Patents review in siRNA delivery for pulmonary disorders

siRNA inhibits protein expression by degrading complementary mRNA sequence and hence, it is widely applicable for the treatment of various diseases where single or multiple gene knock down is necessary. Due to the severity and lethality of pulmonary diseases, siRNA has been focused for improved health in these diseases. Pulmonary accumulation of siRNA can be achieved by different means like intranasal or inhalation administration or intratracheal route which is mainly utilized for in vivo animal studies. However, various pulmonary obstacles and intracellular barriers for siRNA transport challenge this novel therapeutic moiety. Researchers have utilized different viral and non-viral delivery vectors for intracellular delivery of siRNA to knock down target mRNA. The promise of RNA interference, mediated by siRNAs, has revolutionized the prospects for modulating gene expression as a way to achieve therapeutic aims in disease treatment. This review focuses on patents describing the siRNA delivery either in naked form or along with a single/multiple delivery vectors. Many inventors have shown promising results for pulmonary utilization of siRNA and more concentration on delivery system may make this genomic approach available to the clinics soon.     

Critical issues in delivery of RNAi therapeutics in vivo

RNA interference (RNAi) is a fundamental mechanism of gene regulation and has been harnessed to produce a new class of drugs for treatment of various diseases. A key issue in these applications is how to effectively deliver RNAi therapeutics into target cells. This review is focused on advances in RNA delivery in vivo. To achieve it, novel strategies have been developed to enhance stability of RNA in cells and tissues, overcome barriers to transport of RNA or its carriers in the body, and reduce immunogenicity and cytotoxicity of treatment. Approaches to RNA delivery are divided into three categories in this review: biological, chemical, and physical. Advantages and disadvantages of each method are discussed. At present, effective delivery of RNAi therapeutics in vivo is still a challenge although significant advances have been made in this field.     

小干涉RNA导入哺乳动物体内的方法与策略研究进展

小干涉RNA(small interfering RNA,siRNA)是目前研究的比较活跃的一种小RNA。利用siRNA使基因沉寂从而调节目的蛋白的功能,在疾病的基因治疗中有巨大的应用潜力。该文从siRNA在哺乳动物整体水平研究的角度,对近年来siRNA的载体策略以及siRNA导入体内的方法和途径等方面的研究进展作一综述,并讨论了siRNA在体内应用的优势和可能存在的问题。     

Efficient delivery of sticky siRNA and potent gene silencing in a prostate cancer model using a generation 5 triethanolamine-core PAMAM dendrimer

Successful achievement of RNA interference in therapeutic applications requires safe and efficient vectors for siRNA delivery. In the present study, we demonstrate that a triethanolamine (TEA)-core PAMAM dendrimer of generation 5 (G(5)) is able to deliver sticky siRNAs bearing complementary A(n)/T(n) 3'-overhangs effectively to a prostate cancer model in vitro and in vivo and produce potent gene silencing of the heat shock protein 27, leading to a notable anticancer effect. The complementary A(n)/T(n) (n = 5 or 7) overhangs characteristic of these sticky siRNA molecules help the siRNA molecules self-assemble into "gene-like" longer double-stranded RNAs thus endowing a low generation dendrimer such as G(5) with greater delivery capacity. In addition, the A(n)/T(n) (n = 5 or 7) overhangs act as protruding molecular arms that allow the siRNA molecule to enwrap the dendrimer and promote a better interaction and stronger binding, ultimately contributing toward the improved delivery activity of G(5). Consequently, the low generation dendrimer G(5) in combination with sticky siRNA therapeutics may constitute a promising gene silencing-based approach for combating castration-resistant prostate tumors or other cancers and diseases, for which no effective treatment currently exists.     

