Non-viral nucleic acid containing nanoparticles as cancer therapeutics

ABSTRACT

Introduction: The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors.

Areas covered: In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery.

Expert opinion: The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic.     

Delivery of nucleic acid therapeutics by genetically engineered hematopoietic stem cells

Several populations of adult human stem cells have been identified, but only a few of these are in routine clinical use. The hematopoietic stem cell (HSC) is arguably the most well characterized and the most routinely transplanted adult stem cell. Although details regarding several aspects of this cell's phenotype are not well understood, transplant of HSCs has advanced to become the standard of care for the treatment of a range of monogenic diseases and several types of cancer. It has also proven to be an excellent target for genetic manipulation, and clinical trials have already demonstrated the usefulness of targeting this cell as a means of delivering nucleic acid therapeutics for the treatment of several previously incurable diseases. It is anticipated that additional clinical trials will soon follow, such as genetically engineering HSCs with vectors to treat monogenic diseases such as hemophilia A. In addition to the direct targeting of HSCs, induced pluripotent stem (iPS) cells have the potential to replace virtually any engineered stem cell therapeutic, including HSCs. We now know that for the broad use of genetically modified HSCs for the treatment of non-lethal diseases, e.g. hemophilia A, we must be able to regulate the introduction of nucleic acid sequences into these target cells. We can begin to refine transduction protocols to provide safer approaches to genetically manipulate HSCs and strategies are being developed to improve the overall safety of gene transfer. This review focuses on recent advances in the systemic delivery of nucleic acid therapeutics using genetically modified stem cells, specifically focusing on i) the use of retroviral vectors to genetically modify HSCs, ii) the expression of fVIII from hematopoietic stem cells for the treatment of hemophilia A, and iii) the use of genetically engineered hematopoietic cells generated from iPS cells as treatment for disorders of hematopoiesis.     

MagnetofectionTM platform: from magnetic nanoparticles to novel nucleic acid therapeutics

Nucleic acid delivery to cells to make them produce a desired protein or to shut down the expression of endogenous genes opens unique possibilities for research and therapy. During the last decade, to realize the potential of this approach, nanomagnetic methods for delivering and targeting nucleic acids have been developed, methods which are often referred to as Magnetofection. Our research group at the Institute of Experimental Oncology and Therapy Research, located at the University Hospital Klinikum rechts der Isar in the center of Munich, Germany, develops new magnetic nanomaterials and, their formulations with gene-delivery vectors and technologies to allow localized and efficient gene delivery in vitro and in vivo for a variety of research, diagnostic and therapeutic applications.     

Progress on the development of therapeutics against West Nile virus

A decade has passed since the appearance of West Nile virus (WNV) in humans in the Western Hemisphere in New York City. During this interval, WNV spread inexorably throughout North and South America and caused millions of infections ranging from a sub-clinical illness, to a self-limiting febrile syndrome or lethal neuroinvasive disease. Its entry into the United States triggered intensive research into the basic biology of WNV and the elements that comprise a protective host immune response. Although no therapy is currently approved for use in humans, several strategies are being pursued to develop effective prophylaxis and treatments. This review describes the current state of knowledge on epidemiology, clinical presentation, pathogenesis, and immunobiology of WNV infection, and highlights progress toward an effective therapy.     

Challenges in the pharmaceutical development of lipid-based short interfering ribonucleic acid therapeutics

INTRODUCTION: Harnessing RNA interference as a therapeutic approach has the potential to significantly expand the druggable target space, offering new hope for treatment of diseases that cannot be addressed with existing classes of drugs. A number of siRNA therapeutics have already progressed into preclinical and clinical development. Of these, lipid-based systems have emerged as one of the most mature classes of systemic delivery technologies. Despite tremendous advances in development, a number of significant challenges must still be addressed to enable commercialization of a lipid-based siRNA pharmaceutical product. AREAS COVERED: This review addresses specific challenges inherent to the pharmaceutical development of lipid-based siRNA therapeutics. Focus is placed on the development of a robust manufacturing process, the setting of appropriate product specifications and controls, development of strategies to assess and ensure product stability, and the evaluation of product comparability throughout development. EXPERT OPINION: Discovering and developing a lipid-based siRNA therapeutic that can be commercialized requires engineering a particle that selectively and efficiently delivers the cargo to the target tissue and cellular compartment. The particle assembly must be strictly controlled and physical properties thoroughly characterized to successfully develop an understanding of particle attributes that influence in vivo pharmaceutical properties. Correlation of particle physio-chemical properties to product performance is the foundation for advancements in discovery and assuring quality in a commercial drug product. Although difficult, we believe these development challenges can be addressed with appropriate scientific resources and that the industry will continue to progress siRNA therapeutic candidates through clinical development.     

