Nucleic Acid Therapeutics Research Articles

Implications of glioblastoma-derived exosomes in modifying the immune system: state-of-the-art and challenges.

Glioblastoma is a brain cancer with a poor prognosis. Failure of classical chemotherapy and surgical treatments indicates that new therapeutic approaches are needed. Among cell-free options, exosomes are versatile extracellular vesicles (EVs) that carry important cargo across barriers suchas the blood-brain barrier (BBB) to their target cells. Thismakes exosomes an interesting option for the treatment ofglioblastoma. Moreover, exosomes can comprise many therapeutic cargos, including lipids, proteins, and nucleic acids,sampled from special intercellular compartments of their origin cell. Cells exposed to various immunomodulatory stimulican generate exosomes enriched in specific therapeutic molecules. Notably, the secretion of exosomes could modify theimmune response in innate and adaptive immune systems. For instance, glioblastoma-associated exosomes (GBex) uptake bymacrophages could influence macrophage dynamics (e.g., shifting CD markers expression). Expression of critical immunoregulatory proteins such as cytotoxic T-lymphocyte antigen-1 (CTLA1) and programmed death-1 (PD-1) on GBex indicates the direct crosstalk of these nano-size vesicles with the immune system. The present study reviews the role of exosomes in immune system cells, including Bcells, Tcells, natural killer (NK) cells, and dendritic cells (DCs), as well as novel technologies in the field.

Alkylated RALA-Derived Peptides for Efficient Gene Delivery.

RALA is an amphipathic cationic peptide demonstrated to be a low-toxicity and high-efficiency delivery platform for the systemic delivery of nucleic acid therapeutics. This work reports three RALA-derived peptides modified with N-terminal palmitic acid, engineered through amino acid substitutions and truncated sequences. All three peptides have good nucleic acid encapsulation, release and uptake, biocompatibility, and endolysosome escape. The siRNA transfection efficiency is about 90%, and the silencing rate of GA (C16-GLFWHHHARLARALARHLARALRA) exceeds that of lipofectamine 2000 (siRNA concentration = 50 nM). Truncating the peptide chain while retaining a certain amount of arginine ensures an effective particle size. Replacing glutamic acid with three histidines ensures an effective zeta potential and accelerates the endosome escape process through the proton sponge phenomenon. Introducing phenylalanine enhances the carrier-cell interaction. We believe that they are powerful carriers of siRNA therapy and may have good application prospects in treating various diseases.

Amphiphilic Oligonucleotide Derivatives—Promising Tools for Therapeutics

Recent advances in genetics and nucleic acid chemistry have created fundamentally new tools, both for practical applications in therapy and diagnostics and for fundamental genome editing tasks. Nucleic acid-based therapeutic agents offer a distinct advantage of selectively targeting the underlying cause of the disease. Nevertheless, despite the success achieved thus far, there remain unresolved issues regarding the improvement of the pharmacokinetic properties of therapeutic nucleic acids while preserving their biological activity. In order to address these challenges, there is a growing focus on the study of safe and effective delivery methods utilising modified nucleic acid analogues and their lipid bioconjugates. The present review article provides an overview of the current state of the art in the use of chemically modified nucleic acid derivatives for therapeutic applications, with a particular focus on oligonucleotides conjugated to lipid moieties. A systematic analysis has been conducted to investigate the ability of amphiphilic oligonucleotides to self-assemble into micelle-like structures, as well as the influence of non-covalent interactions of such derivatives with serum albumin on their biodistribution and therapeutic effects.

PAMAM-Calix-Dendrimers: Third Generation Synthesis and Impact of Generation and Macrocyclic Core Conformation on Hemotoxicity and Calf Thymus DNA Binding

