|
1
|
Tiwade PB, Fung V, VanKeulen-Miller R, Narasipura EA, Ma Y, Fenton OS. Non-Viral RNA Therapies for Non-Small Cell Lung Cancer and Their Corresponding Clinical Trials. Mol Pharm 2025; 22:1752-1774. [PMID: 40131145 DOI: 10.1021/acs.molpharmaceut.4c00871] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/26/2025]
Abstract
Ribonucleic acid (RNA)-based therapies represent a promising class of drugs for the treatment of non-small cell lung cancer (NSCLC) due to their ability to modulate gene expression. Therapies leveraging small interfering RNA (siRNA), messenger RNA (mRNA), microRNA (miRNA), and antisense oligonucleotides (ASOs) offer various advantages over conventional treatments, including the ability to target specific genetic mutations and the potential for personalized medicine approaches. However, the clinical translation of these therapeutics for the treatment of NSCLC faces challenges in delivery due to their immunogenicity, negative charge, and large size, which can be mitigated with delivery platforms. In this review, we provide a description of the pathophysiology of NSCLC and an overview of RNA-based therapeutics, specifically highlighting their potential application in the treatment of NSCLC. We discuss relevant classes of RNA and their therapeutic potential for NSCLC. We then discuss challenges in delivery and non-viral delivery strategies such as lipid- and polymer-based nanoparticles that have been developed to address these issues in preclinical models. Furthermore, we provide a summary table of clinical trials that leverage RNA therapies for NSCLC [which includes their National Clinical Trial (NCT) numbers] to highlight the current progress in NSCLC. We also discuss how these NSCLC therapies can be integrated with existing treatment modalities to enhance their efficacy and improve patient outcomes. Overall, we aim to highlight non-viral strategies that tackle RNA delivery challenges while showcasing RNA's potential as a next-generation therapy for NSCLC treatment.
Collapse
MESH Headings
- Humans
- Carcinoma, Non-Small-Cell Lung/therapy
- Carcinoma, Non-Small-Cell Lung/genetics
- Carcinoma, Non-Small-Cell Lung/drug therapy
- Lung Neoplasms/genetics
- Lung Neoplasms/therapy
- Lung Neoplasms/drug therapy
- RNA, Small Interfering/genetics
- RNA, Small Interfering/therapeutic use
- RNA, Small Interfering/administration & dosage
- Oligonucleotides, Antisense/therapeutic use
- Oligonucleotides, Antisense/genetics
- Oligonucleotides, Antisense/administration & dosage
- Clinical Trials as Topic
- Animals
- Nanoparticles/chemistry
- MicroRNAs/genetics
- MicroRNAs/therapeutic use
- RNA, Messenger/genetics
- Genetic Therapy/methods
- Drug Delivery Systems/methods
Collapse
Affiliation(s)
- Palas Balakdas Tiwade
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - Vincent Fung
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - Rachel VanKeulen-Miller
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - Eshan Amruth Narasipura
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - Yutian Ma
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| | - Owen S Fenton
- Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
| |
Collapse
|
|
2
|
Qin ZX, Zuo L, Zeng Z, Ma R, Xie W, Zhu X, Zhou X. GalNac-siRNA conjugate delivery technology promotes the treatment of typical chronic liver diseases. Expert Opin Drug Deliv 2025; 22:455-469. [PMID: 39939158 DOI: 10.1080/17425247.2025.2466767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 01/26/2025] [Accepted: 02/10/2025] [Indexed: 02/14/2025]
Abstract
INTRODUCTION Nucleic acid-based therapeutics have become a key pillar of the 'third wave' of modern medicine, following the eras of small molecule inhibitors and antibody drugs. Their rapid progress is heavily dependent on delivery technologies, with the development of N-acetylgalactosamine (GalNAc) conjugates marking a breakthrough in targeting liver diseases. This technology has gained significant attention for its role in addressing chronic conditions like chronic hepatitis B (CHB) and nonalcoholic steatohepatitis (NASH), which are challenging to treat with conventional methods. AREAS COVERED This review explores the origins, mechanisms, and advantages of GalNAc-siRNA delivery systems, highlighting their ability to target hepatocytes via the asialoglycoprotein receptor (ASGPR). The literature reviewed covers preclinical and clinical advancements, particularly in CHB and NASH. Key developments in stabilization chemistry and conjugation technologies are examined, emphasizing their impact on enhancing therapeutic efficacy and patient compliance. EXPERT OPINION GalNAc-siRNA technology represents a transformative advancement in RNA interference (RNAi) therapies, addressing unmet needs in liver-targeted diseases. While significant progress has been made, challenges remain, including restricted targeting scope and scalability concerns. Continued innovation is expected to expand applications, improve delivery efficiency, and overcome limitations, establishing GalNAc-siRNA as a cornerstone for future nucleic acid-based treatments.