Engineering RNA for Targeted siRNA Delivery and Medical Application

RNA engineering for nanotechnology and medical applications is an exciting emerging research field. RNA has intrinsically defined features on the nanometre scale and is a particularly interesting candidate for such applications due to its amazing diversity, flexibility and versatility in structure and function. Specifically, the current use of siRNA to silence target genes involved in disease has generated much excitement in the scientific community. The intrinsic ability to sequence-specifically downregulate gene expression in a temporally- and spatially controlled fashion has led to heightened interest and rapid development of siRNA-based therapeutics. Although methods for gene silencing have been achieved with high efficacy and specificity in vitro, the effective delivery of nucleic acids to specific cells in vivo has been a hurdle for RNA therapeutics. This article covers different RNA-based approaches for diagnosis, prevention and treatment of human disease, with a focus on the latest developments of non-viral carriers of siRNA for delivery in vivo. The applications and challenges of siRNA therapy, as well as potential solutions to these problems, the approaches for using phi29 pRNA-based vectors as polyvalent vehicles for specific delivery of siRNA, ribozymes, drugs or other therapeutic agents to specific cells for therapy will also be addressed.     

Pulmonary delivery of therapeutic siRNA

Small interfering RNA (siRNA) has a huge potential for the treatment or prevention of various lung diseases. Once the RNA molecules have successfully entered the target cells, they could inhibit the expression of specific gene sequence through RNA interference (RNAi) mechanism and generate therapeutic effects. The biggest obstacle to translating siRNA therapy from the laboratories into the clinics is delivery. An ideal delivery agent should protect the siRNA from enzymatic degradation, facilitate cellular uptake and promote endosomal escape inside the cells, with negligible toxicity. Lung targeting could be achieved by systemic delivery or pulmonary delivery. The latter route of administration could potentially enhance siRNA retention in the lungs and reduce systemic toxic effects. However the presence of mucus, the mucociliary clearance actions and the high degree branching of the airways present major barriers to targeted pulmonary delivery. The delivery systems need to be designed carefully in order to maximize the siRNA deposition to the diseased area of the airways. In most of the pulmonary siRNA therapy studies in vivo, siRNA was delivered either intratracheally or intranasally. Very limited work was done on the formulation of siRNA for inhalation which is believed to be the direction for future development. This review focuses on the latest development of pulmonary delivery of siRNA for the treatment of various lung diseases.     

Cyclodextrin-based siRNA delivery nanocarriers: a state-of-the-art review

INTRODUCTION: The discovery of synthetic small interfering RNA (siRNA) has led to a surge of interest in harnessing RNA interference (RNAi) technology for biomedical applications and drug development. Even though siRNA can be a powerful therapeutic drug, its delivery remains a major challenge, due to the difficulty in its cellular uptake. Naked siRNA has a biological half-life of less than an hour in human plasma. To increase the lifetime and improve its therapeutic efficacy, non-viral vectors have been developed. As a natural evolution, cyclodextrins (CDs), which are natural cyclic oligosaccharides, have recently been applied as delivery vehicles for siRNA, and this in turn, has led to a surge of interest in this area. AREAS COVERED: This review discusses the recent advances made in the design of delivery strategies for siRNA, focusing on CD-based delivery vectors, because these have demonstrated clinical success. The methods of preparation of CD-based vectors, their characterization, transfection efficiencies, cellular toxicity, preclinical and clinical trials are also addressed, as well as future therapeutic applications. EXPERT OPINION: siRNA-mediated RNAi therapeutics is beginning to transform healthcare, particularly, for the treatment of solid tumors. For example, CALAA01, a targeted, self-assembling nanoparticle system based on CD complexed with siRNA has been effective in phase I clinical trials. Although siRNA therapeutics suffers from problems related to off-target effects and non-specific gene silencing, these problems can be overcome by reducing the nanoparticle size, improving the targeting efficiency and by modifying the primary sequence of the siRNA.     

