Recent advances in aerosol gene delivery systems using non-viral vectors for lung cancer therapy

ABSTRACT

Introduction: Lung cancer commonly occurs at a high incidence worldwide. Application of aerosol gene delivery systems using various kinds of vectors can improve the patient’s quality of life by prolonging the survival rate.

Areas covered: This review provides a recent update on aerosol gene delivery strategies using various kinds of vectors and gene-modification technologies. Peptide-mediated gene therapy achieves specific targeting of cells and highly improves efficacy. Promoter-operating expression and the CRISPR/Cas9 system are novel gene therapy strategies for effective lung cancer treatment. Furthermore, hybrid systems with a combination of vectors or drugs have been recently applied as new trends in gene therapy.

Expert opinion: Although aerosol gene delivery has many advantages, physiological barriers in the lungs pose formidable challenges. Targeted gene delivery and gene-editing technology are promising strategies for lung cancer therapy. These strategies may allow the development of safety and high efficiency for clinical application. Recently, hybrid gene therapy combining novel and specific vectors has been developed as an advanced strategy. Although gene therapy for lung cancer is being actively researched, aerosol gene therapy strategies are currently lacking, and further studies on aerosol gene therapy are needed to treat lung cancer.     

基因治疗药物递送系统研究进展

基因治疗在癌症及许多遗传性疾病治疗方面有着重要意义。由于基因药物在体内很容易降解,为了有效地将基因药物递送至靶组织、靶细胞及靶细胞器发挥作用,开发安全高效的基因药物载体体系是十分必要的。随着基因治疗的发展和进一步完善,各类非病毒和病毒载体材料应运而生。针对各类基于非病毒和病毒载体的基因递送系统研究及其临床试验中的进展进行汇总。

     

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。     

Recent developments in cationic lipid-mediated gene delivery and gene therapy

Gene therapy will revolutionize medicine, helping us to cure and prevent diseases at their core level. Until becoming a widespread reality, the problem of efficient gene transfer and expression (transfection) must be solved. Cationic lipids represent a safer alternative than viral vectors, which, although more efficient, have the drawback of immunogenicity and propagation risks. Additionally, cationic lipids and cationic liposomes allow the delivery of larger plasmids and may be GMP manufactured and stored in bulk quantities. However, their specific transfection efficiency must be improved in order to reach the performance of biological vectors. In recent years, new structures have been released and tested, with designs adapted to recent findings in lipid-mediated transfection mechanisms. Another trend is the increased use of natural, biodegradable, building blocks in the backbone of these compounds. Here we review the very recent developments in the field of cationic lipids, both from industry and academia. Physicochemical characteristics, insights of transfection mechanisms, as well as therapeutic applications are also presented. Finally, some future prospects and trends are proposed.     

Viral delivery for gene therapy against cell movement in cancer

Viral delivery for cancer gene therapy is a promising approach, where traditional radiotherapy or chemotherapy to limit proliferation and movement of cancer cells has met resistance. Based on the new understanding of the biology of the viral vectors, therapeutic viral vectors for cancer gene therapy have been improved for greater safety and efficacy as well as transitioned from being non-replicating to replication-competent. Traditional oncolytic vectors have focused on eliminating tumor growth, while novel vectors simultaneously target epithelial-to-mesenchymal transition (EMT) in cancer cells, which could further prevent and reverse the aggressive tumor progression. In this review, we highlight the illustrative examples of cancer gene therapy in clinical trials as well as preclinical data and include proposals on methods to further enhance the safety and efficacy of oncolytic viral vectors in cancer gene therapy.     

Gene delivery using cationic liposomes

The development of cationic liposomes for gene delivery has been ongoing for almost 20 years; however, despite extensive efforts to develop a successful therapeutic agent, there has been limited progress towards generating an effective pharmaceutical product. Since the introduction of N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride, an immense number of different cationic lipids have been synthesised and used to formulate cationic liposome–DNA complexes. Structural modification of the cationic lipids and the addition of components within the delivery system that can facilitate the fusion, cellular uptake and targeting of liposome–DNA complexes have all been used in a bid to enhance their transfection efficiency. Unfortunately, the overall impact of these improvements is still nominal, with the vast majority of clinical trials (～ 85%) continuing to rely on more potent viral delivery of DNA despite their associated toxicity issues. Key characteristics of the most effective cationic liposomes for the delivery of plasmid DNA (from a consensus of the literature) is identified here and the problems of converting these attributes into an effective pharmaceutical product are outlined.     