Collapse
Affiliation(s)
- Zhen-Xin Qin
- Department of Immunology, School of Medicine, Nantong University, Nantong, China
- The Second Affiliated Hospital, Guangdong Medical University, Zhanjiang, China
| | - Ling Zuo
- Department of Immunology, School of Medicine, Nantong University, Nantong, China
| | - Ziran Zeng
- The Second Affiliated Hospital, Guangdong Medical University, Zhanjiang, China
| | - Rongguan Ma
- The Second Affiliated Hospital, Guangdong Medical University, Zhanjiang, China
| | - Wenyan Xie
- The Second Affiliated Hospital, Guangdong Medical University, Zhanjiang, China
| | - Xiao Zhu
- The Second Affiliated Hospital, Guangdong Medical University, Zhanjiang, China
- The Marine Biomedical Research Institute of Guangdong Zhanjiang, School of Ocean and Tropical Medicine, Guangdong Medical University, Zhanjiang, China
| | - Xiaorong Zhou
- Department of Immunology, School of Medicine, Nantong University, Nantong, China
| |
Collapse
|
|
3
|
Umezu T, Takanashi M, Fujita K, Ishikawa A, Harada Y, Matsumoto Y, Kuroda M, Murakami Y. Development of novel nucleic acid therapy aimed at directly controlling liver fibrosis. MOLECULAR THERAPY. NUCLEIC ACIDS 2025; 36:102438. [PMID: 39877003 PMCID: PMC11773475 DOI: 10.1016/j.omtn.2024.102438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/29/2024] [Accepted: 12/18/2024] [Indexed: 01/31/2025]
Abstract
Currently, no drugs directly treat liver fibrosis. Previously, we have shown that treatment with miR-29a-3p improved liver fibrosis in a mouse model. To investigate the effectiveness of nucleic acid therapy at a lower dose, a modified nucleic acid was prepared based on miR-29a-3p. The original microRNA was changed to an RNA-DNA hybrid structure: the 2' position of the RNA was modified with a fluorine base, and locked nucleic acid and phosphorothioate were crosslinked (hereafter called modified nucleic acid). In a mouse model of chronic liver disease treated with carbon tetrachloride (CCl4), the inhibitory effect on liver fibrosis was evaluated with oral administration of the modified nucleic acid. The modified nucleic acid was detected in the liver and gastrointestinal tract within 15 min of oral administration. After 5 weeks of stimulation with CCl4, oral administration of the modified nucleic acid for 2 weeks improved liver fibrosis; CCl4 stimulation was continued during this period as well. This treatment also suppressed the worsening of liver fibrosis. We developed a method to improve liver fibrosis orally using nuclease-resistant nucleic acids without using a drug delivery system. This method may be used as a new treatment for inhibiting the progression of liver fibrosis.
Collapse
Affiliation(s)
- Tomohiro Umezu
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Masakatsu Takanashi
- Department of Medical Technology, School of Life and Environmental Science, Azabu University, 1-17-71, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan
| | - Koji Fujita
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Akio Ishikawa
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yuichirou Harada
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yoshinari Matsumoto
- Department of Nutrition, Graduate School of Human Life and Ecology, Osaka Metropolitan University, 3-7-30 Habikino, Habikino-shi, Osaka 583-8555, Japan
| | - Masahiko Kuroda
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
| | - Yoshiki Murakami
- Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
- Faculty of Dentistry, Asahi University, 1851 Hozumi, Muzuho, Gifu 501-0296, Japan
| |
Collapse
|
|
4
|
Li X, Hu H, Wang H, Liu J, Jiang W, Zhou F, Zhang J. DNA nanotechnology-based strategies for minimising hybridisation-dependent off-target effects in oligonucleotide therapies. MATERIALS HORIZONS 2025; 12:1388-1412. [PMID: 39692461 DOI: 10.1039/d4mh01158a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2024]
Abstract
Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.
Collapse
Affiliation(s)
- Xiaoyu Li
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
| | - Huanhuan Hu
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
| | - Hailong Wang
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
- School of Materials Science and Chemical Engineering, Ningbo University, Ningbo, China
| | - Jia Liu
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
| | - Wenting Jiang
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
- Cixi Biomedical Research Institute, Wenzhou Medical University, Zhejiang, China
| | - Feng Zhou
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
| | - Jiantao Zhang
- Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, China.