Self-Assembled and Nanostructured siRNA Delivery Systems

A wide range of organic and inorganic materials have been used in the development of nano-scale self-assembling gene delivery systems to improve the therapeutic efficacy of nucleic acid drugs. Small interfering RNA (siRNA) has recently been recognized as a promising and potent nucleic acid medicine for the treatment of incurable genetic disorders including cancer; however, siRNA-based therapeutics suffer from the same delivery problems as conventional nucleic acid drugs such as plasmid DNA and antisense oligonucleotides. Many of the delivery strategies developed for nucleic acid drugs have been applied to siRNA therapeutics, but they have not produced satisfactory in vivo gene silencing efficiencies to warrant clinical trials. This review discusses recent progress in the development of self-assembled and nanostructured delivery systems for efficient siRNA-induced gene silencing and their potential application in clinical settings.     

Physicochemical characterization techniques for lipid based delivery systems for siRNA

siRNA based therapeutics is an emerging class of molecules with a high potential for fulfilling the promise of gene medicine. The high selectivity of siRNAs for their targets and subsequent gene ablation has been effectively demonstrated in a wide range of pre-clinical models. siRNA delivery in vivo has been most successfully achieved using lipid-based drug delivery systems. These lipid based formulations are designed to entrap siRNA molecules, ensure stability in in vitro and in vivo milieu, facilitate uptake, enhance cellular targeting, and facilitate delivery in the desired intracellular compartment. As more siRNA-based therapeutics enters the clinic with the associated regulatory scrutiny, there is a clear need to develop well-characterized systems that ensure consistent quality and thus reliable performance. Early clinical trials can be conducted using formulations with limited short-term stability manufactured on a small scale. However, a thorough understanding of the factors that influence the structure and stability of these particulate formulations is required to prevent any issues with optimization of large-scale industrial manufacturing, scale-up, and long-term shelf-life required to support large clinical trials and eventual market use. As newer targets for siRNA are identified and novel lipids are synthesized to optimize their in vivo efficiency, concomitant development of bio-physical methodologies that can improve understanding of the assembly and stability of these complex systems is critical. Along with bio-physical characterization, these assays are also required to reliably design, screen, develop and optimize formulations. Physicochemical characterization thus forms the basis of developing an effective analytical control strategy for siRNA delivery systems. In this review, analytical techniques used to characterize lipid-based siRNA delivery systems are discussed in detail. The importance of these physicochemical characterization techniques and analytical assays is explained. Case studies illustrating their use in siRNA formulation development and optimization are presented.     

Pseudovirions as Vehicles for the Delivery of siRNA

Over the last two decades, small interfering RNA (siRNA)-mediated gene silencing has quickly become one of the most powerful techniques used to study gene function in vitro and a promising area for new therapeutics. Delivery remains a significant impediment to realizing the therapeutic potential of siRNA, a problem that is also tied to immunogenicity and toxicity. Numerous delivery vehicles have been developed, including some that can be categorized as pseudovirions: these are vectors that are directly derived from viruses but whose viral coding sequences have been eliminated, preventing their classification as viral vectors. Characteristics of the pseudovirions discussed in this review, namely phagemids, HSV amplicons, SV40 in vitro-packaged vectors, influenza virosomes, and HVJ-Envelope vectors, make them attractive for the delivery of siRNA-based therapeutics. Pseudovirions were shown to deliver siRNA effector molecules and bring about RNA interference (RNAi) in various cell types in vitro, and in vivo using immune-deficient and immune-competent mouse models. Levels of silencing were not always determined directly, but the duration of siRNA-induced knockdown lasted at least 3 days. We present examples of the use of pseudovirions for the delivery of synthetic siRNA as well as the delivery and expression of DNA-directed siRNA.     

