Scalable production of siRNA-encapsulated extracellular vesicles for the inhibition of KRAS-mutant cancer using acoustic shock waves.

Extracellular vesicles (EVs) have emerged as a potential delivery vehicle for nucleic-acid-based therapeutics, but challenges related to their large-scale production and cargo-loading efficiency have limited their therapeutic potential. To address these issues, we developed a novel "shock wave extracellular vesicles engineering technology" (SWEET) as a non-genetic, scalable manufacturing strategy that uses shock waves (SWs) to encapsulate siRNAs in EVs. Here, we describe the use of the SWEET platform to load large quantities of KRASG12C-targeting siRNA into small bovine-milk-derived EVs (sBMEVs), with high efficiency. The siRNA-loaded sBMEVs effectively silenced oncogenic KRASG12C expression in cancer cells; they inhibited tumour growth when administered intravenously in a non-small cell lung cancer xenograft mouse model. Our study demonstrates the potential for the SWEET platform to serve as a novel method that allows large-scale production of cargo-loaded EVs for use in a wide range of therapeutic applications.

Don't shake it! Mechanical stress testing of mRNA-lipid nanoparticles.

Shaking stress studies are typically performed during formulation development to test the liability of a drug product towards interfacial stress occurring during transport, especially if a liquid formulation is desired. We evaluated various shaking procedures using a polyA-surrogate solution and verified our findings by eGFP-LNP cell-expression experiments. Shaking on an orbital shaker in vertical and horizontal orientations at increasing speeds from 300 to 600 rpm resulted in decreasing levels of encapsulated nucleic acid content, larger LNP sizes, and decreasing PDI. We report that vertical and horizontal shaking of both polyA- and eGFP-LNPs led to white deposits on the inner glass vial surface, depending on time, rpm, and temperature. Increasing the fill volume/smaller headspace (0.3 versus 0.9 mL fill) did not mitigate this phenomenon in the studied configuration, and the use of hydrophobic primary packaging even accelerated the formation of white deposits. In contrast, we demonstrated that a lyophilized polyA-LNP dosage form was less susceptible to shaking and maintained cake integrity and product properties. Multiple vortexing steps resulted in an increase in LNP size, PDI, and a decrease in encapsulated polyA content. We conclude that shaking experiments of nucleic acid-loaded LNPs in their final configuration at intended transport conditions need to be considered during technical development.

Next-generation therapeutics for rare genetic disorders.

The therapeutic potential of the human genome has been explored through the development of next-generation therapeutics, which have had a high impact on treating genetic disorders. Classical treatments have traditionally focused on common diseases that require repeated treatments. However, with the recent advancements in the development of nucleic acids, utilizing DNA and RNA to modify or correct gene expression in genetic disorders, there has been a paradigm shift in the treatment of rare diseases, offering more potential one-time cure options. Advanced technologies that use CRISPR-Cas 9, antisense oligonucleotides, siRNA, miRNA, and aptamers are promising tools that have achieved successful breakthroughs in the treatment of various genetic disorders. The advancement in the chemistry of these molecules has improved their efficacy, reduced toxicity, and expanded their clinical use across a wide range of tissues in various categories of human disorders. However, challenges persist regarding the safety and efficacy of these advanced technologies in translating into clinical practice. This review mainly focuses on the potential therapies for rare genetic diseases and considers how next-generation techniques enable drug development to achieve long-lasting curative effects through gene inhibition, replacement, and editing.

Long non-coding RNA-targeting therapeutics: discovery and development update.