Background/Objectives: Current promising treatments for many diseases are based on the use of therapeutic nucleic acids, including DNA. However, the list of nanocarriers is limited due to their low biocompatibility, high cost, and toxicity. The design of synthetic building blocks for creating universal delivery systems for genetic material is an unsolved problem. In this work, we propose PAMAM dendrimers with rigid thiacalixarene core in various conformations, i.e., PAMAM-calix-dendrimers, as a platform for a supramolecular universal constructor for nanomedicine. Results: Third generation PAMAM dendrimers with a macrocyclic core in three conformations (cone, partial cone, and 1,3-alternate) were synthesized for the first time. The obtained dendrimers were capable of binding and compacting calf thymus DNA, whereby the binding efficiency improved with increasing generation, while the influence of the macrocyclic core was reduced. A dramatic effect of the macrocyclic core conformation on the hemolytic activity of PAMAM-calix-dendrimers was observed. Specifically, a notable reduction in hemotoxicity was associated with a decrease in compound amphiphilicity. Conclusions: We hope the results will help reduce financial and labor costs in developing new drug delivery systems based on dendrimers.

Is it Time for Multi-Drug Therapy with Combination of Therapeutic Nucleic Acids?

Therapeutic nucleic acids (TNAs) are a new class of drugs that exhibit different properties and mechanisms of action from those of small molecules or biological drugs. Over twenty oligonucleotide drugs and several COVID-19 vaccines have received regulatory approval for clinical use. A characteristic feature of these TNAs is that they are directed against one specific biological target and one specific RNA or DNA sequence. Consequently, TNAs currently used are administered as monotherapy. Due to the known advantages of multidrug therapy with low molecular weight drugs, it may be time to intensify work on such a treatment protocol, also in the case of TNAs.

Unlocking the Potential of Chemically Modified Nucleic Acid Therapeutics

AbstractNucleic acid therapeutics have demonstrated tremendous potential for treating diseases by targeting the genetic underpinnings at the transcriptomic level. However, their efficacy hinges on robust strategies to protect nucleic acids from degradation during circulation and to facilitate precise delivery to diseased tissues and cells. Here the critical roles of chemical modification and bioconjugation in advancing nucleic acid therapeutics for improved binding affinity, enhanced stability, and targeted delivery are reviewed. Commencing diverse applications, the significance of different chemical modifications is discussed based on recent literature and clinical products, on oligonucleotides. These modifications encompass backbone, ribose, base alterations and bioconjugation techniques such as N‐acetylgalactosamine (GAlNac), aptamers, antibodies, and cell‐penetrating peptides (CPPs). Supported by a clinical perspective, diverse applications and ongoing developments are highlighted. Furthermore, the current landscape of nucleic acid therapeutics and their potential in addressing genetic disorders with multiple cellular/organelle targeting is discussed. Here the promising prospect of combining chemical innovation and bioconjugation strategies is underscored to propel the development of more effective nucleic acid therapeutics.

Recent Advances and Prospects of Nucleic Acid Therapeutics for Anti-Cancer Therapy

Nucleic acid therapeutics are promising alternatives to conventional anti-cancer therapy, such as chemotherapy and radiation therapy. While conventional therapies have limitations, such as high side effects, low specificity, and drug resistance, nucleic acid therapeutics work at the gene level to eliminate the cause of the disease. Nucleic acid therapeutics treat diseases in various forms and using different mechanisms, including plasmid DNA (pDNA), small interfering RNA (siRNA), anti-microRNA (anti-miR), microRNA mimics (miRNA mimic), messenger RNA (mRNA), aptamer, catalytic nucleic acid (CNA), and CRISPR cas9 guide RNA (gRNA). In addition, nucleic acids have many advantages as nanomaterials, such as high biocompatibility, design flexibility, low immunogenicity, small size, relatively low price, and easy functionalization. Nucleic acid therapeutics can have a high therapeutic effect by being used in combination with various nucleic acid nanostructures, inorganic nanoparticles, lipid nanoparticles (LNPs), etc. to overcome low physiological stability and cell internalization efficiency. The field of nucleic acid therapeutics has advanced remarkably in recent decades, and as more and more nucleic acid therapeutics have been approved, they have already demonstrated their potential to treat diseases, including cancer. This review paper introduces the current status and recent advances in nucleic acid therapy for anti-cancer treatment and discusses the tasks and prospects ahead.