Targeted gene delivery to the lung

Gene therapy holds promise for the treatment of a range of inherited pulmonary disorders. However, efficient delivery and expression of the therapeutic transgene at levels sufficient to result in phenotypic correction of the diseased state has proved elusive. This review focuses on the development of gene delivery strategies for the lungs. One of the principal prerequisites for successful gene therapy is the delivery of gene vectors to the target area within a tissue and to target cells within that area. Physical and biological targeting of the gene vectors and its application in various models is discussed. Subsequently, both viral and non-viral vectors are addressed with respect to their transfection efficiency in different lung cells, the longevity of expression and their immunogenicity. Also, the various methods for pulmonary gene delivery are evaluated for their merits and limitations.     

Gelatin-based delivery systems for cancer gene therapy

Gelatin is a natural, biocompatible, nontoxic, edible, and inexpensive macromolecule. These properties result in its wide application in pharmaceutical, medical, and cosmetic products. Recently, it has been used for the delivery of such gene therapeutic entities as plasmid DNA. This review discusses the in vivo and in vitro studies using gelatin for delivery of therapeutic genes to cancerous cells. Recent studies show that present cancer gene therapy using gelatin is lacking in both efficiency and specificity in comparison with viral vectors, whereas complexes of therapeutic DNA with modified gelatin possibly offer a safe and efficient strategy for systemic administration of therapeutic genes to solid tumors compared to injection of naked plasmid DNA. The future of these promising approaches lies in the development of better techniques for preparing gelatin–gene complexes with the aim of a gelatin-based cancer gene therapy with comparable efficiency to viral vectors but with the added advantage of biosafety for patients.     

Dendrosome-dendriplex inside liposomes: as a gene delivery system

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue^® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.     

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.     

精蛋白在改进非病毒载体基因转染中的应用

非病毒基因载体的出现,为基因治疗提供了低毒、易于大规模制备的载体。但与病毒载体相比,非病毒基因载体的转染效率仍然偏低,阻碍了非病毒基因载体的临床应用。本文旨在探讨精蛋白在改进非病毒基因载体方面的应用,希望通过合理的载体设计与优化,制备出一种高效、低毒的基因载体。     

Aerosol delivery of spermine-based poly(amino ester)/Akt1 shRNA complexes for lung cancer gene therapy

Polyethylenimine (PEI) has been commonly used as a cationic polymeric gene carrier due to high transfection efficiency, however, its cytotoxicity has hindered the practical application. In this study, we report the development of poly(amino ester) (PAE) based on glycerol propoxylate triacrylate (GPT) and spermine (SPE) as an alternative gene carrier for lung cancer therapy. GPT-SPE copolymer was prepared by Michael addition reaction between GPT and SPE, and the efficacy was evaluated using shAkt1 as a model therapeutic gene. The molecular weight and composition were characterized using gel permeability chromatography (GPC) and ¹H-nuclear magnetic resonance (¹H-NMR), respectively. The GPT-SPE could effectively condense DNA with about 163 nm size and protect the DNA from nucleases. GPT-SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo. Furthermore, aerosol delivery of GPT-SPE/Akt1 shRNA complexes significantly suppressed lung tumorigenesis in K-ras^LA1 lung cancer model mice. These results suggest that GPT-SPE can be used in shRNA-based lung cancer gene therapy.     

Nanostructured lipid carriers as novel drug delivery system for lung cancer gene therapy

Context: Nanostructured lipid carriers (NLC) are potentially good colloidal drug carriers for gene delivery. They are advised to be the second lifetime of lipid nanocarriers.