INTRODUCTION: lncRNAs are major players in regulatory networks orchestrating multiple cellular functions, such as 3D chromosomal interactions, epigenetic modifications, gene expression and others. Due to progress in the development of nucleic acid-based therapeutics, lncRNAs potentially represent easily accessible therapeutic targets. AREAS COVERED: Currently, significant efforts are directed at studies that can tap the enormous therapeutic potential of lncRNAs. This review describes recent developments in this field, particularly focusing on clinical applications. EXPERT OPINION: Extensive druggable target range of lncRNA combined with high specificity and accelerated development process of nucleic acid-based therapeutics open new prospects for treatment in areas of extreme unmet medical need, such as genetic diseases, aggressive cancers, protein deficiencies, and subsets of common diseases caused by known mutations. Although currently wide acceptance of lncRNA-targeting nucleic acid-based therapeutics is impeded by the need for parenteral or direct-to-CNS administration, development of less invasive techniques and orally available/BBB-penetrant nucleic acid-based therapeutics is showing early successes. Recently, mRNA-based COVID-19 vaccines have demonstrated clinical safety of all aspects of nucleic acid-based therapeutic technology, including multiple chemical modifications of nucleic acids and nanoparticle delivery. These trends position lncRNA-targeting drugs as significant players in the future of drug development, especially in the area of personalized medicine.

The Promise of PCSK9 and Lipoprotein(a) as Targets for Gene Silencing Therapies.

PURPOSE: High plasma concentrations of LDL and lipoprotein(a) (Lp[a]) are independent and causal risk factors for atherosclerotic cardiovascular disease (ASCVD). There is an unmet therapeutic need for high-risk patients with elevated levels of LDL-C and/or Lp(a). Recent advances in the development of nucleic acids for gene silencing (ie, triantennary N-acetylgalactosamine conjugated antisense-oligonucleotides [ASOs] and small interfering RNA [siRNA]) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and Lp(a) offer effective and sustainable therapies. METHODS: Related articles in the English language were identified through a search for original and review articles in the PubMed database using the following key terms: cardiovascular disease, dyslipidemia, PCSK9 inhibitors, Lp(a), LDL-cholesterol, familial hypercholesterolemia, siRNA, and antisense oligonucleotide and clinical trials (either alone or in combination). FINDINGS: Inclisiran, the most advanced siRNA-treatment targeting hepatic PCSK9, is well tolerated, producing a >30% reduction on LDL-C levels in randomized controlled trials. Pelacarsen is the most clinical advanced ASO, whereas olpasiran and SLN360 are the 2 siRNAs directed against the mRNA of the LPA gene. Evidence suggests that all Lp(a)-targeting agents are safe and well tolerated, with robust and sustained reduction in plasma Lp(a) concentration up to 70% to 90% in individuals with elevated Lp(a) levels. IMPLICATIONS: Cumulative evidence from clinical trials supports the value of ASO and siRNA therapies targeting the synthesis of PCSK9 and Lp(a) for lowering LDL-C and Lp(a) in patients with established ASCVD or high risk of ASCVD. Further research is needed to examine whether gene silencing therapy could improve clinical outcomes in patients with elevated LDL and/or Lp(a) levels. Confirmation of the tolerability and cost-effectiveness of long-term inhibition of PCSK9 and Lp(a) with this approach is essential.

Up-regulated Circular RNAs in Colorectal Cancer: New Entities for Therapy and Tools for Identification of Therapeutic Targets.

Patients with disseminated colorectal cancer have a dismal prognosis with a 5-year survival rate of only 13%. In order to identify new treatment modalities and new targets, we searched the literature for up-regulated circular RNAs in colorectal cancer which induce tumor growth in corresponding preclinical in vivo models. We identified nine circular RNAs that mediate resistance against chemotherapeutic agents, seven that up-regulate transmembrane receptors, five that induce secreted factors, nine that activate signaling components, five which up-regulate enzymes, six which activate actin-related proteins, six which induce transcription factors and two which up-regulate the MUSASHI family of RNA binding proteins. All of the circular RNAs discussed in this paper induce the corresponding targets by sponging microRNAs (miRs) and can be inhibited by RNAi or shRNA in vitro and in xenograft models. We have focused on circular RNAs with demonstrated activity in preclinical in vivo models because the latter is an important milestone in drug development. All circular RNAs with in vitro activity only data are not referenced in this review. The translational impact of inhibition of these circular RNAs and of the identified targets for treatment of colorectal cancer (CRC) are discussed.

