RNA-Based Therapeutics: Current Progress and Future Prospects
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Mrna Vaccine Era—Mechanisms, Drug Platform and Clinical Prospection
International Journal of Molecular Sciences Review mRNA Vaccine Era—Mechanisms, Drug Platform and Clinical Prospection 1, 1, 2 1,3, Shuqin Xu y, Kunpeng Yang y, Rose Li and Lu Zhang * 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200438, China; [email protected] (S.X.); [email protected] (K.Y.) 2 M.B.B.S., School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China; [email protected] 3 Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, China * Correspondence: [email protected]; Tel.: +86-13524278762 These authors contributed equally to this work. y Received: 30 July 2020; Accepted: 30 August 2020; Published: 9 September 2020 Abstract: Messenger ribonucleic acid (mRNA)-based drugs, notably mRNA vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. The extraordinary advantages associated with mRNA vaccines, including their high efficacy, a relatively low severity of side effects, and low attainment costs, have enabled them to become prevalent in pre-clinical and clinical trials against various infectious diseases and cancers. Recent technological advancements have alleviated some issues that hinder mRNA vaccine development, such as low efficiency that exist in both gene translation and in vivo deliveries. mRNA immunogenicity can also be greatly adjusted as a result of upgraded technologies. In this review, we have summarized details regarding the optimization of mRNA vaccines, and the underlying biological mechanisms of this form of vaccines. Applications of mRNA vaccines in some infectious diseases and cancers are introduced. It also includes our prospections for mRNA vaccine applications in diseases caused by bacterial pathogens, such as tuberculosis. -
Patenting Interfering RNA
Patenting Interfering RNA J. Douglas Schultz SPE Art Unit 1635 (571) 272-0763 [email protected] Oligonucleotide Inhibitors: Mechanisms of Action RNAi - Mechanism of Action • dsRNA induces sequence-specific degradation of homologous gene transcripts • RISC metabolizes dsRNA to small 21-23- nucleotide siRNAs – RISC contains dsRNase (“Dicer”), ssRNase (Argonaut 2 or Ago2) • RISC utilizes antisense strand as “guide” to find cleavable target siRNA Mechanism of Action miRNA Mechanism of Action Interfering RNA Glossary of Terms • RNAi – RNA interference • dsRNA – double stranded RNA • siRNA – small interfering RNA, double stranded, 21-23 nucleotides • shRNA – short hairpin RNA (doubled stranded by virtue of a ssRNA folding back on itself) • miRNA – micro RNA • RISC – RNA-induced silencing complex – Dicer – RNase endonuclease siRNA miRNA • Exogenously delivered • Endogenously produced • 21-23mer dsRNA • 21-23mer dsRNA • Acts through RISC • Acts through RISC • Induces homologous target • Induces homologous target cleavage cleavage • Perfect sequence match • Imperfect sequence match – Results in target degradation – Results in translation arrest RNAi Patentability issues Sample Claims: • A siRNA that inhibits expression of a nucleic acid encoding protein X. OR • A siRNA comprising a 2’-modification, wherein said modification comprises 2’-fluoro, 2’-O-methyl, or 2’- deoxy. (Note: no target recited) OR • A method of reducing tumor cell growth comprising administering siRNA targeting protein X. RNAi Patentability Issues 35 U.S.C. 101 – Utility • Credible/Specific/Substantial/Well Established. • Used to attempt modulation of gene expression in human diseases • Routinely investigate gene function in a high throughput fashion or to (see Rana RT, Nat. Rev. Mol. Cell Biol. 2007, Vol. 8:23-36). -
(12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub
US 20040O86860A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0086860 A1 Sohail (43) Pub. Date: May 6, 2004 (54) METHODS OF PRODUCING RNAS OF Publication Classification DEFINED LENGTH AND SEQUENCE (51) Int. Cl." .............................. C12Q 1/68; C12P 19/34 (76) Inventor: Muhammad Sohail, Marston (GB) (52) U.S. Cl. ............................................... 