Nanocarrier systems for delivery of siRNA to ovarian cancer tissues

INTRODUCTION: Novel therapeutic strategies have been investigated for ovarian cancer to reduce toxicity and to improve outcomes for patients. Short interfering RNA (siRNA), which directs the sequence-specific degradation of target mRNA and provides specificity of gene knockdown, represents a unique class of potential therapeutics for ovarian cancer. However, siRNA molecules are rapidly degraded in plasma and are unable to passively diffuse through cellular membranes. Nanocarriers can efficiently protect siRNA from in vivo degradation and are able to deliver these active macromolecules to tumor cells even after intravenous administration. AREAS COVERED: Strategies of gene therapy and the role of siRNA in ovarian cancer treatment are introduced, followed by an overview of nanocarriers for siRNA delivery, the advantages of the systems and the types of targeting to tumor cells. Classes of nanocarriers for delivery of siRNA, their functionalities and modalities are discussed with emphasis on the promising vehicles. EXPERT OPINION: Gene silencing therapy based on siRNA represents a possible opportunity for treatment of ovarian cancer patients. However, this approach requires selection of suitable nanocarriers that can safely and effectively deliver siRNA to the target site to induce its effect. Very little work has been done in this field; therefore, it is a good direction for future development.     

Cyclodextrin-based siRNA delivery nanocarriers: a state-of-the-art review

Introduction: The discovery of synthetic small interfering RNA (siRNA) has led to a surge of interest in harnessing RNA interference (RNAi) technology for biomedical applications and drug development. Even though siRNA can be a powerful therapeutic drug, its delivery remains a major challenge, due to the difficulty in its cellular uptake. Naked siRNA has a biological half-life of less than an hour in human plasma. To increase the lifetime and improve its therapeutic efficacy, non-viral vectors have been developed. As a natural evolution, cyclodextrins (CDs), which are natural cyclic oligosaccharides, have recently been applied as delivery vehicles for siRNA, and this in turn, has led to a surge of interest in this area.

Areas covered: This review discusses the recent advances made in the design of delivery strategies for siRNA, focusing on CD-based delivery vectors, because these have demonstrated clinical success. The methods of preparation of CD-based vectors, their characterization, transfection efficiencies, cellular toxicity, preclinical and clinical trials are also addressed, as well as future therapeutic applications.

Expert opinion: siRNA-mediated RNAi therapeutics is beginning to transform healthcare, particularly, for the treatment of solid tumors. For example, CALAA01, a targeted, self-assembling nanoparticle system based on CD complexed with siRNA has been effective in phase I clinical trials. Although siRNA therapeutics suffers from problems related to off-target effects and non-specific gene silencing, these problems can be overcome by reducing the nanoparticle size, improving the targeting efficiency and by modifying the primary sequence of the siRNA.     

Inhalable siRNA: potential as a therapeutic agent in the lungs

RNA interference (RNAi) is gaining increasing popularity both as a molecular biology tool and as a potential therapeutic agent. RNAi is a naturally occurring gene regulatory mechanism, which has a number of advantages over other gene/antisense therapies including specificity of inhibition, potency, the small size of the molecules and the diminished risk of toxic effects, e.g., immune responses. Targeted, local delivery of RNAi to the lungs via inhalation offers a unique opportunity to treat a range of previously untreatable or poorly controlled respiratory conditions. In this timely review we look at the potential applications of RNAi in the lungs for the treatment of a range of diseases including inflammatory and immune conditions, cystic fibrosis, infectious disease and cancer. In 2006 Alnylam initiated the first phase 1 clinical study of an inhaled siRNA for the treatment of respiratory syncytial virus. If its potential as a therapeutic is to be realized, then safe and efficient means of targeted delivery of small interfering RNA (siRNA) to the lungs must be developed. Therefore in this review we also present the latest developments in siRNA delivery to airway cells in vitro and the work to date on in vivo delivery of siRNA to the lungs for the treatment of a range of diseases.     