Future promise of siRNA and other nucleic acid based therapeutics for the treatment of chronic HCV

RNA interference (RNAi) is gaining favor as a potential therapeutic option for the treatment of Hepatitis C virus infections. RNAi, first discovered in plants, induces sequence specific degradation of messenger RNA following the introduction of short interference RNA (siRNA). RNAi is a natural defense mechanism used by plants to combat viral infections, and the discovery of RNAi activity in mammalian cells has prompted several drug companies to investigate and exploit RNAi based drugs as a potential therapy against HCV infections. A number of research groups have demonstrated that strong RNAi activity can be induced against HCV using synthetic siRNA duplexes as triggers, or by expressing short hairpin RNAs from plasmid or viral vectors. However, much work remains to improve delivery, maintain specificity and limit the development of virus resistance. HCV is capable of evading RNAi activity through the incorporation escape mutations within the siRNA target sequence, highlighting the importance of implementing strategies to limit the development of resistance. Other nucleic acid based therapies such as antisense oligonucleotides, RNA aptamers and ribozymes have also been considered for use as HCV therapeutics, and we will outline the potential opportunities and obstacles to their use as well as RNAi.     

Recent advances in lipid nanoparticles for delivery of nucleic acid,mRNA, and gene editing-based therapeutics

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.     

Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics

Branched polyethylenimine (PEI; 25 kDa) as a nonviral vector exhibits high transfection efficiency and is a potential candidate for efficient gene delivery. However, the cytotoxicity of PEI limits its application in vivo. PEI was ionically interacted with hexametaphosphate, a compact molecule with high anionic charge density, to obtain nanoparticles (PEI-HMP). Nanoparticles were assessed for their efficacy in protecting complexed DNA against nucleases. The intracellular trafficking of nanoparticles was monitored by confocal microscopy. The cytotoxicity and transfection efficiency of PEI-HMP nanoparticles were evaluated in vitro. In vitro transfection efficiency of PEI-HMP (7.7%) was ～1.3- to 6.4-folds higher than that of the commercial reagents GenePORTER 2^TM, Fugene^TM, and Superfect^TM. Also, PEI-HMP (7.7%) delivered green fluorescent protein (GFP)-specific small interfering ribonucleic acid (siRNA) in culture cells leading to >80% suppression in GFP gene expression. PEI-HMP nanoparticles protected complexed DNA against DNase for at least 2 hours. A time-course uptake of PEI-HMP (7.7%) nanoparticles showed the internalization of nanoparticles inside the cell nucleus in 2 hours. Thus, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as nonviral vectors for gene delivery.From the Clinical EditorBranched polyethylenimine (PEI) as a non-viral vector exhibits high transfection efficiency for gene delivery, but its cytotoxicity limits its applications. PEI hexametaphosphate nanoparticles (PEI-HMP) demonstrated a 1.3-6.4 folds higher transfection rate compared to commercial reagents. Overall, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as non-viral vectors for gene delivery.     

基于CRISPR-Cas系统的新型冠状病毒核酸检测技术进展

新型冠状病毒（severe acute respiratory syndrome coronavirus 2,SARS-CoV-2）核酸检测是迅速发现传染源、锁定管控目标,进而切断传播途径的关键有效手段。成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeat,CRISPR）及其相关蛋白（CRISPR associated protein,Cas）系统的定向基因编辑技术在病原微生物检测方面有广阔的应用前景,尤其是分别靶向切割RNA和DNA的Cas13和Cas12,依靠其附带切割特性,结合荧光报告系统,已应用于SARS-CoV-2的核酸检测,在灵敏度、专一性、便携性、经济性和易操作性等多方面表现出色。此文系统总结了基于CRISPR-Cas系统的SARS-CoV-2核酸检测技术研究进展,包括作用原理、改进的方案和优势,以评价推广应用的可行性,也为基于CRISPR-Cas系统研发病原体快速检测方法提供参考。     

肽核酸单体骨架修饰的研究进展

肽核酸(PNA)是DNA模拟物,能与DNA和RNA以序列特异性方式结合,在基因研究和基因治疗方面有广泛的应用前景。为了优化肽核酸的性质(如水溶性、对细胞的透膜能力、杂交专一性),许多骨架修饰的PNA被合成出来。该文综述了近年来肽核酸单体骨架修饰的合成研究进展。指出设计新型的PNA结构是改良PNA性能、拓宽DNA应用的主要途径。     

Progress in nanoparticulate systems for peptide, proteins and nucleic acid drug delivery

Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape.     