- Ningbo Cixi Institute of Biomedical Engineering, Ningbo, China
| |
Collapse
|
|
5
|
Andrade JFM, Verbinnen A, Bakst A, Cunha-Filho M, Gelfuso GM, Gratieri T. An update on nanocarriers for follicular-targeted drug delivery for androgenetic alopecia topical treatment. Expert Opin Drug Deliv 2025; 22:367-381. [PMID: 39841606 DOI: 10.1080/17425247.2025.2457950] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2024] [Revised: 12/23/2024] [Accepted: 01/21/2025] [Indexed: 01/24/2025]
Abstract
INTRODUCTION Androgenic alopecia is a multifactorial disease with a high incidence and a great psychological burden on patients. The current FDA-approved treatment is topical minoxidil or oral finasteride. However, both present significant limitations. While the systemic absorption of finasteride causes serious sexual side effects, minoxidil's low solubility imposes a challenge in obtaining a non-irritative and effective formulation. One way to solve such limitations is by using nanocarriers targeting the drug delivery to the hair follicles upon topical application. AREAS COVERED Here, we review which advancements have been made to achieve a more effective treatment for androgenic alopecia, focusing on nanocarriers for the topical drug delivery systems developed to target hair follicles. EXPERT OPINION The results from multiple reviewed studies demonstrate the potential of incorporating drugs into different nanocarriers to improve follicular targeting in drug delivery for androgenic alopecia treatment. However, many studies fail to perform the proper controls. Most studies also do not quantify the drug accumulation in all skin layers, especially in hair follicles, which avoids comparisons between different nanocarriers and, hence, reliable conclusions. Future experiments with a broader nanocarrier size range, suitable skin models and controls, and clinical tests to assess the safety of developed formulations will improve the androgenic alopecia treatment.
Collapse
Affiliation(s)
- Jayanaraian F M Andrade
- Laboratory of Food, Drugs, and Cosmetics (LTMAC), University of Brasilia (UnB), Brasília, DF, Brazil
| | | | | | - Marcilio Cunha-Filho
- Laboratory of Food, Drugs, and Cosmetics (LTMAC), University of Brasilia (UnB), Brasília, DF, Brazil
| | - Guilherme M Gelfuso
- Laboratory of Food, Drugs, and Cosmetics (LTMAC), University of Brasilia (UnB), Brasília, DF, Brazil
| | - Taís Gratieri
- Laboratory of Food, Drugs, and Cosmetics (LTMAC), University of Brasilia (UnB), Brasília, DF, Brazil
| |
Collapse
|
|
6
|
Balaraman AK, Afzal M, Moglad E, Babu MA, Priya GP, Bansal P, Rajotiya S, Kondapavuluri BK, Kazmi I, Alzarea SI, Goyal K, Ali H. The interplay of p16INK4a and non-coding RNAs: bridging cellular senescence, aging, and cancer. Biogerontology 2025; 26:50. [PMID: 39907830 DOI: 10.1007/s10522-025-10194-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2024] [Accepted: 01/23/2025] [Indexed: 02/06/2025]
Abstract
p16INK4a is a crucial tumor suppressor and regulator of cellular senescence, forming a molecular bridge between aging and cancer. Dysregulated p16INK4a expression is linked to both premature aging and cancer progression, where non-coding RNAs (ncRNAs) such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and small interfering RNAs (siRNAs) play key roles in modulating its function. These ncRNAs interact with p16INK4a through complex post-transcriptional and epigenetic mechanisms, influencing pathways critical to senescence and tumor suppression. In this review, we explore ncRNAs, including ANRIL, MIR31HG, UCA1, MALAT1, miR-24, miR-30, and miR-141, which collectively regulate p16INK4a expression, promoting or inhibiting pathways associated with cancer and aging. ANRIL and MIR31HG modulate p16INK4a silencing via interactions with polycomb repressive complexes (PRC), while miRNAs such as miR-24 and miR-30 target p16INK4a to influence cellular proliferation and senescence. This regulatory interplay underscores the therapeutic potential of ncRNA-targeted strategies to restore p16INK4a function. We summarize recent studies supporting that ncRNAs that control p16INK4a may be diagnostic biomarkers and therapeutic targets for age-related diseases and cancer.
Collapse
Affiliation(s)
- Ashok Kumar Balaraman
- Research and Enterprise, University of Cyberjaya, Persiaran Bestari, Cyber 11, 63000, Cyberjaya, Selangor, Malaysia
| | - Muhammad Afzal
- Department of Pharmaceutical Sciences, Pharmacy Program, Batterjee Medical College, P.O. Box 6231, 21442, Jeddah, Saudi Arabia
| | - Ehssan Moglad
- Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, 11942, Al Kharj, Saudi Arabia
| | - M Arockia Babu
- Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh, India
| | - G Padma Priya
- Department of Chemistry and Biochemistry, School of Sciences, JAIN (Deemed to Be University), Bangalore, Karnataka, India
| | - Pooja Bansal
- Department of Allied Healthcare and Sciences, Vivekananda Global University, Jaipur, Rajasthan, 303012, India
| | - Sumit Rajotiya
- NIMS Institute of Pharmacy, NIMS University, Jaipur, Rajasthan, India
| | - Benod Kumar Kondapavuluri
- Department of General Surgery, Consultant Head and Neck Surgical Oncology, Dr.D.Y.Patil Medical College, Hospital and Research Centre, Pimpri, Pune, India
| | - Imran Kazmi
- Department of Biochemistry, Faculty of Science, King Abdulaziz University, 21589, Jeddah, Saudi Arabia
| | - Sami I Alzarea
- Department of Pharmacology, College of Pharmacy, Jouf University, 72341, Sakaka, Al-Jouf, Saudi Arabia
| | - Kavita Goyal
- Department of Biotechnology, Graphic Era (Deemed to Be University), Clement Town, Dehradun, 248002, India.