Supplementation with ions enhances the efficiency of nucleic acid delivery with cell-penetrating peptides

The successful delivery of therapeutic nucleic acids (NAs) into eukaryotic cells is essential for numerous biomedical applications, including gene therapy, gene silencing, and genome editing. Cell-penetrating peptides (CPPs) have claimed significant attention as delivery vehicles due to their inherent ability to penetrate cellular membranes and efficiently transport cargo, including NAs, into the cells. However, further optimization and a deeper understanding of underlying mechanisms are necessary for such transfection methods. Previous studies have demonstrated that Ca2+ ions can significantly enhance NA delivery efficiency when included in transfection media or CPP/NA nanoparticles during preparation. Similar effects have been observed for Mg2+, but the impact of other ions in this context has not been thoroughly investigated. In this study, we supplemented the CPP/NA formulations with various inorganic biocompatible ions by introducing solutions of the respective salts to colloidal nanoparticles at the preparation stage. Our results indicated that supplementing the CPP/NA formulations with certain salt solutions enhanced the biological effect achieved with NAs while also influencing nanoparticle size, surface charge, complexation stability, and, to some extent, the internalization route. Our findings offer valuable insights for optimizing the formation of CPP nanoparticles to improve NA delivery efficiency.

Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.

Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NAther). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NAhc) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NAhc need to be removed effectively before loading the VLP with NAther. Various techniques reported in literature for this NAhc removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NAhc removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NAhc removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NAhc loadings were subjected to evaluation. Process performance was thoroughly examined through NAhc removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NAhc removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NAhc removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NAhc removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NAhc removal and VLP protein recovery.

Rosalind Franklin Society Proudly Announces the 2023 Award Recipient for Nucleic Acid Therapeutics.

Rosalind Franklin Society Proudly Announces the 2023 Award Recipient for Nucleic Acid Therapeutics.

Self-assembled nanoparticles of PEG and poly(2-oxazoline) based lactide block copolymers

Access to self-assembled nanoparticles has become increasingly vital for the development of next generation adjuvants for the delivery of nucleic acid therapeutics. However, the block ratio of amphiphilic block copolymers plays a significant role in achieving the desired architectures and in some cases coformulation of two different block copolymers is needed. Herein, we introduce an elegant approach to self-assembled stomatocytes and cubosomes through coformulation of PEG and poly(2-oxazoline) (POx) based lactide diblock copolymers. A series of well-defined POx macroinitiators and their block copolymers with D,L-lactide (PDLLA) has been synthesized and achieved narrow polydispersity indices at high monomer conversions. Thermal analysis of block copolymers indicated tunable glass transition temperatures (Tg) ranging from 33 °C to 56 °C. Other critical factors influencing the structure of the nanoparticle included the ratio of POx-PDLLA and PEG-PDLLA blocks as well as the hydrophobicity of the POx block. Moreover, DLS and cryo-TEM analysis revealed the formation of diverse nanostructures, namely stomatocytes, pseudo-vesicles, and possibly cubosomes. This versatile platform allows for precise control over nanoparticle shapes by adjusting block lengths and coformulation ratios. This highlights the potential of using coformulations in biomedical applications, enabling the rational design of advanced nanomaterials with tailored functionalities for specific targets.

Quinine-Based Polymers Are Versatile and Effective Vehicles for Intracellular pDNA, mRNA, and Cas9 Protein Delivery.

Quinine-based polymers have previously demonstrated promising performance in delivering pDNA in cells owing to their electrostatic as well as the nonelectrostatic interactions with pDNA. Herein, we evaluate whether quinine-based polymers are versatile for delivery of mRNA and Cas9-sgRNA complexes, especially in a serum-rich environment. Both mRNA and the Cas9-sgRNA complex are potent therapeutics that are structurally, chemically, and functionally very different from pDNA. By exploring a family of 7 quinine-based polymers that vary in monomer structure and polymer composition, we tested numerous formulations (42 with pDNA, 96 with mRNA, and 48 with Cas9-sgRNA) for payload-polymer complexation and delivery to compare payload-dependent structure-activity relationships. Several formulations demonstrated performance comparable to or better than the commercially available transfection agent jetPEI. The results of this study demonstrate the potential of quinine-based as a versatile carrier platform for delivering a wide range of nucleic acid therapeutics and serving the drug delivery needs in the field genetic medicine.

