Tumor therapeutics by design: targeting and activation of death receptors

Due to their strong apoptosis-inducing capacity, the death receptor ligands CD95L, TNF and TRAIL have been widely viewed as potential cancer therapeutics. While clinical data with CD95L and TRAIL are not yet available, TNF is a registered drug, albeit only for loco-regional application in a limited number of indications. The TNF experience has told us that specific delivery and restricted action is a major challenge in the development of multifunctional, pleiotropically acting cytokines into effective cancer therapeutics. Thus, gene-therapeutic approaches and new cytokine variants have been designed over the last 10 years with the aim of increasing anti-tumoral activity and reducing systemic side effects. Here, we present our current view of the therapeutic potential of the death receptor ligands TNF, CD95L and TRAIL and of the progress made towards improving their efficacy by tumor targeting, use of gene therapy and genetic engineering. Results generated with newly designed fusion proteins suggest that enhanced tumor-directed activity and prevention of undesirable actions of death receptor ligands is possible, thereby opening up a useful therapeutic window for all of the death receptor ligands, including CD95L.     

Utilizing endocrine secretory pathways in salivary glands for systemic gene therapeutics

Mammalian salivary glands are commonly used models of exocrine secretion. However, there is substantial experimental evidence showing the physiological existence of endocrine secretory pathways in these tissues. The use of gene transfer technology in vivo has allowed the unambiguous demonstration of these endocrine pathways. We and others have exploited such findings and evaluated salivary glands as possible target tissues for systemic applications of gene therapeutics. Salivary glands present numerous advantages for this purpose, including being well encapsulated, which limits extra-glandular vector dissemination, and having the luminal membranes of almost all parenchymal cells accessible via intraoral delivery of vectors through the main excretory ducts. Existing studies suggest that clinical benefits will result from salivary gland targeted systemic gene therapeutics.     

The therapeutic potential of RNA interference

In recent years, we have witnessed the discovery of a new mechanism of gene regulation called RNA interference (RNAi), which has revitalized interest in the development of nucleic acid-based technologies for therapeutic gene suppression. This review focuses on the potential therapeutic use of RNAi, discussing the theoretical advantages of RNAi-based therapeutics over previous technologies as well as the challenges involved in developing RNAi for clinical use. Also reviewed, are the in vivo proof-of principle experiments that provide the preclinical justification for the continued development of RNAi-based therapeutics.     

Smart Nanocarriers for the Delivery of Nucleic Acid-Based Therapeutics: A Comprehensive Review

Nucleic acid-based therapies are promising therapeutics for the treatment of several systemic disorders, and they offer an exciting opportunity to address emerging biological challenges. The scope of nucleic acid-based therapeutics in the treatment of multiple disease states including cancers has been widened by recent progress in Ribonucleic acids (RNA) biology. However, cascades of systemic and intracellular barriers, including rapid degradation, renal clearance, and poor cellular uptake, hinder the clinical effectiveness of nucleic acid-based therapies. These barriers can be circumvented by utilizing advanced smart nanocarriers that efficiently deliver and release the encapsulated nucleic acids into the target tissues. This review describes the current status of clinical trials on nucleic acid-based therapeutics and highlights representative examples that provide an overview on the current and emerging trends in nucleic acid-based therapies. A better understanding of the design of advanced nanocarriers is essential to promote the translation of therapeutic nucleic acids into a clinical reality.     

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.     

Current progress of siRNA/shRNA therapeutics in clinical trials

Through a mechanism known as RNA interference (RNAi), small interfering RNA (siRNA) molecules can target complementary mRNA strands for degradation, thus specifically inhibiting gene expression. The ability of siRNAs to inhibit gene expression offers a mechanism that can be exploited for novel therapeutics. Indeed, over the past decade, at least 21 siRNA therapeutics have been developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders. Like other biological drugs, RNAi-based therapeutics often require a delivery vehicle to transport them to the targeted cells. Thus, the clinical advancement of numerous siRNA drugs has relied on the development of siRNA carriers, including biodegradable nanoparticles, lipids, bacteria, and attenuated viruses. Most therapies permit systemic delivery of the siRNA drug, while others use ex vivo delivery by autologous cell therapy. Advancements in bioengineering and nanotechnology have led to improved control of delivery and release of some siRNA therapeutics. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules. Here, we provide an overview of siRNA therapeutics in clinical trials, including their clinical progress, the challenges they have encountered, and the future they hold in the treatment of human diseases.     

Coding and non-coding nucleotides': The future of stroke gene therapeutics

Stroke is the foremost cause of death ranked after heart disease and cancer. It is the fatal life-threatening event that requires immediate medical admissions to overcome following morbidity and mortality. The therapeutic advances in stroke therapy have been manipulated with diverse paths for last 5 years. Recent research and clinical trials have investigated a variety of anti-stroke agents including anti-coagulants, cerebro-protective agents, antiplatelet therapy, stem-cell therapy, and specified gene therapy. In recent advanced studies, genetic therapies including noncoding RNAs (ncRNAs), long non-coding RNAs (LncRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), Piwi interacting RNAs (PiWi RNAs) have shown better potential as targeted future therapeutics with a better outcome than conventional stroke therapeutics. The potential of targeted gene therapy is much more advanced in not only the induction of neuroprotection but also safer non-toxic targeted therapeutics. In the current state of the art review, we have focused on the recent advancements made towards the stroke with RNA modifications and targeted gene therapeutics.     