| | - Haider Ali
- Centre for Global Health Research, Saveetha Medical College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India
| |
Collapse
|
|
7
|
Andersson P, Burel SA, Estrella H, Foy J, Hagedorn PH, Harper TA, Henry SP, Hoflack JC, Holgersen EM, Levin AA, Morrison E, Pavlicek A, Penso-Dolfin L, Saxena U. Assessing Hybridization-Dependent Off-Target Risk for Therapeutic Oligonucleotides: Updated Industry Recommendations. Nucleic Acid Ther 2025; 35:16-33. [PMID: 39912803 DOI: 10.1089/nat.2024.0072] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2025] Open
Abstract
Hybridization-dependent off-target (OffT) effects, occurring when oligonucleotides bind via Watson-Crick-Franklin hybridization to unintended RNA transcripts, remain a critical safety concern for oligonucleotide therapeutics (ONTs). Despite the importance of OffT assessment of clinical trial ONT candidates, formal guidelines are lacking, with only brief mentions in Japanese regulatory documents (2020) and US Food and Drug Administration (FDA) recommendations for hepatitis B virus treatments (2022). This article presents updated industry recommendations for assessing OffTs of ONTs, building upon the 2012 Oligonucleotide Safety Working Group (OSWG) recommendations and accounting for recent technological advancements. A new OSWG subcommittee, comprising industry experts in RNase H-dependent and steric blocking antisense oligonucleotides and small interfering RNAs, has developed a comprehensive framework for OffT assessment. The proposed workflow encompasses five key steps: (1) OffT identification through in silico complementarity prediction and transcriptomics analysis, (2) focus on cell types with relevant ONT activity, (3) in vitro verification and margin assessment, (4) risk assessment based on the OffT biological role, and (5) management of unavoidable OffTs. The authors provide detailed considerations for various ONT classes, emphasizing the importance of ONT-specific factors such as chemistry, delivery systems, and tissue distribution in OffT evaluation. The article also explores the potential of machine learning models to enhance OffT prediction and discusses strategies for experimental verification and risk assessment. These updated recommendations aim to improve the safety profile of ONTs entering clinical trials and to manage unavoidable OffTs. The authors hope that these recommendations will serve as a valuable resource for ONT development and for the forthcoming finalization of the FDA draft guidance and the International Council for Harmonization S13 guidance on Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics.
Collapse
Affiliation(s)
| | | | | | | | | | | | | | - Jean-Christophe Hoflack
- Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
| | | | | | | | | | | | - Utsav Saxena
- Dicerna Pharmaceuticals, a Novo Nordisk Company, Lexington, Massachusetts, USA
| |
Collapse
|
|
8
|
Murphree-Terry M, Keith JD, Oden AM, Birket SE. Normalization of Muc5b ameliorates airway mucus plugging during persistent Pseudomonas aeruginosa infection in the CFTR -/- rat. Am J Physiol Lung Cell Mol Physiol 2024; 327:L672-L683. [PMID: 39316674 PMCID: PMC11563644 DOI: 10.1152/ajplung.00381.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 07/19/2024] [Accepted: 09/02/2024] [Indexed: 09/26/2024] Open
Abstract
In cystic fibrosis, the airway gel-forming mucin MUC5B accumulates in the airways, preventing clearance of pathogens like Pseudomonas aeruginosa (PA). The cystic fibrosis transmembrane conductance regulator (CFTR)-/- (KO) rat model exhibits a similar accumulation of Muc5b. Our lab has shown that increased Muc5b precipitates the development of chronic PA infection. We hypothesized that reducing Muc5b in the KO rat airway would prevent occlusive mucus plugs and development of persistent PA infection. Six-month-old KO rats received Muc5b or scramble siRNA via intratracheal instillation. Rats were then inoculated with 106 colony-forming units of mucoid P. aeruginosa isolate PAM57-15 and euthanized at 3- or 14-days post infection (dpi) to assess acute and persistent infection. At 14 dpi, Muc5b siRNA-treated KO rats had increased weight, decreased neutrophilic inflammation, and reduced mucus plugging in the small airways compared with scramble-treated KO and WT rats. These results indicate that pharmacological intervention of Muc5b reduces mucus plugging during persistent PA infection.NEW & NOTEWORTHY Although highly effective modulator therapies for cystic fibrosis (CF) have improved mucus-related outcomes of disease for people with CF, eradication of Pseudomonas aeruginosa (PA) infection has not been achieved in this population. In addition, current therapies for CF do not target mucin hypersecretion directly. Here, we show that a novel approach of normalizing airway Muc5b hypersecretion ameliorates infection-induced mucus plugging and neutrophilic inflammation during persistent PA infection in CFTR-/- rats.