Natural antisense transcripts as drug targets.

The recent discovery of vast non-coding RNA-based regulatory networks that can be easily modulated by nucleic acid-based drugs has opened numerous new therapeutic possibilities. Long non-coding RNA, and natural antisense transcripts (NATs) in particular, play a significant role in networks that involve a wide variety of disease-relevant biological mechanisms such as transcription, splicing, translation, mRNA degradation and others. Currently, significant efforts are dedicated to harnessing these newly emerging NAT-mediated biological mechanisms for therapeutic purposes. This review will highlight the recent clinical and pre-clinical developments in this field and survey the advances in nucleic acid-based drug technologies that make these developments possible.

Intranasal delivery: An attractive route for the administration of nucleic acid based therapeutics for CNS disorders.

The etiologies of several cardiovascular, inflammatory, neurological, hereditary disorders, cancer, and infectious diseases have implicated changes in the genetic set up or genetic mutations as the root cause. Nucleic acid based therapeutics (NBTs) is a new class of biologics that are known to regulate gene expression at the transcriptional and post-transcriptional level. The NBTs include oligonucleotides, nucleosides, antisense RNA, small interfering RNAs, micro RNA etc. In recent times, this new category of biologics has found enormous potential in the management of cardiovascular, inflammatory, neurological disorders, cancer, infectious diseases and organ transplantation. However, the delivery of NBTs is highly challenging in terms of target specificity (intracellular delivery), mononuclear phagocyte system uptake, stability and biodistribution. Additionally, management of the above mentioned disorders require regular and intrusive therapy making non-invasive routes preferable in comparison to invasive routes like parenteral. The nasal route is garnering focus in delivery of NBTs to the brain in the management of several CNS disorders due to the associated merits such as non-invasiveness, possibility of chronic delivery, improved patient compliance, avoidance of hepatic and gastrointestinal metabolism as well as ability to bypass the BBB. Hence in recent times, this route has been sought by the reserachers as an alternative to parenteral therapy for the delivery of several NBTs. This review shall focus on an array of NBTs delivered through nasal route, their challenges, applications and opportunities. The novel delivery systems for incorporating NBTs; their targeting strategies shall be critically reviewed. The challenges towards regulatory approvals and commercialization shall also be discussed at large. Comparison of learnings derived from the success and barriers in nasal delivery of NBTs will help in identification of futuristic opportunities for their translation from bench to bedside.

RNA Interference in Aquaculture: A Small Tool for Big Potential.

For decades, the tight regulatory functions of DNA and RNA have been the focus of extensive research with the goal of harnessing RNA molecules (e.g., microRNA and small interfering RNA) to control gene expression and to study biological functions. RNA interference (RNAi) has shown evidence of mediating gene expression, has been utilized to study functional genomics, and recently has potential in therapeutic agents. RNAi is a natural mechanism and a well-studied tool that can be used to silence specific genes. This method is also used in aquaculture as a research tool and to enhance immune responses. RNAi methods do have their limitations (e.g., immune triggering); efficient and easy-to-use RNAi methods for large-scale applications need further development. Despite these limitations, RNAi methods have been successfully used in aquaculture, in particular shrimp. This review discusses the uses of RNAi in aquaculture, such as immune- and production-related issues and the possible limitations that may hinder the application of RNAi in the aquaculture industry. Our challenge is to develop a highly potent in vivo RNAi delivery platform that could complete the desired action with minimal side effects and which can be applied on a large-scale with relatively little expense in the aquaculture industry.

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.

Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation.

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.

Therapeutic efficacy of liposomal Grb2 antisense oligodeoxynucleotide (L-Grb2) in preclinical models of ovarian and uterine cancer.