Objective: The aim of this study is to develop novel modified NLC as nanomedicine for delivery of plasmid-containing enhanced green fluorescence protein (pEGFP). This system could target the lung cancer cells through receptor-mediated pathways to increase the nuclear uptake of genetic materials.

Methods: In the present study, pEGFP-loaded NLC (NLC/pEGFP) were prepared. Transferrin (Tf) containing ligands were used for the surface coating of the vectors. In vitro transfection efficiency of the modified vectors was evaluated in human alveolar adenocarcinoma cell line (A549 cells) and in vivo transfection efficiency of the modified vectors was evaluated on mice bearing A549 cells model.

Results: Tf-modified NLC/pEGFP (Tf-NLC/pEGFP) has a particle size of 157?nm, and ～82% of gene loading quantity. Tf-NLC/pEGFP displayed remarkably higher transfection efficiency than non-modified NLC/pEGFP both in vitro and in vivo.

Conclusion: The results demonstrate that the novel NLC gene delivery system offers an effective strategy for lung cancer gene therapy.     

Tumor-targeted delivery of siRNA by non-viral vector: safe and effective cancer therapy

RNA interference technology has been developed as a potential therapeutic agent for many indications, including cancer. Silencing a specific oncogene in tumor cells brings about cell death both in vitro and in vivo. However, there is a great need for powerful delivery strategies to enhance the therapeutic effect of small interfering RNA (siRNA). This review summarizes different signaling pathways inhibited by siRNA and the advantages of targeted siRNA as a delivery system.     

Solid lipid nanoparticles for applications in gene therapy: a review of the state of the art

Importance of the field: Gene therapy represents a new paradigm in the prevention and treatment of many inherited and acquired diseases, including genetic disorders, such as cystic fibrosis, haemophilia and many somatic diseases, such as tumours, neurodegenerative diseases and viral infections, such as AIDS.

Areas covered in this review: Among a large array of non-viral transfection agents used for in-vitro applications, cationic SLNs are the topic of this review, being recently proposed as an alternative carrier for DNA delivery, due to many technological advantages such as large-scale production from substances generally recognized as safe, good storage stability and possibility of steam sterilization and lyophilisation.

What the reader will gain: The authors give some information on the knowledge of intracellular trafficking and SLNs-DNA complex chemical-physical properties reported until now in the literature.

Take home message: The future success of cationic SLNs for administration of genetic material will depend on their ability to efficiently cross the physiological barriers, selectively targeting a specific cell type in vivo and expressing therapeutic genes.     

Anionic liposomal delivery system for DNA transfection

The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17:83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofectAmine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.     

Antisense strategies and non-viral gene therapy for cancer

Effective gene therapy for cancer remains an elusive goal, even after more than a decade of intensive research. There has been, however, tremendous progress in the development of increasingly sophisticated non-viral (or synthetic) delivery vectors for local and systemic administration of nucleic acids. Recent clinical data has also indicated the feasibility of using antisense oligonucleotides to inhibit inappropriately expressed or mutated genes in human cancers. The purpose of this review is to provide an update of the patent literature on the development of non-viral approaches for cancer gene therapy. In particular, patents on lipoplexes and polyplexes for delivery of therapeutic genes and antisense oligonucleotides are reviewed. The diverse range of antisense strategies being developed and recent clinical data are also highlighted.     

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。     

Pharmaceutical emulsions: a new approach for gene therapy

The concept of gene therapy involves the experimental transfer of a therapeutic gene into an individual’s cells and tissues to replace an abnormal gene aiming to treat a disease, or to use the gene to treat a disease just like a medicine, improving the clinical status of a patient. The achievement of a foreigner nucleic acid into a population of cells requires its transfer to the target. Therefore, it is essential to create carriers (vectors) that transfer and protect the nucleic acid until it reaches the target. The obvious disadvantages of the use of viral vectors have directed the research for the development of a nonviral organized system such as emulsions. In fact, recently, there has been an increase of interest in its use in biotechnology as a nonviral vector for gene therapy. This review focuses on the progress of cationic emulsions and the improvement of the formulations, as a potential delivery system for gene therapy.