Novel lentiviral vectors for human gene therapy

This review summarises an emerging viral vector system for use in human gene therapy - lentiviral vectors. Lentiviral vectors have several advantages over existing viral vectors. They can stably express transgenes in non-dividing cells in vivo without provoking a significant immune response. They can be produced to a high titre and can be pseudotyped with heterologous envelope proteins to confer broad tropism. Although not without safety concerns, the properties of lentiviral vectors makes them an attractive choice for human gene therapy.     

Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed lentivirus encoding Rab9 cDNA. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1-GAG, pPACKH1-REV and pVSV-G into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 h, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with lentivirus encoding Rab9 cDNA, which evidenced a satisfactory increasing effect of this virus. Administration of lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulated the death rate of Purkinje neurons. It is concluded that the packaging of lentivirus encoding Rab9 cDNA was successful. lentivirus encoding Rab9 can increase the expression of Rab9 cDNA gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.     

大鼠HSP27基因RNA干扰慢病毒载体的构建与鉴定

目的构建大鼠的热休克蛋白27(heat shock protein27,HSP27)RNA干扰慢病毒载体。方法设计并合成3对互补针对大鼠HSP27 mRNA的oligoDNA片段。磷酸化和退火后,分别克隆入pSUPER-basic质粒载体,获重组的pSU-PER-HSP27-oligoDNA。将其中表达shRNA的结构酶切插入慢病毒转移质粒pNL-IRES2-EGFP,产生pNL-HSP27-IRES2-EGFP,在293T细胞中与VSVG、pHelper包装产生慢病毒。48 h后收集上清液,离心过滤后进行病毒滴度测定。用构建正确的慢病毒质粒载体感染血管平滑肌细胞(vascularsmooth muscle cells,VSMCs),检测干扰效果。结果酶切和测序两种方法结果证明3种质粒载体的插入序列完全正确,慢病毒载体构建成功并获得相应的慢病毒,病毒悬液的滴度为3.12×109fu.L-1。重组慢病毒质粒pNL-HSP27-IRES2-EGFP-1的RNA干扰效率最高,为0.771。结论成功构建靶向大鼠HSP27基因RNAi慢病毒载体。为进一步研究HSP27功能和用慢病毒进行基因治疗奠定基础。     

Polymers for viral gene delivery

Background: The development of viral vectors capable of providing efficient gene transfer in diseased tissues without causing any pathogenic effects is pivotal for overcoming the many challenges facing gene therapy. Objective: Immune responses against viral vectors, inadequate gene expression and inefficient targeting to specific cells in vivo are some of the major problems limiting the clinical utility of viral gene therapy. Methods: This review will focus on recent progress in strategic polymer-based modifications to improve the performance and biocompatibility of a variety of viral vectors. We will discuss the preclinical development of four approaches involving injectable polymers, polyelectrolytes, polymer microspheres and polymer–virus conjugates. Results/conclusion: Much progress has been made in creating ‘hybrid’ gene delivery vectors that combine the strengths of polymers and viruses. With further optimization, these hybrid vectors, which may be safer and more effective, are likely to succeed in clinical applications.     

Future prospects for gene delivery systems

Introduction: Gene therapy is the challenging area of biotechnology. Despite its promise for critical diseases, it has serious safety and efficiency issues, particularly with regards to gene transfer systems.

Areas covered: We examined the current situation with gene transfer systems and addressed problems this technology. We then searched patent applications about in the area from the Patentscope online system, the international patent database. We analyzed the data obtained to get a general idea about gene delivery systems designed for future use and assessed approaches for more efficient, safer and valid delivery systems.

Expert opinion: When quality assurance terms are fulfilled, some of these issues (genetic changes, mutations) could be minimized during the production process. Modification of vectors for improving their efficiency and safety or development of alternative transfer systems could be the solutions for these problems. Gene transfer technologies are important for gene therapy and should demonstrate effective, target-specific and acceptable safety profiles. For this reason, searching for alternatives to current systems is a necessity.     

Drug delivery in soft tissue engineering

Introduction: Tissue defects, sustained through disease or trauma, present enormous challenges in regenerative medicine. Modern tissue engineering (TE) aims at replacing or repairing these defects through a combined approach of biodegradable scaffolds, suitable cell sources and appropriate environmental cues, such as biomolecules presented on scaffold surfaces or sustainably released from within.