Collapse
Affiliation(s)
- Mikayla Murphree-Terry
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States
| | - Johnathan D Keith
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States
| | - Ashley M Oden
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States
| | - Susan E Birket
- Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States
| |
Collapse
|
|
9
|
Wei PS, Thota N, John G, Chang E, Lee S, Wang Y, Ma Z, Tsai YH, Mei KC. Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies. J Control Release 2024; 375:366-388. [PMID: 39179112 PMCID: PMC11972657 DOI: 10.1016/j.jconrel.2024.08.030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2024] [Revised: 07/25/2024] [Accepted: 08/19/2024] [Indexed: 08/26/2024]
Abstract
Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between preclinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.
Collapse
Affiliation(s)
- Pu-Sheng Wei
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Nagasri Thota
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Greshma John
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Evelyn Chang
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Sunjae Lee
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Yuanjun Wang
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Zitao Ma
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Yu-Hsuan Tsai
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA
| | - Kuo-Ching Mei
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, State University of New York at Binghamton, Johnson City, New York, NY 13790, USA.
| |
Collapse
|
|
10
|
Wang TT, Hirons A, Doerflinger M, Morris KV, Ledger S, Purcell DFJ, Kelleher AD, Ahlenstiel CL. Current State of Therapeutics for HTLV-1. Viruses 2024; 16:1616. [PMID: 39459949 PMCID: PMC11512412 DOI: 10.3390/v16101616] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 10/07/2024] [Accepted: 10/11/2024] [Indexed: 10/28/2024] Open
Abstract
Human T cell leukaemia virus type-1 (HTLV-1) is an oncogenic retrovirus that causes lifelong infection in ~5-10 million individuals globally. It is endemic to certain First Nations populations of Northern and Central Australia, Japan, South and Central America, Africa, and the Caribbean region. HTLV-1 preferentially infects CD4+ T cells and remains in a state of reduced transcription, often being asymptomatic in the beginning of infection, with symptoms developing later in life. HTLV-1 infection is implicated in the development of adult T cell leukaemia/lymphoma (ATL) and HTLV-1-associated myelopathies (HAM), amongst other immune-related disorders. With no preventive or curative interventions, infected individuals have limited treatment options, most of which manage symptoms. The clinical burden and lack of treatment options directs the need for alternative treatment strategies for HTLV-1 infection. Recent advances have been made in the development of RNA-based antiviral therapeutics for Human Immunodeficiency Virus Type-1 (HIV-1), an analogous retrovirus that shares modes of transmission with HTLV-1. This review highlights past and ongoing efforts in the development of HTLV-1 therapeutics and vaccines, with a focus on the potential for gene therapy as a new treatment modality in light of its successes in HIV-1, as well as animal models that may help the advancement of novel antiviral and anticancer interventions.
Collapse
Affiliation(s)
- Tiana T. Wang
- Kirby Institute, University of New South Wales, Sydney, NSW 2052, Australia; (T.T.W.); (S.L.); (A.D.K.)
| | - Ashley Hirons
- The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3052, Australia; (A.H.); (D.F.J.P.)
| | - Marcel Doerflinger
- Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia;
- Department of Medical Biology, University of Melbourne, Melbourne, VIC 3050, Australia
| | - Kevin V. Morris
- Centre for Genomics and Personalised Health, School of Biomedical Sciences, Queensland University of Technology, Kelvin Grove, QLD 4059, Australia;
| | - Scott Ledger
- Kirby Institute, University of New South Wales, Sydney, NSW 2052, Australia; (T.T.W.); (S.L.); (A.D.K.)
| | - Damian F. J. Purcell
- The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3052, Australia; (A.H.); (D.F.J.P.)
| | - Anthony D. Kelleher
- Kirby Institute, University of New South Wales, Sydney, NSW 2052, Australia; (T.T.W.); (S.L.); (A.D.K.)
- UNSW RNA Institute, University of New South Wales, Sydney, NSW 2052, Australia
| | - Chantelle L. Ahlenstiel
- Kirby Institute, University of New South Wales, Sydney, NSW 2052, Australia; (T.T.W.); (S.L.); (A.D.K.)
- UNSW RNA Institute, University of New South Wales, Sydney, NSW 2052, Australia
| |
Collapse
|
|
11
|
Li Q, Dong M, Chen P. Advances in structural-guided modifications of siRNA. Bioorg Med Chem 2024; 110:117825. [PMID: 38954918 DOI: 10.1016/j.bmc.2024.117825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2024] [Revised: 06/27/2024] [Accepted: 06/27/2024] [Indexed: 07/04/2024]
Abstract
To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery.