435/6; 435/91.2 Correspondence Address: MINTZ, LEVIN, COHN, FERRIS, GLOWSKY (57) ABSTRACT AND POPEO, PC. ONE FINANCIAL CENTER Methods of making RNA duplexes and single-stranded BOSTON, MA 02111 (US) RNAS of a desired length and Sequence based on cleavage of RNA molecules at a defined position, most preferably (21) Appl. No.: 10/264,748 with the use of deoxyribozymes. Novel deoxyribozymes capable of cleaving RNAS including a leader Sequence at a (22) Filed: Oct. 4, 2002 Site 3' to the leader Sequence are also described. Patent Application Publication May 6, 2004 Sheet 1 of 2 US 2004/0086860 A1 DNA Oligonucleotides T7 Promoter -TN-- OR 2N-2-N-to y Transcription Products GGGCGAAT-N-UU GGGCGAAT-N-UU w N Deoxyribozyme Cleavage - Q GGGCGAAT -------' Racction GGGCGAAT N-- UU N-UU ssRNA products N-UU Anneal ssRNA UU S-2N- UU siRNA product FIGURE 1: Flowchart summarising the procedure for siRNA synthesis. Patent Application Publication May 6, 2004 Sheet 2 of 2 US 2004/0086860 A1 Full-length transcript 3'-digestion product 5'-digestion product (5'GGGCGAATA) A: Production of single-stranded RNA templates by in vitro transcription and digestion With a deoxyribozyme V 2- 2 V 22inv 22 * 2 &3 S/AS - 88.8x, *...* or as IGFR -- is as 4. -
Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas Reinhardtii
University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of Spring 2013 Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii Xinrong Ma University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Biology Commons, Cellular and Molecular Physiology Commons, Microbiology Commons, and the Molecular Genetics Commons Ma, Xinrong, "Small Interfering RNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii" (2013). Dissertations and Theses in Biological Sciences. 51. https://digitalcommons.unl.edu/bioscidiss/51 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII by Xinrong Ma A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences Under the Supervision of Professor Heriberto Cerutti Lincoln, Nebraska May, 2013 SMALL INTERFERING RNA-MEDIATED TRANSLATION REPRESSION ALTERS RIBOSOME SENSITIVITY TO INHIBITION BY CYCLOHEXIMIDE IN CHLAMYDOMONAS REINHARDTII Xinrong Ma, Ph.D. University of Nebraska, 2013 Advisor: Heriberto Cerutti RNA interference (RNAi) is an evolutionarily conserved gene silencing mechanism in eukaryotes, with regulatory roles in a variety of biological processes, including cell cycle, cell differentiation, physiological and metabolic pathways, and stress responses. -
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DNA and RNA Nanotechnology 2015; 2: 42–52 Mini review Open Access Martin Panigaj*, Jakob Reiser Aptamer guided delivery of nucleic acid-based nanoparticles DOI 10.1515/rnan-2015-0005 Evolution of Ligands by Exponential enrichment) [4,5]. Received July 15, 2015; accepted October 3, 2015 Nucleic acid-based aptamers are especially well suited Abstract: Targeted delivery of bioactive compounds is a for the delivery of nucleic acid-based therapeutics. Any key part of successful therapies. In this context, nucleic nucleic acid with therapeutic potential can be linked acid and protein-based aptamers have been shown to to an aptamer sequence [6], resulting in a bivalent bind therapeutically relevant targets including receptors. molecule endowed with a targeting aptamer moiety and In the last decade, nucleic acid-based therapeutics a functional RNA/DNA moiety like a small interfering coupled to aptamers have emerged as a viable strategy for RNA (siRNA), a micro RNA (miRNA), a miRNA antagonist cell specific delivery. Additionally, recent developments (antimiR), deoxyribozymes (DNAzymes), etc. In addition in nucleic acid nanotechnology offer an abundance of to the specific binding, many aptamers upon receptor possibilities to rationally design aptamer targeted RNA recognition elicit antagonistic or agonistic responses that, or DNA nanoparticles involving combinatorial use of in combination with conjugated functional nucleic acids various intrinsic functionalities. Although a host of issues have the potential of synergism. Since the first report including stability, safety and intracellular trafficking describing an aptamer-siRNA delivery approach in 2006 remain to be addressed, aptamers as simple functional many functional RNAs and DNAs conjugated to aptamer chimeras or as parts of multifunctional self-assembled sequences have been tested in vitro and in vivo [7-9]. -
Retroviral Delivery of Small Interfering RNA Into Primary Cells