Targeting adenosine receptors in the development of cardiovascular therapeutics

Adenosine receptor stimulation has negative inotropic and dromotropic actions, reduces cardiac ischemia–reperfusion injury and remodeling, and prevents cardiac arrhythmias. In the vasculature, adenosine modulates vascular tone, reduces infiltration of inflammatory cells and generation of foam cells, and may prevent the development of atherosclerosis as a result. Modulation of insulin sensitivity may further add to the anti-atherosclerotic properties of adenosine signaling. In the kidney, adenosine plays an important role in tubuloglomerular feedback and modulates tubular sodium reabsorption. The challenge is to take advantage of the beneficial actions of adenosine signaling while preventing its potential adverse effects, such as salt retention and sympathoexcitation. Drugs that interfere with adenosine formation and elimination or drugs that allosterically enhance specific adenosine receptors seem to be most promising to meet this challenge.     

阿片类药物依赖的治疗学进展

介绍阿片类物质依赖的主要药物治疗学进展，主要包括三个步骤，重点介绍阿片受体激动药和非阿片受体激动药的使用，此外尚有非药物疗法及免疫疗法等。     

Practical considerations in the development of hemoglobin-based oxygen therapeutics

The development of hemoglobin based oxygen therapeutics (HBOCs) requires consideration of a number of factors. While the enabling technology derives from fundamental research on protein biochemistry and biological interactions, translation of these research insights into usable medical therapeutics demands the application of considerable technical expertise and consideration and reconciliation of a myriad of manufacturing, medical, and regulatory requirements. The HBOC development challenge is further exacerbated by the extremely high intravenous doses required for many of the indications contemplated for these products, which in turn implies an extremely high level of purity is required. This communication discusses several of the important product configuration and developmental considerations that impact the translation of fundamental research discoveries on HBOCs into usable medical therapeutics.     

Targeting adenosine receptors in the development of cardiovascular therapeutics

Adenosine receptor stimulation has negative inotropic and dromotropic actions, reduces cardiac ischemia-reperfusion injury and remodeling, and prevents cardiac arrhythmias. In the vasculature, adenosine modulates vascular tone, reduces infiltration of inflammatory cells and generation of foam cells, and may prevent the development of atherosclerosis as a result. Modulation of insulin sensitivity may further add to the anti-atherosclerotic properties of adenosine signaling. In the kidney, adenosine plays an important role in tubuloglomerular feedback and modulates tubular sodium reabsorption. The challenge is to take advantage of the beneficial actions of adenosine signaling while preventing its potential adverse effects, such as salt retention and sympathoexcitation. Drugs that interfere with adenosine formation and elimination or drugs that allosterically enhance specific adenosine receptors seem to be most promising to meet this challenge.     

Advances in the development of siRNA-based therapeutics for cancer

Short interfering (si)RNA represents a promising novel therapeutic modality for the treatment of cancer. However, delivering siRNA to tumors via clinically relevant means is a major technical hurdle that has impeded the successful development of siRNA-based cancer therapeutics. Significant progress has been made in recent years for the delivery of siRNA to tumors, and several promising siRNA delivery platforms have begun to emerge. These platforms include liposomes, in which siRNA is encapsulated in a lipid vesicle; polyplexes, in which a cationic carrier is used to bind siRNA to form siRNA-containing nanoparticles; liposome-polycation-DNA (LPD) complexes, in which an siRNA-containing polyplex is encapsulated in a lipid vesicle; and siRNA conjugates, in which siRNA is coupled to a targeting moiety that carries the siRNA into target cells via receptor-mediated endocytosis. An unbiased, independent evaluation of various tumor-delivery approaches will be a critical starting point in identifying the most promising delivery platforms for the development of siRNA-based cancer therapeutics.