Collapse
Affiliation(s)
- Qiang Li
- Department of Medicinal Chemistry, School of Pharmacy, Qingdao University, Qingdao 266021, China; Research and Development Department, NanoPeptide (Qingdao) Biotechnology Ltd., Qingdao, China.
| | - Mingxin Dong
- Department of Medicinal Chemistry, School of Pharmacy, Qingdao University, Qingdao 266021, China.
| | - Pu Chen
- Research and Development Department, NanoPeptide (Qingdao) Biotechnology Ltd., Qingdao, China; Department of Chemical Engineering and Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON, Canada.
| |
Collapse
|
|
12
|
Vatanparast M, Merkel L, Amari K. Exogenous Application of dsRNA in Plant Protection: Efficiency, Safety Concerns and Risk Assessment. Int J Mol Sci 2024; 25:6530. [PMID: 38928236 PMCID: PMC11204322 DOI: 10.3390/ijms25126530] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 06/10/2024] [Accepted: 06/11/2024] [Indexed: 06/28/2024] Open
Abstract
The use of double-stranded RNA (dsRNA) for plant protection shows great potential as a sustainable alternative to traditional pesticides. This review summarizes the current state of knowledge on using exogenous dsRNA in plant protection and includes the latest findings on the safety and efficiency of this strategy. The review also emphasizes the need for a cautious and comprehensive approach, considering safety considerations such as off-target effects and formulation challenges. The regulatory landscape in different regions is also discussed, underscoring the need for specific guidelines tailored to dsRNA-based pesticides. The review provides a crucial resource for researchers, regulators, and industry stakeholders, promoting a balanced approach incorporating innovation with thorough safety assessments. The continuous dialog emphasized in this review is essential for shaping the future of dsRNA-based plant protection. As the field advances, collaboration among scientists, regulators, and industry partners will play a vital role in establishing guidelines and ensuring the responsible, effective, and sustainable use of dsRNA in agriculture.
Collapse
Affiliation(s)
| | | | - Khalid Amari
- Julius Kühn Institute (JKI), Federal Research Centre for Cultivated Plant, Institute for Biosafety in Plant Biotechnology, D-06484 Quedlinburg, Germany
| |
Collapse
|
|
13
|
Sun WD, Zhu XJ, Li JJ, Mei YZ, Li WS, Li JH. Nicotinamide N-methyltransferase (NNMT): a novel therapeutic target for metabolic syndrome. Front Pharmacol 2024; 15:1410479. [PMID: 38919254 PMCID: PMC11196770 DOI: 10.3389/fphar.2024.1410479] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Accepted: 05/21/2024] [Indexed: 06/27/2024] Open
Abstract
Metabolic syndrome (MetS) represents a constellation of metabolic abnormalities, typified by obesity, hypertension, hyperglycemia, and hyperlipidemia. It stems from intricate dysregulations in metabolic pathways governing energy and substrate metabolism. While comprehending the precise etiological mechanisms of MetS remains challenging, evidence underscores the pivotal roles of aberrations in lipid metabolism and insulin resistance (IR) in its pathogenesis. Notably, nicotinamide N-methyltransferase (NNMT) has recently surfaced as a promising therapeutic target for addressing MetS. Single nucleotide variants in the NNMT gene are significantly correlated with disturbances in energy metabolism, obesity, type 2 diabetes (T2D), hyperlipidemia, and hypertension. Elevated NNMT gene expression is notably observed in the liver and white adipose tissue (WAT) of individuals with diabetic mice, obesity, and rats afflicted with MetS. Knockdown of NNMT elicits heightened energy expenditure in adipose and hepatic tissues, mitigates lipid accumulation, and enhances insulin sensitivity. NNMT catalyzes the methylation of nicotinamide (NAM) using S-adenosyl-methionine (SAM) as the donor methyl group, resulting in the formation of S-adenosyl-l-homocysteine (SAH) and methylnicotinamide (MNAM). This enzymatic process results in the depletion of NAM, a precursor of nicotinamide adenine dinucleotide (NAD+), and the generation of SAH, a precursor of homocysteine (Hcy). Consequently, this cascade leads to reduced NAD+ levels and elevated Hcy levels, implicating NNMT in the pathogenesis of MetS. Moreover, experimental studies employing RNA interference (RNAi) strategies and small molecule inhibitors targeting NNMT have underscored its potential as a therapeutic target for preventing or treating MetS-related diseases. Nonetheless, the precise mechanistic underpinnings remain elusive, and as of yet, clinical trials focusing on NNMT have not been documented. Therefore, further investigations are warranted to elucidate the intricate roles of NNMT in MetS and to develop targeted therapeutic interventions.
Collapse
Affiliation(s)
| | | | | | | | | | - Jiang-Hua Li
- Key Lab of Aquatic Training Monitoring and Intervention of General Administration of Sport of China, Physical Education College, Jiangxi Normal University, Nanchang, China
| |
Collapse
|
|
14
|
Tang Q, Khvorova A. RNAi-based drug design: considerations and future directions. Nat Rev Drug Discov 2024; 23:341-364. [PMID: 38570694 PMCID: PMC11144061 DOI: 10.1038/s41573-024-00912-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/14/2024] [Indexed: 04/05/2024]
Abstract
More than 25 years after its discovery, the post-transcriptional gene regulation mechanism termed RNAi is now transforming pharmaceutical development, proved by the recent FDA approval of multiple small interfering RNA (siRNA) drugs that target the liver. Synthetic siRNAs that trigger RNAi have the potential to specifically silence virtually any therapeutic target with unprecedented potency and durability. Bringing this innovative class of medicines to patients, however, has been riddled with substantial challenges, with delivery issues at the forefront. Several classes of siRNA drug are under clinical evaluation, but their utility in treating extrahepatic diseases remains limited, demanding continued innovation. In this Review, we discuss principal considerations and future directions in the design of therapeutic siRNAs, with a particular emphasis on chemistry, the application of informatics, delivery strategies and the importance of careful target selection, which together influence therapeutic success.