BACKGROUND: Adaptor proteins such as growth factor receptor-bound protein-2 (Grb2) play important roles in cancer cell signaling. In the present study, we examined the biological effects of liposomal antisense oligodeoxynucleotide that blocks Grb2 expression (L-Grb2) in gynecologic cancer models. MATERIALS AND METHODS: Murine orthotopic models of ovarian (OVCAR5 and SKOV3ip1) and uterine (Hec1a) cancer were used to study the biological effects of L-Grb2 on tumor growth. In vitro experiments (cell viability assay, Western blot analysis, siRNA transfection, and reverse phase protein array) were carried out to elucidate the mechanisms and potential predictors of tumor response to L-Grb2. FINDINGS: Treatment with L-Grb2 decreased tumor growth and metastasis in orthotopic models of ovarian cancer (OVCAR5, SKOV3ip1) by reducing angiogenesis and increasing apoptosis at a dose of 15 mg/kg with no effect on mouse body weight. Treatment with L-Grb2 and paclitaxel led to the greatest decrease in tumor weight (mean ± SEM, 0.17 g ± 0.10 g) compared with that in control mice (0.99 g ± 0.35 g). We also observed a reduction in tumor burden after treatment with L-Grb2 and the anti-VEGF antibody B-20 (86% decrease in tumor weight compared with that in controls). Ovarian cancer cells with ErbB2 amplification (OVCAR8 and SKOV3ip1) were the most sensitive to Grb2 downregulation. Reverse phase protein array analysis identified significant dysregulation of metabolites (LDHA, GAPDH, and TCA intermediates) in ovarian cancer cells after Grb2 downregulation. INTERPRETATION: L-Grb2 has therapeutic efficacy in preclinical models of ovarian and uterine cancer. These findings support further clinical development of L-Grb2.

Expanding the Biotherapeutics Realm via miR-34a: "Potent Clever Little" Agent in Breast Cancer Therapy.

BACKGROUND: One of the most prevalent cancers befell to women is considered to be breast cancer (BC). It is also the deadliest among the female population after lung cancer. Additionally, several studies have demonstrated that there is an association between microRNA34-a and breast cancer. METHODS: We searched PubMed, Web of Science, and Google Scholar up to December 2018. Those studies which have been studied miR-34a and its tumor-suppressing capabilities were considered as the most important topics. Moreover, we extracted articles which were solely focused on microRNA-34a in breast cancer therapy. Finally, 80 articles were included. RESULTS: In comparison with the normal tissues, down-regulation of miR-34a expression is shown considerably in tumor cells. Overexpression of miR-34a acts as a tumor suppressor by transcriptional regulating one of the signaling pathways (TP53), NOTCH, and transforming growth factor beta (TGF-ß), Bcl- 2 and SIRT1genes, HDAC1 and HDAC7, Fra-1, TPD52, TLR Via CXCL10. Moreover, drug resistance declines which lead to the apoptosis, cell cycle arrest and senescence. As a result, the proliferation, invasion and metastasis of the tumor are suppressed. The Mrx34 drug contains miR-34a mimic and a lipid vector. MiR-34a as the active ingredient portrays the role of a tumor suppressor. This drug has recently entered the clinical trials studies. CONCLUSION: These findings suggest a robust cause for developing miR-34a as a therapeutic agent to target BC. In that scenario, miR-34a is strongly useful to introduce new therapeutic goals for BC. Moreover, this review aims to confirm the signal pathways, therapeutic and diagnostic values of miR- 34a in BC and beyond.

Glutathione-triggered activation of the model of pro-oligonucleotide with benzyl protecting groups at the internucleotide linkage.

We have examined substituted benzyl protecting groups for the phosphodiester in oligodeoxyribonucleotides. Stability of the protecting groups in buffer and rates of deprotection by glutathione (GSH) were strongly influenced by benzyl ring substituents.

Bioreductive deprotection of 4-nitrobenzyl group on thymine base in oligonucleotides for the activation of duplex formation.

Oligonucleotides containing 4-O-(4-NO2-benzyl)thymine residues were synthesized to assess potential prodrug-type action against hypoxic cells. These modified oligonucleotides were incapable of stable duplex formation under non-hypoxic conditions. However, following deprotection of the thymine residues under bioreductive conditions, the deprotected oligonucleotides were able to form stable duplexes with target oligonucleotides.