Development of Organ Targeting Lipid Nanoparticles with Low Immunogenicity and Their Application in the Treatment of Pulmonary Fibrosis

AbstractAs the most advanced non‐viral delivery system, lipid nanoparticles (LNPs) were approved by the FDA, propelling the advancements of gene therapy. However, their clinical applications are hampered by the potential immunogenicity of the lipid components that trigger immune‐related adverse events, like inflammation and allergy. Herein, we formulate various dLNPs with diminished immunogenicity by incorporating dexamethasone (Dex) into liver‐, spleen‐, and lung‐targeting LNPs formulations that exhibit excellent abilities to target specific organs and deliver various types of RNA, such as mRNA and siRNA. In vivo investigations demonstrate unparalleled advantages in safety as compared to conventional LNPs, showing promising potential in the development of RNA therapeutics. Intriguingly, the encapsulation of runt‐related transcription factor‐1 siRNA (siRUNX1) into lung‐targeting dLNPs (dLNPs@siRUNX1) demonstrates remarkable advantages in the treatment of pulmonary fibrosis through the synergy of gene therapy and drug therapy. This research establishes secure and universal platforms for the precise delivery of nucleic acid therapeutics, showcasing promising clinical applications in gene therapy.

Development of Organ Targeting Lipid Nanoparticles with Low Immunogenicity and Their Application in the Treatment of Pulmonary Fibrosis.

As the most advanced non-viral delivery system, lipid nanoparticles (LNPs) were approved by the FDA, propelling the advancements of gene therapy. However, their clinical applications are hampered by the potential immunogenicity of the lipid components that trigger immune-related adverse events, like inflammation and allergy. Herein, we formulate various dLNPs with diminished immunogenicity by incorporating dexamethasone (Dex) into liver-, spleen-, and lung-targeting LNPs formulations that exhibit excellent abilities to target specific organs and deliver various types of RNA, such as mRNA and siRNA. In vivo investigations demonstrate unparalleled advantages in safety as compared to conventional LNPs, showing promising potential in the development of RNA therapeutics. Intriguingly, the encapsulation of runt-related transcription factor-1 siRNA (siRUNX1) into lung-targeting dLNPs (dLNPs@siRUNX1) demonstrates remarkable advantages in the treatment of pulmonary fibrosis through the synergy of gene therapy and drug therapy. This research establishes secure and universal platforms for the precise delivery of nucleic acid therapeutics, showcasing promising clinical applications in gene therapy.

Oral delivery of stabilized lipid nanoparticles for nucleic acid therapeutics.

Gastrointestinal disorders originate in the gastrointestinal tract (GIT), and the therapies can benefit from direct access to the GIT achievable through the oral route. RNA molecules show great promise therapeutically but are highly susceptible to degradation and often require a carrier for cytoplasmic access. Lipid nanoparticles (LNPs) are clinically proven drug-delivery agents, primarily administered parenterally. An ideal Orally Delivered (OrD) LNP formulation should overcome the diverse GI environment, successfully delivering the drug to the site of action. A versatile OrD LNP formulation has been developed to encapsulate and deliver siRNA and mRNA in this paper. The formulations were prepared by the systematic addition of cationic lipid to the base LNP formulation, keeping the total of cationic lipid and ionizable lipid to 50mol%. Biorelevant media stability depicted increased resistance to bile salt mediated destabilization upon the addition of the cationic lipid, however the in vitro efficacy data underscored the importance of the ionizable lipid. Based on this, OrD LNP was selected comprising of 20% cationic lipid and 30% ionizable lipid. Further investigation revealed the enhanced efficacy of OrD LNP in vitro after incubation in different dilutions of fasted gastric, fasted intestinal media, and mucin. Confocal imaging and flow cytometry confirmed uptake while in vivo studies demonstrated efficacy with siRNA and mRNA as payloads. Taken together, this research introduces OrD LNP to deliver nucleic acid locally to the GIT.

Lipid nanoparticle-mediated RNA delivery for immune cell modulation.