Areas covered: This review provides a brief overview of the various drugs and bioactive molecules of interest to TE, as well as a selection of materials that have been proposed for TE scaffolds and matrices in the past. It then proceeds to discuss encapsulation, immobilization and controlled release strategies for bioactive proteins, before discussing recent advances in this area with a special focus on soft TE.

Expert opinion: Overall, minimal clinical success has been achieved so far in using growth factor, morphogen, or adhesion factor modified scaffolds and matrices; only one growth factor delivery system (Regranex Gel), has been approved by the FDA for clinical use, with only a handful of other growth factors being approved for human use so far. However, many more growth factors are currently in clinical Phase I – II or preclinical trials and many delivery systems utilize materials already approved by the FDA for other purposes. With respect to drug delivery in soft TE, a combination of increased research efforts in hydrogel and support material development as well as growth factor development is needed before clinical success is realized.     

聚合物用作基因治疗载体的研究进展

综述了近年来聚合物用作非病毒基因治疗载体的研究进展,包括聚氨基葡糖基系统、聚氮丙啶、树枝状聚合物、明胶基系统等,简述了它们各自的特点和体内转染效果。     

Viral-mediated gene delivery for cell-based assays in drug discovery

Background: Adenovirus, retrovirus and lentivirus-based vectors, originally engineered and optimized for in vivo and ex vivo gene therapy, have become increasingly useful for viral-mediated gene delivery to support in vitro cell-based assays. Viral vectors underpin functional genomics screening of cDNA, shRNA and aptamer libraries, are used for a variety of target validation studies and importantly, for high-throughput cell-based drug discovery and compound profiling assays. The baculovirus/insect cell expression system had gained prevalence as a tool for recombinant protein production when it was observed that recombinant baculovirus vectors too could serve as efficient gene delivery vehicles for a wide range of mammalian cells. Although the use of baculovirus vectors in vivo has lagged behind retroviral, adenoviral and lentiviral vectors, they have gained prominence for development of in vitro cell-based assays due to the ease of generation, broad host range and excellent biosafety profile. There is an increasing emphasis on cell-based assays in high-throughput automated drug discovery laboratories and a variety of commercially available viral-vectors can be used for supporting these assays. Objective: We compare and contrast the current viral-mediated gene delivery vector systems and highlight their suitability for cell-based drug discovery assays. Conclusion: Viral-mediated gene delivery is increasingly being used in support of genome scale target validation studies and cell-based assay development for specific drug target genes such as ion channels, G protein-coupled receptors and intracellular enzymes. The choice of a delivery system over another for a particular application is largely dictated by the cell types and cell lines in use, virus cellular tropism, assay throughput, safety requirements and ease/cost of reagent generation.     

Viral-based gene delivery and regulated gene expression for targeted cancer therapy

Importance of the field: Cancer is both a major health concern and a care-cost issue in the US and the rest of the world. It is estimated that there will be a total of 1,479,350 new cancer cases and 562,340 cancer deaths in 2009 within the US alone. One of the major obstacles in cancer therapy is the ability to target specifically cancer cells. Most existing chemotherapies and other routine therapies (such as radiation therapy and hormonal manipulation) use indiscriminate approaches in which both cancer cells and non-cancerous surrounding cells are treated equally by the toxic treatment. As a result, either the cancer cell escapes the toxic dosage necessary for cell death and consequently resumes replication, or an adequate lethal dose that kills the cancer cell also causes the cancer patient to perish. Owing to this dilemma, cancer- or organ/tissue-specific targeting is greatly desired for effective cancer treatment and the reduction of side effect cytotoxicity within the patient.

Areas covered in this review: In this review, the strategies of targeted cancer therapy are discussed, with an emphasis on viral-based gene delivery and regulated gene expression.

What the reader will gain: Numerous approaches and updates in this field are presented for several common cancer types.

Take home message: A summary of existing challenges and future directions is also included.     

Vectors for airway gene delivery

Delivery of genes to the airway epithelium for therapeutic purposes seemed easy at first, because the epithelial cells interface with the environment and are therefore accessible. However, problems encountered were more substantial than were originally expected. Nonviral systems may be preferred for long-term gene expression, for they can be dosed repeatedly. Two nonviral gene transfer systems have been in clinical trials, lipid-mediated gene transfer and DNA nanoparticles. Both have sufficient efficiency to be candidates for correction of the cystic fibrosis defect, and both can be dosed repeatedly. However, lipid-mediated gene transfer in the first generation provokes significant inflammatory toxicity, which may be engineered out by adjustments of the lipids, the plasmid CpG content, or both. Both lipid-mediated gene transfer and DNA nanoparticles in the first generation have short duration of expression, but reengineering of the plasmid DNA to contain mostly eukaryotic sequences may address this problem. Considerable advances in the understanding of the cellular uptake and expression of these agents and in their practical utility have occurred in the last few years; these advances are reviewed here.     

