Methodological optimization of polyethylenimine (PEI)-based gene delivery to the lungs of mice via aerosol application

BACKGROUND: Polyethylenimine (PEI) has been successfully used for gene delivery to the lungs of mice via aerosol application using a whole body nebulization device. In this report we optimized the design of such an aerosol device. METHODS: Aerosol devices were constructed as either serial inhalation apparatus or as a whole body nebulization chamber connected to an aerosol spacer placed in a horizontal or vertical position. PEI-based gene vectors were nebulized using a standard jet nebulizer and luciferase gene expression of various tissues was examined. RESULTS: Using a whole body aerosol device resulted in luciferase gene expression in the lungs of mice at the same level as compared with a serial inhalation apparatus. Whereas gene expression was enhanced in the presence of 5% CO(2)-in-air, anesthesia of mice strongly decreased gene expression even when mice were intubated with an intravascular cannula. Reduction of the median mass aerodynamic diameter (MMAD) of the aerosol from 3.4 to 0.27 microm by interposition of an aerosol spacer increased gene expression significantly 3-fold. Drying of the aerosol by silica gel additionally increased gene delivery significantly 3-fold. Reporter gene expression mediated by branched PEI 25 kDa was 9- and 15-fold higher as compared with linear PEIs of 22 and 25 kDa, respectively, and was dependent on the DNA concentration. Gene expression was detectable as soon as 6 h after gene vector application and reached a maximum after 72 h but was still detectable after 14 days. The presence of Zn(2+) did not increase gene expression. CONCLUSION: We propose aerosol drying as a novel and simple method of optimizing PEI-based gene delivery to the lungs.     

Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration.

Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein; the replacement of a missing or inactive gene; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.     

无机纳米粒子作为基因载体的研究进展

转染是将具生物功能的核酸转移、运送到细胞内,并使其在细胞内维持生物功能的过程。作为现代生物化学和分子生物学中的一种主要技术手段,转染对于基因治疗有重要的意义。无机纳米粒子作为基因载体受到人们日益广泛的关注,其具有易于制备,可进行多样化的表面修饰等多种优势。本文将概述无机纳米粒子作为基因载体的现状及其对基因表达的影响。     

Nanotechnology and pharmaceutical inhalation aerosols

Pharmaceutical inhalation aerosols have been playing a crucial role in the health and well being of millions of people throughout the world for many years. The technology's continual advancement, the ease of use and the more desirable pulmonary-rather-than-needle delivery for systemic drugs has increased the attraction for the pharmaceutical aerosol in recent years. But administration of drugs by the pulmonary route is technically challenging because oral deposition can be high, and variations in inhalation technique can affect the quantity of drug delivered to the lungs. Recent advances in nanotechnology, particularly drug delivery field have encouraged formulation scientists to expand their reach in solving tricky problems related to drug delivery. Moreover, application of nanotechnology to aerosol science has opened up a new category of pharmaceutical aerosols (collectively known as nanoenabled-aerosols) with added advantages and effectiveness. In this review, some of the latest approaches of nano-enabled aerosol drug delivery system (including nano-suspension, trojan particles, bioadhesive nanoparticles and smart particle aerosols) that can be employed successfully to overcome problems of conventional aerosol systems have been introduced.     

基因治疗的应用及研究进展

基因治疗是一种新的治疗手段,可用于癌症、遗传性疾病、感染性疾病、心血管疾病和自身免疫性疾病等的治疗。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,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特异的细胞。简要综述了基因治疗研究和应用的进展、困难及其发展前景。     

Calculating expected lung deposition of aerosolized administration of AAV vector in human clinical studies

BACKGROUND: Cystic fibrosis is an autosomal recessive disease affecting approximately 1 in 2500 live births. Introducing the cDNA that codes for normal cystic fibrosis transmembrane conductance regulator (CFTR) to the small airways of the lung could result in restoring the CFTR function. A number of vectors for lung gene therapy have been tried and adeno-associated virus (AAV) vectors offer promise. The vector is delivered to the lung using a breath-actuated jet nebulizer. The purpose of this project was to determine the aerosolized AAV (tgAAVCF) particle size distribution (PSD) in order to calculate target doses for lung delivery. METHODS: A tgAAVCF solution was nebulized using the Pari LC Plus (n = 3), and the PSD was determined by coupling laser diffraction and inertial impaction (NGI) techniques. The NGI allowed for quantification of the tgAAVCF at each stage of impaction, ensuring that rAAV-CFTR vector is present and not empty particles. Applying the results to mathematical algorithms allowed for the calculation of expected pulmonary deposition. RESULTS: The mass median diameter (MMD) for the tgAAVCF was 2.78 +/- 0.43 microm. If the system works ideally and the patient only receives aerosol on inspiration, the patient would receive 47 +/- 0% of the initial dose placed in the nebulizer, with 72 +/- 0.73% of this being deposited beyond the vocal cords. CONCLUSIONS: This technology for categorizing the pulmonary delivery system for lung gene therapy vectors can be adapted for advanced aerosol delivery systems or other vectors.     