Collapse
Affiliation(s)
- Qi Tang
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
- Department of Dermatology, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Anastasia Khvorova
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA.
| |
Collapse
|
|
15
|
Kyslík J, Born-Torrijos A, Holzer AS, Kosakyan A. RNAi-directed knockdown in the cnidarian fish blood parasite Sphaerospora molnari. Sci Rep 2024; 14:3545. [PMID: 38347054 PMCID: PMC10861503 DOI: 10.1038/s41598-024-54171-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 02/09/2024] [Indexed: 02/15/2024] Open
Abstract
RNA interference (RNAi) is an effective approach to suppress gene expression and monitor gene regulation. Despite its wide application, its use is limited in certain taxonomic groups, including cnidarians. Myxozoans are a unique group of cnidarian parasites that diverged from their free-living ancestors about 600 million years ago, with several species causing acute disease in farmed and wild fish populations. In this pioneering study we successfully applied RNAi in blood stages of the myxozoan Sphaerospora molnari, combining a dsRNA soaking approach, real-time PCR, confocal microscopy, and Western blotting. For proof of concept, we knocked down two unusual actins, one of which is known to play a critical role in S. molnari cell motility. We observed intracellular uptake of dsRNA after 30 min and accumulation in all cells of the typical myxozoan cell-in-cell structure. We successfully knocked down actin in S. molnari in vitro, with transient inhibition for 48 h. We observed the disruption of the cytoskeletal network within the primary cell and loss of the characteristic rotational cell motility. This RNAi workflow could significantly advance functional research within the Myxozoa, offering new prospects for investigating therapeutic targets and facilitating drug discovery against economically important fish parasites.
Collapse
Affiliation(s)
- Jiří Kyslík
- Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic.
| | - Ana Born-Torrijos
- Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic
- Department of Coastal Systems, NIOZ Royal Netherlands Institute for Sea Research, Den Burg, PO Box 59, 1790 AB, Texel, The Netherlands
| | - Astrid S Holzer
- Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic
- Fish Health Division, University of Veterinary Medicine, Vienna, Austria
| | - Anush Kosakyan
- Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic
- Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
- National Biodiversity Future Center (NBFC), Palermo, Italy
| |
Collapse
|
|
16
|
Prescott MA, Moulton H, Pastey MK. An alternative strategy to increasing influenza virus replication for vaccine production in chicken embryo fibroblast (DF-1) cells by inhibiting interferon alpha and beta using peptide-conjugated phosphorodiamidate morpholino oligomers. J Med Microbiol 2024; 73. [PMID: 38353513 DOI: 10.1099/jmm.0.001807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/16/2024] Open
Abstract
Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-β) in chicken embryo fibroblast (DF-1) cells.Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-β proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-β PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-β PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-β PPMO compared to virus titre when treated with JAK/STAT inhibitors.Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.
Collapse
Affiliation(s)
- Meagan A Prescott
- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis Oregon 97331, USA
- Department of Microbiology, College of Science, Oregon State University, Corvallis Oregon 97331, USA
| | - Hong Moulton
- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis Oregon 97331, USA
| | - Manoj K Pastey
- Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis Oregon 97331, USA
| |
Collapse
|
|
17
|
Le Huy B, Bui Thi Phuong H, Luong Xuan H. Advantages and disadvantages of RNA therapeutics. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2024; 203:151-164. [PMID: 38359996 DOI: 10.1016/bs.pmbts.2023.12.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/17/2024]
Abstract
RNA therapeutics is an innovative and rapidly evolving field at the forefront of medical research and biotechnology. Recently, many studies have shown that diverse RNA types play important roles in cells. Besides the protein translation coding, they also express and regulate a variety of cellular pathways. Indeed, along with the research and studies, many drugs and vaccines were developed from RNAs, including both coding and non-coding RNA. Some cases were approved to be medicines or under clinical trial. After years of use and application, they have shown a bright opportunity to prevent and treat many fatal and rare diseases with many strong points, such as fast production and long-term effects. Besides, they still have some drawbacks that need to be overcome, like stability or delivery to become the new generation of medicine. Therefore, this chapter focuses on providing an overview of the advantages and disadvantages of RNA therapeutics as well as some crucial points for future development.