Immunological and hematological toxicities challenging clinical translation of nucleic acid-based therapeutics.

INTRODUCTION: Nucleic acid-based therapeutics (NATs) are proven agents in correcting disorders caused by gene mutations, as treatments against cancer, microbes and viruses, and as vaccine adjuvants. Although many traditional small molecule NATs have been approved for clinical use, commercialization of macromolecular NATs has been considerably slower, and only a few have successfully reached the market. Preclinical and clinical evaluation of macromolecular NATs has revealed many assorted challenges in immunotoxicity, hematotoxicity, pharmacokinetics (PKs), toxicology and formulation. Extensive review has been given to the PK and toxicological concerns of NATs including approaches designed to overcome these issues. Immunological and hematological issues are a commonly reported side effect of NAT treatment; however, literature exploring the mechanistic background of these effects is sparse. AREAS COVERED: This review focuses on the immunomodulatory properties of various types of therapeutic nucleic acid concepts. The most commonly observed immunological and hematological toxicities are described for various NAT classes, with citations of how to circumvent these toxicities. EXPERT OPINION: Although some success with overcoming immunological and hematological toxicities of NATs has been achieved in recent years, immunostimulation remains the main dose-limiting factor challenging clinical translation of these promising therapies. Novel delivery vehicles should be considered to overcome this challenge.

Development of a photolabile protecting group for phosphodiesters in oligonucleotides.

A photolabile protecting group, consisting of an o-nitrobenzyl group and a 3-(2'-hydroxy-3',6'-dimethylphenyl)-2,2-dimethylpropyl moiety, was developed for phosphodiesters in oligodeoxyribonucleotides. Deprotection was triggered by photoirradiation and subsequent spontaneous cyclization to release the naked oligonucleotide.

A new nucleic acid-based agent inhibits cytotoxic T lymphocyte-mediated immune disorders.

BACKGROUND: Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and graft-versus-host disease (GVHD) are distinct immune reactions elicited by drugs or allogeneic antigens; however, they share a pathomechanism with the activation of cytotoxic T lymphocytes (CTLs). CTLs produce cytotoxic proteins, cytokines, chemokines, or immune alarmins, such as granulysin (GNLY), leading to the extensive tissue damage and systemic inflammation seen in patients with SJS/TEN or GVHD. Currently, there is no effective therapeutic agent specific for CTL-mediated immune disorders. OBJECTIVES: By targeting GNLY(+) CTLs, we aimed to develop a nucleic acid-based agent consisting of an anti-CD8 aptamer with GNLY small interfering RNA (siRNA). METHODS: We performed systematic evolution of ligands using exponential enrichment to select and identify effective anti-CD8 aptamers. We developed an aptamer-siRNA chimera using a "sticky bridge" method by conjugating the aptamer with siRNA. We analyzed the inhibitory effects of the aptamer-siRNA chimera on CTL responses in patients with SJS/TEN or GVHD. RESULTS: We identified a novel DNA aptamer (CD8AP17s) targeting CTLs. This aptamer could be specifically internalized into human CTLs. We generated the CD8AP17s aptamer-GNLY siRNA chimera, which showed a greater than 79% inhibitory effect on the production of GNLY by drug/alloantigen-activated T cells. The CD8AP17s aptamer-GNLY siRNA chimera decreased cytotoxicity in in vitro models of both SJS/TEN (elicited by drug-specific antigen) and GVHD (elicited by allogeneic antigens). CONCLUSIONS: Our results identified a new nucleic acid-based agent (CD8 aptamer-GNLY siRNA chimera) that can significantly inhibit CTL-mediated drug hypersensitivity, such as that seen in patients with SJS/TEN, as well as the alloreactivity seen in patients with GVHD. This study provides a novel therapeutic strategy for CTL-mediated immune disorders.

Prospects for nucleic acid-based therapeutics against hepatitis C virus.

In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.