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.     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。     

Hot topics in adeno-associated virus as a gene transfer vector

Adeno-associated virus (AAV) is a promising viral vector in treating many kinds of hereditary diseases. The broad host range, low level of immune response, and longevity of gene expression observed with this vector have enabled the initiation of a number of clinical trials using this gene delivery system. Another potential benefit of AAV vectors is their ability to integrate site-specifically in the presence of Rep proteins. However, this virus is not well characterized. To obtain high level, persistent expression of the foreign gene, some problems should be solved. In this article, we will describe the advances in some fields of recombinant AAV technology that overcome certain limitations of the vector as a gene delivery system, such as the transduction efficiency, the production, the package capacity, and elimination of immune responses, as well as the applications involving these recombinant vectors for the treatment of some diseases.     

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.     

Gene therapy: light is finally in the tunnel

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.     

Cas9/sgRNA递送技术及其研究进展

在与CRISPR/Cas9基因编辑技术相关的临床应用中,Cas9/sgRNA的递送是决定基因编辑治疗效果的关键技术之一。无需转录和翻译过程的Cas9蛋白/sgRNA复合物直接递送形式可能能够提供更高的特异性和安全性。文中通过对Cas9/sgRNA递送技术的研究现状及其在基因相关疾病治疗中的进展进行简要综述,为新型药物载体的设计和基因治疗的临床应用提供新思路。     

Polymers for gene delivery: current status and future perspectives

Gene therapy is a hope for curing many diseases and pathological conditions which are relatively difficult to treat. However lack of proper gene delivery vehicle is the main limiting step in this direction. Though viral vectors still lead as the major vehicle used in gene therapy clinical trials, their immunogenicity and low capacity restrict their wide use. Hence there is a need for developing non-viral vectors which can really be used for clinical applications. Polymers are a versatile group of molecules which can be modified and designed or engineered according to the end needs of the applications. The objective of this review is to summarize the recent advances in the development of polymeric vectors for gene delivery applications reported in patents and scientific journals.     

Critical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing

Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno-associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients’ access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV-mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state-of-the-art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.     

Helper virus-free gutless adenovirus (HF-GLAd): a new platform for gene therapy

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replication-competent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases.     

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。     

Gene transfer to the vasculature: historical perspective and implication for future research objectives

Cardiovascular diseases are a major cause of fatality, disability, and economic burden in Western civilization. Although the pharmaceutical industry has delivered a plethora of drugs for treatment of diverse cardiovascular complaints, there remain many conditions for which pharmacological regimens are either nonexistent or largely ineffective. In contrast, remarkable progress has been made in the field of vascular gene transfer in the last decade. The vast majority of studies are preclinical, although a number of high profile vascular gene therapy clinical trials are in progress. In principle, vascular gene therapy represents an unprecedented opportunity to treat a host of cardiovascular diseases in humans although many scientific, clinical, and ethical obstacles remain. Here we discuss the rapid progress in preclinical vascular gene therapy, highlight the most appropriate gene delivery vectors, and discuss the advances toward the ultimate goal of an efficient and safe gene therapy for diverse cardiovascular diseases.     

Raman characterization and chemical imaging of biocolloidal self-assemblies, drug delivery systems, and pulmonary inhalation aerosols: A review

This review presents an introduction to Raman scattering and describes the various Raman spectroscopy, Raman microscopy, and chemical imaging techniques that have demonstrated utility in biocolloidal self-assemblies, pharmaceutical drug delivery systems, and pulmonary research applications. Recent Raman applications to pharmaceutical aerosols in the context of pulmonary inhalation aerosol delivery are discussed. The "molecular fingerprint" insight that Raman applications provide includes molecular structure, drug-carrier/excipient interactions, intramolecular and intermolecular bonding, surface structure, surface and interfacial interactions, and the functional groups involved therein. The molecular, surface, and interfacial properties that Raman characterization can provide are particularly important in respirable pharmaceutical powders, as these particles possess a higher surface-area-to-volume ratio; hence, understanding the nature of these solid surfaces can enable their manipulation and tailoring for functionality at the nanometer level for targeted pulmonary delivery and deposition. Moreover, Raman mapping of aerosols at the micro- and nanometer level of resolution is achievable with new, sophisticated, commercially available Raman microspectroscopy techniques. This noninvasive, highly versatile analytical and imaging technique exhibits vast potential for in vitro and in vivo molecular investigations of pulmonary aerosol delivery, lung deposition, and pulmonary cellular drug uptake and disposition in unfixed living pulmonary cells.     

超声空化效应和超声微泡在生物医学中的应用

近年来,随着超声技术在医疗领域的广泛应用及超声造影剂研制的进展,空化效应和超声造影剂协同运用作为一种高效、安全、操作简单,且具有一定靶向性的无创治疗手段,在基因治疗、药物输送、溶栓和治栓,以及炎症与肿瘤的靶向诊断与治疗方面显示了巨大的运用潜力。简要综述了超声空化效应和超声微泡的治疗机制及应用。     

综述——纳米材料与药物输递：基因治疗药物输递系统的研究现状及发展趋势

基因治疗是一种有效的治疗方法,可用于治疗多种严重威胁人类健康的疾病．然而,裸露的基因治疗药物存在易被核酶降解、细胞内吞效果差和细胞靶向能力差等缺点．因此,需要寻求合适的载体,将基因治疗药物有效地输递到靶细胞,实现高效的基因治疗．本文主要综述了近年来基因治疗药物输递系统的研究进展,分别总结和阐述了病毒载体,脂质体、聚合物和树状大分子等非病毒载体,以及具有示踪功能的输递系统的特点及研究和发展现状．