Retroviral delivery of small interfering RNA into primary cells Gregory M. Barton and Ruslan Medzhitov* Section of Immunobiology and The Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520 Communicated by Peter Cresswell, Yale University School of Medicine, New Haven, CT, October 2, 2002 (received for review August 9, 2002) RNA interference is an evolutionarily conserved process in which gene was replaced with the human CD4 gene, which was cloned recognition of double-stranded RNA ultimately leads to posttran- from splenic cDNA. To eliminate any potential signaling, a scriptional suppression of gene expression. This suppression is premature stop codon was introduced after amino acid 425, just mediated by short (21- to 22-nt) small interfering RNAs (siRNAs), after the transmembrane domain. The human H1 promoter was which induce degradation of mRNA based on complementary base cloned from genomic DNA and inserted either upstream of the pairing. The silencing of gene expression by siRNAs is emerging cytomegalovirus (CMV) promoter (RVH1) by using previously rapidly as a powerful method for genetic analysis. Recently, several introduced XhoI and EcoRI sites or within the 3Ј LTR by groups have reported systems designed to express siRNAs in using SalI. The oligonucleotides encoding the human p53 mammalian cells through transfection of either oligonucleotides or siRNA, described by Brummelkamp et al. (6), were 5Ј-GATC- plasmids encoding siRNAs. Because these systems rely on trans- CCCGACTCCAGTGGTAATCTACTTCAAGAGAGTA- fection for delivery, the cell types available for study are restricted GATTACCACTGGAGTCTTTTTGGAAC-3Ј and 5Ј-TCGA- generally to transformed cell lines. Here, we describe a retroviral GTTCCAAAAAGACTCCAGTGGTAATCTACTCTCTTG- system for delivery of siRNA into cells. -
Viral Vectors Applied for Rnai-Based Antiviral Therapy
viruses Review Viral Vectors Applied for RNAi-Based Antiviral Therapy Kenneth Lundstrom PanTherapeutics, CH1095 Lutry, Switzerland; [email protected] Received: 30 July 2020; Accepted: 21 August 2020; Published: 23 August 2020 Abstract: RNA interference (RNAi) provides the means for alternative antiviral therapy. Delivery of RNAi in the form of short interfering RNA (siRNA), short hairpin RNA (shRNA) and micro-RNA (miRNA) have demonstrated efficacy in gene silencing for therapeutic applications against viral diseases. Bioinformatics has played an important role in the design of efficient RNAi sequences targeting various pathogenic viruses. However, stability and delivery of RNAi molecules have presented serious obstacles for reaching therapeutic efficacy. For this reason, RNA modifications and formulation of nanoparticles have proven useful for non-viral delivery of RNAi molecules. On the other hand, utilization of viral vectors and particularly self-replicating RNA virus vectors can be considered as an attractive alternative. In this review, examples of antiviral therapy applying RNAi-based approaches in various animal models will be described. Due to the current coronavirus pandemic, a special emphasis will be dedicated to targeting Coronavirus Disease-19 (COVID-19). Keywords: RNA interference; shRNA; siRNA; miRNA; gene silencing; viral vectors; RNA replicons; COVID-19 1. Introduction Since idoxuridine, the first anti-herpesvirus antiviral drug, reached the market in 1963 more than one hundred antiviral drugs have been formally approved [1]. Despite that, there is a serious need for development of novel, more efficient antiviral therapies, including drugs and vaccines, which has become even more evident all around the world today due to the recent coronavirus pandemic [2]. -
Advances in Oligonucleotide Drug Delivery
REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured -
Ribozyme-Enhanced Single-Stranded Ago2-Processed Interfering RNA Triggers Efficient Gene Silencing with Fewer Off-Target Effects
ARTICLE Received 9 Dec 2014 | Accepted 21 Aug 2015 | Published 12 Oct 2015 DOI: 10.1038/ncomms9430 OPEN Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects Renfu Shang1,2,3, Fengjuan Zhang1,2,3, Beiying Xu1,2,3, Hairui Xi4, Xue Zhang1,2,3, Weihua Wang1,2,3 & Ligang Wu1,2,3 Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 30 end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 30 overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. 1 National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 2 Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 201204, China. -
Ribonucleic Acid (RNA)
AccessScience from McGraw-Hill Education Page 1 of 11 www.accessscience.com Ribonucleic acid (RNA) Contributed by: Michael W. Gray, Ann L. Beyer Publication year: 2014 One of the two major classes of nucleic acid, mainly involved in translating the genetic information carried in deoxyribonucleic acid (DNA) into proteins. Various types of ribonucleic acids (RNAs) [see table] function in protein synthesis: transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) function in the synthesis of all proteins, whereas messenger RNAs (mRNAs) are a diverse set, with each member specifically directing the synthesis of one protein. Messenger RNA is the intermediate in the usual biological pathway of DNA → RNA → protein. However, RNA