The gene encoding interleukin-13: a susceptibility locus for asthma and related traits

Respirable antisense oligonucleotides (RASONs), which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.     

Respirable antisense oligonucleotides: a new drug class for respiratory disease

Respirable antisense oligonucleotides (RASONs), which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.     

Development of new RNAi therapeutics

RNAi-mediated gene inactivation has become a cornerstone of the present day gene function studies that are the foundation of mechanism and target based drug discovery and development, which could potentially shorten the otherwise long process of drug development. In particular, the coming of age of "RNAi drug" could provide new promising therapeutics bypassing traditional approaches. However, there are technological hurdles need to overcome and the biological limitations need to consider for achieving effective therapeutics. Major hurdles include the intrinsic poor pharmacokinetic property of siRNA and major biological restrictions include off-target effects, interferon response and the interference with endogenous miRNA. Recent innovations in nucleic acid chemistry, formulations and delivery methods have gradually rendered it possible to develop effective RNAi-based therapeutics. Careful design based on the newest RNAi/miRNA biology can also help to minimize the potential tissue toxicity. If successful with systemic application, RNAi drug will no doubt revolutionize the whole drug development process. This review attempts to describe the progress in this area, including applications in preclinical models and recent favorable experience in a number of human trials of local diseases, along with the discussion on the potential limitations of RNAi therapeutics.     

Current advances in adenovirus nanocomplexes: more specificity and less immunogenicity

An often overlooked issue in the field of adenovirus (Ad)-mediated cancer gene therapy is its limited capacity for effective systemic delivery. Although primary tumors can be treated effectively with intralesional injection of conventional Ad vectors, systemic metastasis is difficult to cure. Systemic administration of conventional naked Ads leads to acute accumulation of Ad particles in the liver, induction of neutralizing antibody, short blood circulation half-life, non-specific biodistribution in undesired organs, and low selective accumulation in the target disease site. Versatile strategies involving the modification of viral surfaces with polymers and nanomaterials have been designed for the purpose of maximizing Ad anti-tumor activity and specificity by systemic administration. Integration of viral and non-viral nanomaterials will substantially advance both fields, creating new concepts in gene therapeutics. This review focuses on current advances in the development of smart Ad hybrid nanocomplexes based on various design-based strategies for optimal Ad systemic administration.     

Modulation of angiogenesis with siRNA inhibitors for novel therapeutics

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.     

microRNA治疗在癌症及其他疾病中的研究进展*

microRNAs(miRNAs)是一类内源性、非编码小分子RNAs(约22 nt),在基因表达调控中发挥关键作用。已有研究表明,miRNAs失调是造成多种人类疾病的原因,如癌症、病毒感染及自身免疫性疾病等。补充或抑制miRNAs功能与活性已成为多种疾病治疗的新策略,抗肿瘤miR-34 mimics、治疗HCV感染的anti-miR-122等基于miRNAs的治疗方案已进入临床试验。重点就miRNAs治疗在癌症及其他疾病中的最新研究进展进行综述,并对目前开发安全有效miRNAs治疗策略所面临的挑战进行分析。     

MicroRNA replacement therapy in cancer

Despite the recent progress in cancer management approaches, the mortality rate of cancer is still growing and there are lots of challenges in the clinics in terms of novel therapeutics. MicroRNAs (miRNA) are regulatory small noncoding RNAs and are already confirmed to have a great role in regulating gene expression level by targeting multiple molecules that affect cell physiology and disease development. Recently, miRNAs have been introduced as promising therapeutic targets for cancer treatment. Regulatory potential of tumor suppressor miRNAs, which enables regulation of entire signaling networks within the cells, makes them an interesting option for developing cancer therapeutics. In this regard, over recent decades, scientists have aimed at developing powerful and safe targeting approaches to restore these suppressive miRNAs in cancerous cells. The present review summarizes the function of miRNAs in tumor development and presents recent findings on how miRNAs have served as therapeutic agents against cancer, with a special focus on tumor suppressor miRNAs (mimics). Moreover, the latest investigations on the therapeutic strategies of miRNA delivery have been presented.     

Epigenetic drug discovery: targeting DNA methyltransferases

Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine, respectively; the oligonucleotides MG98 and miR29a; and a number of reversible inhibitors, some of which appear to be selective against particular DNMT isoforms. Finally, we discuss future opportunities and challenges for next-generation therapeutics.     

Single-chain TNF, a TNF derivative with enhanced stability and antitumoral activity

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.     

Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

Potent sequence selective gene inhibition by siRNA ‘targeted’ therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets.