic effects in animal models with eyes more anatomically similar to those of humans and demonstrating long-term rescue with minimal toxicity.     

基因治疗的现状与展望

自从明确了单基因缺陷的疾病,就提出了基因治疗的概念。基因治疗是直接通过基因传递治疗人类疾病的一种手段,还可以理解成临床症状通过基因物质的传递而行到改善。目前在这个领域已经投入了几十亿美元,开展了超过三百例的临床试验,人们正通过不懈努力,期待着基因治疗的美好前景。     

Clinical translation of gene medicine

Gene therapy has recently witnessed accelerated progress as a new therapeutic strategy with the potential to treat a range of inherited and acquired diseases. Billions of dollars have been invested in basic and clinical research on gene medicine, with ongoing clinical trials focused on cancer, monogenic diseases, cardiovascular diseases and other refractory diseases. Advances addressing the inherent challenges of gene therapy, particularly those related to retaining the delivery efficacy and minimizing unwanted immune responses, provide the basis for the widespread clinical application of gene medicine. Several types of genes delivered by viral or non‐viral delivery vectors have demonstrated encouraging results in both animals and humans. As augmented by clinical indications, gene medicine techniques have rapidly become a promising alternative to conventional therapeutic strategies because of their better clinical benefit and lower toxicities. Their application in the clinic has been extensive as a result of the approval of many gene therapy drugs in recent years. In this review, we provide a comprehensive overview of the clinical translation of gene medicine, focusing on the key events and latest progress made regarding clinical gene therapy products. We also discuss the gene types and non‐viral materials with respect to developing gene therapeutics in clinical trials.     

Model-specific selection of molecular targets for heart failure gene therapy

Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of β-adrenergic receptor signaling, Ca(2+) handling proteins and angiogenesis in the most common extrinsic models of HF.     

Mesenchymal stem cells as carrier of the therapeutic agent in the gene therapy of blood disorders

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.     

基因治疗的发展现状、问题和展望

基因治疗是一种新的治疗手段,可以治疗多种疾病,包括癌症、遗传性疾病、感染性疾病、心血管疾病和自身免疫性疾病。癌症基因治疗是基因治疗的主要应用领域。过去几年里,全球基因治疗临床试验取得了很大的进步。实际上,基因治疗也遇到了很多困难。未来,基因治疗的主要目标是发展安全和高效的基因导入系统,它们能将外源遗传物质靶向性地导入到特异的细胞。本文主要综述基因治疗所取得的突出进展、所遇到的困难和发展前景。     

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.     

In pursuit of new developments for gene therapy of human diseases

Gene therapy aims at transferring a therapeutic gene into human somatic cells in order to treat a disease. Originally addressed to hereditary genetic disorders, gene therapy has found therapeutic applications in cancer, infectious diseases and degenerative disorders, particularly those of the nervous system. Although gene transfer into humans has been demonstrated in several clinical trials, with more than 300 currently underway worldwide, there is still no single outcome that undoubtedly showed a consistent benefit for the patient. Nevertheless, the expectations for gene therapy are still high, and the prospects of future clinical success are increasing together with the growing of the field. The development of better delivery systems specifically tailored to individual diseases, with sustained expression of the therapeutic gene in the appropriate cells, will in the end make possible true therapeutic applications of human gene transfer.     

Lectin functionalized nanocarriers for gene delivery

Gene therapy has emerged as one of the most promising therapeutic methods to treat various diseases. However, inadequate gene transfection efficacy during gene therapy demands further development of more efficient gene delivery strategies. Targeting genetic material to specific sites of action endows numerous advantages over non-targeted delivery. An ample variety of non-viral gene delivery vectors have been developed in recent years owing to the safety issues raised by viral vectors. Non-viral gene delivery vectors containing specific targeting ligands on their surfaces have been reported to enhance the gene transfection efficiency via receptor-mediated endocytosis for gene delivery. Among various targeting moieties investigated, carbohydrates and lectins (carbohydrate-binding proteins) played an essential role in gene delivery via either direct or reverse lectin targeting strategies. Lectins have a specific carbohydrate binding domain that can bind specifically to the carbohydrates. This review sheds light on various gene delivery nanovectors conjugated with either lectins or carbohydrates for enhanced gene transfection.     

Nonhuman primates are relevant models for research in hematology, immunology and virology

Nonhuman primates have been used for biomedical research for several decades. They have proved to be models that are relevant to humans because of the high level of gene homology which underlies physiological and biochemical similarities. The similarity of monkeys to humans has been used to investigate pathophysiological mechanisms in hematology, immunology and virology. New therapeutic procedures can be assessed in primates by using materials, in particular pharmacological reagents, and methods designed for humans. The relevance of these models also relies on the use of species-specific pathogens and the availability of recombinant, homologous cytokines. The introduction of more and more sophisticated cell and gene therapy protocols in hematopoietic cell transplantation and immunotherapy requires the development of preclinical trials similar to clinical settings. For several decades now, baboons and cynomolgus/rhesus monkeys have been the most useful primate models in experimental hematology, and this has contributed to numerous therapeutic advances. Primate models of AIDS have been developed to study the pathogenesis, transmission and immune responses to infection, and to test vaccines and drugs. Primate research should be restricted in quantity, and mainly designed with the aim of removing uncertainty as to the safety and clinical benefit to the patient, of new biomedical protocols.     

Current progress of siRNA/shRNA therapeutics in clinical trials

Through a mechanism known as RNA interference (RNAi), small interfering RNA (siRNA) molecules can target complementary mRNA strands for degradation, thus specifically inhibiting gene expression. The ability of siRNAs to inhibit gene expression offers a mechanism that can be exploited for novel therapeutics. Indeed, over the past decade, at least 21 siRNA therapeutics have been developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders. Like other biological drugs, RNAi-based therapeutics often require a delivery vehicle to transport them to the targeted cells. Thus, the clinical advancement of numerous siRNA drugs has relied on the development of siRNA carriers, including biodegradable nanoparticles, lipids, bacteria, and attenuated viruses. Most therapies permit systemic delivery of the siRNA drug, while others use ex vivo delivery by autologous cell therapy. Advancements in bioengineering and nanotechnology have led to improved control of delivery and release of some siRNA therapeutics. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules. Here, we provide an overview of siRNA therapeutics in clinical trials, including their clinical progress, the challenges they have encountered, and the future they hold in the treatment of human diseases.     

Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles

Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments.     

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.     

Delivery of RNA Interference

Over the last few years, RNA Interference (RNAi), a naturally occurring mechanism of gene regulation conserved in plant and mammalian cells, has opened numerous novel opportunities for basic research across the field of biology. While RNAi has helped accelerate discovery and understanding of gene functions, it also has great potential as a therapeutic and potentially prophylactic modality. Challenging diseases failing conventional therapeutics could become treatable by specific silencing of key pathogenic genes. More specifically, therapeutic targets previously deemed “undruggable” by small molecules, are now coming within reach of RNAi based therapy. For RNAi to be effective and elicit gene silencing response, the double-stranded RNA molecules must be delivered to the target cell. Unfortunately, delivery of these RNA duplexes has been challenging, halting rapid development of RNAi-based therapies. In this review we present current advancements in the field of siRNA delivery methods, including the pros and cons of each method.