Lipid nanoparticles (LNPs) have emerged as the preeminent nonviral drug delivery vehicles for nucleic acid therapeutics, as exemplified by their usage in the mRNA COVID-19 vaccines. As a safe and highly modular delivery platform, LNPs are attractive for a wide range of applications. In addition to vaccines, LNPs are being utilized as platforms for other immunoengineering efforts, especially as cancer immunotherapies by modulating immune cells and their functionality via nucleic acid delivery. In this review, we focus on the methods and applications of LNP-based immunotherapy in five cell types: T cells, NK cells, macrophages, stem cells, and dendritic cells. Each of these cell types has wide-reaching applications in immunotherapy but comes with unique challenges and delivery barriers. By combining knowledge of immunology and nanotechnology, LNPs can be developed for improved immune cell targeting and transfection, ultimately working toward novel clinical therapeutics.

Claisen Self-Condensation of Lactones in the Synthesis of Ionizable Lipids.

The Claisen self-condensation of lactones can be carried out safely and efficiently under Mukaiyama conditions, in the presence of TiCl4 and triethylamine. The primary Claisen products can be elaborated to various derivatives or converted directly into dihydroxyketones. Such compounds are valuable educts for the synthesis of ionizable lipids for the delivery of nucleic acid therapeutics and can now be accessed through a concise, economical, scalable route that avoids more technically challenging reaction sequences.

Lipid Nanoparticles for Nucleic Acid Delivery Beyond the Liver.

Lipid nanoparticles (LNPs) are the most clinically advanced drug delivery system for nucleic acid therapeutics, exemplified by the success of the COVID-19 mRNA vaccines. However, their clinical use is currently limited to hepatic diseases and vaccines due to their tendency to accumulate in the liver upon intravenous administration. To fully leverage their potential, it is essential to understand and address their liver tropism, while also developing strategies to enhance delivery to tissues beyond the liver. Ensuring that these therapeutics reach their target cells while avoiding off-target cells is essential for both their efficacy and safety. There are three potential targeting strategies-passive, active, and endogenous-which can be used individually or in combination to target nonhepatic tissues. In this review, we delve into the recent advancements in LNP engineering for delivering nucleic acid beyond the liver.

Probing the higher order structure of oligonucleotides through anion exchange chromatography

Large synthetic oligonucleotides such as guide ribonucleic acid (gRNA), a critical reagent in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, have complex higher order structures (HOS) inherent in their design. In this study, we first developed a generic anion exchange chromatography (AEX) method for the comprehensive analysis of a 100mer single guide ribonucleic acid (sgRNA) impurity profiling. AEX demonstrated superior resolution compared to other common chromatographic methods employed for sgRNA analysis, such as Ion-Pairing Reversed Phase Liquid Chromatography (IP-RPLC) and Hydrophilic Interaction Chromatography (HILIC). Moreover, we discovered AEX's potential in probing the HOS of RNAs by adjusting the temperature and using organic additives. Our study also highlighted that sgRNA possesses a unique HOS distinctly different from other therapeutic nucleic acids, such as antisense oligonucleotides and messenger RNAs.

Brain Nucleic Acid Delivery and Genome Editing via Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Long-Circulating Nanoparticles.

We introduce a two-pronged strategy comprising focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening and long-circulating biodegradable nanoparticles (NPs) for systemic delivery of nucleic acids to the brain. Biodegradable poly(β-amino ester) polymer-based NPs were engineered to stably package various types of nucleic acid payloads and enable prolonged systemic circulation while retaining excellent serum stability. FUS was applied to a predetermined coordinate within the brain to transiently open the BBB, thereby allowing the systemically administered long-circulating NPs to traverse the BBB and accumulate in the FUS-treated brain region, where plasmid DNA or mRNA payloads produced reporter proteins in astrocytes and neurons. In contrast, poorly circulating and/or serum-unstable NPs, including the lipid NP analogous to a platform used in clinic, were unable to provide efficient nucleic acid delivery to the brain regardless of the BBB-opening FUS. The marriage of FUS-mediated BBB opening and the long-circulating NPs engineered to copackage mRNA encoding CRISPR-associated protein 9 and single-guide RNA resulted in genome editing in astrocytes and neurons precisely in the FUS-treated brain region. The combined delivery strategy provides a versatile means to achieve efficient and site-specific therapeutic nucleic acid delivery to and genome editing in the brain via a systemic route.