is a very versatile molecule. Other types of RNA serve other important functions for cells and viruses, such as the involvement of small nuclear RNAs (snRNAs) in mRNA splicing. In some cases, RNA performs functions typically considered DNA-like, such as serving as the genetic material for certain viruses, or roles typically carried out by proteins, such as RNA enzymes or ribozymes. See also: DEOXYRIBONUCLEIC ACID (DNA); NUCLEIC ACID. Structure and synthesis RNA is a linear polymer of four different nucleotides (Fig. 1). Each nucleotide is composed of three parts: a five-carbon sugar known as ribose; a phosphate group; and one of four bases attached to each ribose, that is, adenine (A), cytosine (C), guanine (G), or uracil (U). The combination of base and sugar constitutes a nucleoside. The structure of RNA is basically a repeating chain of ribose and phosphate moieties, with one of the four bases attached to each ribose. -
Recent Advances in Mrna Vaccine Delivery
Nano Research 2018, 11(10): 5338–5354 https://doi.org/10.1007/s12274-018-2091-z Recent advances in mRNA vaccine delivery Lu Tan and Xun Sun () Key Laboratory of Drug Targeting and Novel Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China Received: 14 March 2018 ABSTRACT Revised: 30 April 2018 In recent years, messenger RNA (mRNA) vaccines have been intensively studied Accepted: 4 May 2018 in the fields of cancer immunotherapy and infectious diseases because of their excellent efficacy and safety profile. Despite significant progress in the rational © Tsinghua University Press design of mRNA vaccines and elucidation of their mechanism of action, their and Springer-Verlag GmbH widespread application is limited by the development of safe and effective Germany, part of Springer delivery systems that protect them from ubiquitous ribonucleases (RNases), Nature 2018 facilitate their entry into cells and subsequent escape from endosomes, and target them to lymphoid organs or particular cells. Some mRNA vaccines based KEYWORDS on lipid carriers have entered clinical trials. Vaccines based on polymers, while messenger RNA (mRNA) not as clinically advanced as lipid vectors, show considerable potentials. In this vaccines, review, we discuss the necessity of formulating mRNA vaccines with delivery delivery systems, systems, and we provide an overview of reported delivery systems. polymer, lipid 1 Introduction and their complex composition can trigger adverse effects [3]. In contrast, mRNA vaccines express well- Messenger RNA (mRNA) vaccines carry transcripts defined antigens that induce focused immune responses encoding antigens, and use the host cell translational specifically against the encoded antigens [4]. -
Plant Non-Coding Rnas: Origin, Biogenesis, Mode of Action and Their Roles in Abiotic Stress
International Journal of Molecular Sciences Review Plant Non-Coding RNAs: Origin, Biogenesis, Mode of Action and Their Roles in Abiotic Stress Joram Kiriga Waititu 1, Chunyi Zhang 1, Jun Liu 2 and Huan Wang 1,* 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] (J.K.W.); [email protected] (C.Z.) 2 National Key Facility for Crop Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China; [email protected] * Correspondence: [email protected] Received: 13 October 2020; Accepted: 4 November 2020; Published: 9 November 2020 Abstract: As sessile species, plants have to deal with the rapidly changing environment. In response to these environmental conditions, plants employ a plethora of response mechanisms that provide broad phenotypic plasticity to allow the fine-tuning of the external cues related reactions. Molecular biology has been transformed by the major breakthroughs in high-throughput transcriptome sequencing and expression analysis using next-generation sequencing (NGS) technologies. These innovations have provided substantial progress in the identification of genomic regions as well as underlying basis influencing transcriptional and post-transcriptional regulation of abiotic stress response. Non-coding RNAs (ncRNAs), particularly microRNAs (miRNAs), short interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs), have emerged as essential regulators of plants abiotic stress response. However, shared traits in the biogenesis of ncRNAs and the coordinated cross-talk among ncRNAs mechanisms contribute to the complexity of these molecules and might play an essential part in regulating stress responses. Herein, we highlight the current knowledge of plant microRNAs, siRNAs, and lncRNAs, focusing on their origin, biogenesis, modes of action, and fundamental roles in plant response to abiotic stresses.