Delivery systems for siRNA drug development in cancer therapy

Since the discovery of the Nobel prize-winning mechanism of RNA interference (RNAi) ten years ago, it has become a promising drug target for the treatment of multiple diseases, including cancer. There have already been some successful applications of siRNA drugs in the treatment of age-related macular degeneration and respiratory syncytial virus infection. However, significant barriers still exist on the road to clinical applications of siRNA drugs, including poor cellular uptake, instability under physiological conditions, off-target effects and possible immunogenicity. The successful application of siRNA for cancer therapy requires the development of clinically suitable, safe and effective drug delivery systems. Herein, we review the design criteria for siRNA delivery systems and potential siRNA drug delivery systems for cancer therapy, including chemical modifications, lipid-based nanovectors, polymer-mediated delivery systems, conjugate delivery systems, and others.     

siRNA delivery systems for cancer treatment

With increasing knowledge on the molecular mechanisms of endogenous RNA interference, small interfering RNAs (siRNAs) have been emerging as innovative nucleic acid medicines for treatment of incurable diseases such as cancers. Although several siRNA candidates for the treatment of ocular and respiratory diseases are undergoing clinical trials, there are challenges inherent in the further development of siRNAs for anti-cancer therapeutics, because systemic administration will be required in most cases. In addition to nonspecific off-target and immune stimulation problems, appropriate delivery remains a major hurdle. The technologies developed for delivery of nucleic acid medicines such as plasmid DNA and antisense oligonucleotides have paved the way to rapid progress for in vivo delivery of siRNAs. Here, we review various in vivo delivery strategies including chemical modification, conjugation, lipid-based techniques, polymer-based nanosystems, and physical methods. Moreover, the current progress in siRNA delivery systems for gynecologic, liver, lung, and prostate cancers is discussed.     

Drug delivery of siRNA therapeutics: potentials and limits of nanosystems

Gene therapy is a promising tool for the treatment of human diseases that cannot be cured by rational therapies. The major limitation for the use of small interfering RNA (siRNA), both in vitro and in vivo, is the inability of naked siRNA to passively diffuse through cellular membranes due to the strong anionic charge of the phosphate backbone and consequent electrostatic repulsion from the anionic cell membrane surface. Therefore, the primary success of siRNA applications depends on suitable vectors to deliver therapeutic genes. Cellular entrance is further limited by the size of the applied siRNA molecule. Multiple delivery pathways, both viral and nonviral, have been developed to bypass these problems and have been successfully used to gain access to the intracellular environment in vitro and in vivo, and to induce RNA interference (RNAi). This review focuses on different pathways for siRNA delivery and summarizes recent progress made in the use of vector-based siRNA technology.     

Recent progress in copolymer-mediated siRNA delivery

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.     

Nanodelivery in airway diseases: Challenges and therapeutic applications

This review describes the challenges and therapeutic applications of nanodelivery systems for treatment of airway diseases. Therapeutic applications of nanodelivery in airway diseases involve targeted delivery of DNA, short interfering RNA, drugs, or peptides to hematopoietic progenitor cells and pulmonary epithelium to control chronic pathophysiology of obstructive and conformational disorders. The major challenges to nanodelivery involve physiologic barriers such as mucus and alveolar fluid. It is necessary for the nanoparticles to be biodegradable and capable of providing sustained drug delivery to the selected cell type. Once inside the cell, the nanoparticle should be capable of escaping the endocytic degradation machinery. In addition, for effective gene delivery, nuclear entry and chromosomal integration are critical. The strategies to overcome these pathophysiologic barriers are discussed as an attempt to synchronize the efforts of pulmonary biologists, chemists, and clinicians to develop novel nanodelivery therapeutics for airway diseases.From the Clinical EditorTherapeutic applications of nano-delivery in airway diseases involve targeted delivery of DNA, siRNA, drugs or peptides to hematopoietic progenitor cells and pulmonary epithelium. These nano-particles must be biodegradable, capable of providing sustained drug delivery to specific cells, and should escape the endocytic degradation machinery. For effective gene-delivery they should also provide nuclear entry and chromosomal integration.     

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.