Cationic liposomes for gene delivery

Cationic liposome–DNA complexes (lipoplexes) constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. This review will focus on various parameters governing lipoplex biological activity, from their mode of formation to invivo behaviour. Particular emphasis is given to the mechanism of interaction of lipoplexes with cells, in an attempt to dissect the different barriers that need to be surpassed for efficient gene expression to occur. Aspects related to new trends in the formulation of lipid-based gene delivery systems aiming at overcoming some of their limitations will be covered. Finally, examples illustrating the potential of ca-tionic liposomes in clinical applications will be provided.     

非病毒型基因传递系统

介绍基因治疗的基本原理与非病毒型基因传递系统的设计方法、作用机制的研究进展 ,对基因传递系统的研究前景进行了展望。     

非病毒基因递送载体的研究进展

目前,基因药物的递送成为药学研究的热点,基因递送载体主要包括病毒载体和非病毒载体。非病毒基因载体的毒性低,生物相容性好,转染效率高,具有潜在的临床应用价值。本文就靶向递送基因载体、多功能基因载体、同时载基因与化疗药物的载体、智能基因载体和脂质体等非病毒基因递送载体的研究进展做一综述。     

阳性脂质体介导基因转染及其研究进展*

基因治疗是一种很有前景的治疗模式,而阳性脂质体介导的基因转染是目前基因治疗的研究热点之一。现综述近5年来有关阳性脂质体的文献,介绍了阳性脂质体的基本组成,并从生物学、理化性质及制剂学等几个方面介绍了影响阳性脂质体/DNA复合物转染效率的主要因素,最后从新的阳性脂质成分及阳性脂质体或阳性脂质体/DNA复合物的表面修饰等方面介绍了近年来有关改善阳性脂质体介导基因转染的研究进展。     

Liposomes and lipopolymeric carriers for gene delivery

In this work we have examined the ability of various lipopolyplexes to deliver genes into liver cancer cells. We evaluated different parameters such as the protocol of preparation, the lipid/DNA molar ratio, and the molecular weight and type of PEI, to optimize the formulation to achieve high transfection activity. Our hypothesis was that the association of PEI with cationic liposomes (lipopolyplexes) would increase luciferase expression compared to lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA) alone.     

Gene doping: gene delivery for olympic victory

With one recently recommended gene therapy in Europe and a number of other gene therapy treatments now proving effective in clinical trials it is feasible that the same technologies will soon be adopted in the world of sport by unscrupulous athletes and their trainers in so called ‘gene doping’. In this article an overview of the successful gene therapy clinical trials is provided and the potential targets for gene doping are highlighted. Depending on whether a doping gene product is secreted from the engineered cells or is retained locally to, or inside engineered cells will, to some extent, determine the likelihood of detection. It is clear that effective gene delivery technologies now exist and it is important that detection and prevention plans are in place.     

Recent advances in the development of polyethylenimine-based gene vectors for safe and efficient gene delivery

Introduction: Despite the great therapeutic potential of gene therapy for treating critical diseases, the clinical application is limited by lack of safe and effective gene delivery vectors. Nonviral gene vectors have attracted tremendous attention due to the favorable loading capacity and facile manufacture. Among them, polyethylenimine-based gene vectors (PEIs) hold great promise for highly efficient gene delivery.

Areas covered: In this review, we outline the multiple biological barriers associated with gene delivery process and point out several challenges exist in the clinical usage of PEIs. We then provide an overview of the most impressive progresses made to overcome such challenges in recent years, including modifications of PEIs (i.e. to enhance biocompatibility, specific targeting effect, and buffering capacity) and stimuli-responsive strategies (i.e. endogenous and exogenous stimuli) for safe and efficient gene delivery.

Expert opinion: Rational modification of PEIs with diverse functionalized segments or the development of stimuli-responsive PEIs is an appealing strategy to meet some requirements involved in gene delivery. Nevertheless, further optimization by combining the two strategies is needed for the maximized transfection efficiency and minimized side effects, shedding new light on the development of nonviral gene delivery for clinical application.     

The potential of adeno-associated viral vectors for gene delivery to muscle tissue

Introduction: Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8⁺ T-cell response against AAV capsid in humans.

Areas covered: In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application.

Expert opinion: Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.