Collapse
Affiliation(s)
- Binh Le Huy
- Center for High Technology Development, Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam; School of Chemical Engineering-Hanọi University of Science and Technology, Hanoi, Vietnam
| | | | - Huy Luong Xuan
- Faculty of Pharmacy, PHENIKAA University, Hanoi, Vietnam; PHENIKAA Institute for Advanced Study (PIAS), PHENIKAA University, Hanoi, Vietnam.
| |
Collapse
|
|
18
|
Henderson ML, Zieba JK, Li X, Campbell DB, Williams MR, Vogt DL, Bupp CP, Edgerly YM, Rajasekaran S, Hartog NL, Prokop JW, Krueger JM. Gene Therapy for Genetic Syndromes: Understanding the Current State to Guide Future Care. BIOTECH 2024; 13:1. [PMID: 38247731 PMCID: PMC10801589 DOI: 10.3390/biotech13010001] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 12/08/2023] [Accepted: 12/21/2023] [Indexed: 01/23/2024] Open
Abstract
Gene therapy holds promise as a life-changing option for individuals with genetic variants that give rise to disease. FDA-approved gene therapies for Spinal Muscular Atrophy (SMA), cerebral adrenoleukodystrophy, β-Thalassemia, hemophilia A/B, retinal dystrophy, and Duchenne Muscular Dystrophy have generated buzz around the ability to change the course of genetic syndromes. However, this excitement risks over-expansion into areas of genetic disease that may not fit the current state of gene therapy. While in situ (targeted to an area) and ex vivo (removal of cells, delivery, and administration of cells) approaches show promise, they have a limited target ability. Broader in vivo gene therapy trials have shown various continued challenges, including immune response, use of immune suppressants correlating to secondary infections, unknown outcomes of overexpression, and challenges in driving tissue-specific corrections. Viral delivery systems can be associated with adverse outcomes such as hepatotoxicity and lethality if uncontrolled. In some cases, these risks are far outweighed by the potentially lethal syndromes for which these systems are being developed. Therefore, it is critical to evaluate the field of genetic diseases to perform cost-benefit analyses for gene therapy. In this work, we present the current state while setting forth tools and resources to guide informed directions to avoid foreseeable issues in gene therapy that could prevent the field from continued success.
Collapse
Affiliation(s)
- Marian L. Henderson
- The Department of Biology, Calvin University, Grand Rapids, MI 49546, USA;
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Jacob K. Zieba
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Xiaopeng Li
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Daniel B. Campbell
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Michael R. Williams
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Daniel L. Vogt
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
| | - Caleb P. Bupp
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
- Medical Genetics, Corewell Health, Grand Rapids, MI 49503, USA
| | | | - Surender Rajasekaran
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
- Office of Research, Corewell Health, Grand Rapids, MI 49503, USA;
- Pediatric Intensive Care Unit, Helen DeVos Children’s Hospital, Corewell Health, Grand Rapids, MI 49503, USA
| | - Nicholas L. Hartog
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
- Allergy & Immunology, Corewell Health, Grand Rapids, MI 49503, USA
| | - Jeremy W. Prokop
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
- Office of Research, Corewell Health, Grand Rapids, MI 49503, USA;
| | - Jena M. Krueger
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, Grand Rapids, MI 48824, USA; (J.K.Z.); (X.L.); (D.B.C.); (M.R.W.); (D.L.V.); (C.P.B.); (S.R.); (N.L.H.)
- Department of Neurology, Helen DeVos Children’s Hospital, Corewell Health, Grand Rapids, MI 49503, USA
| |
Collapse
|
|
19
|
Mahmoud DB, Wölk C, Schulz-Siegmund M. Fabrication of 3D Printed, Core-and-Shell Implants as Controlled Release Systems for Local siRNA Delivery. Adv Healthc Mater 2023; 12:e2301643. [PMID: 37712605 DOI: 10.1002/adhm.202301643] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 08/21/2023] [Indexed: 09/16/2023]
Abstract
The development and clinical translation of small interfering RNA (siRNA) therapies remains challenging owing to their poor pharmacokinetics. 3D printing technology presents a great opportunity to fabricate personalized implants for local and sustained delivery of siRNA. Hydrogels can mimic the mechanical properties of tissues, avoiding the problems associated with rigid implants. Herein, a thermoresponsive composite hydrogel suitable for extrusion 3D-printing is formulated to fabricate controlled-release implants loaded with siRNA-Lipofectamine RNAiMAX complexes. A hydrogel matrix mainly composed of uncharged agarose to protect siRNA from decomplexation is selected. Additionally, pluronic F127 and gelatin are added to improve the printability, degradation, and cell adhesion to the implants. To avoid exposing siRNA to thermal stress during the printing process, a core-and-shell design is set up for the implants in which a core of siRNA-complexes loaded-pluronic F127 is printed without heat and enclosed with a shell comprising the thermoresponsive composite hydrogel. The release profile of siRNA-complexes is envisioned to be controlled by varying the printing patterns. The results reveal that the implants sustain siRNA release for one month. The intactness of the released siRNA-complexes is proven until the eighth day. Furthermore, by changing the printing patterns, the release profiles can be tailored.
Collapse
Affiliation(s)
- Dina B Mahmoud
- Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine, Leipzig University, 04317, Leipzig, Germany
- Department of Pharmaceutics, Egyptian Drug Authority, Giza, 11553, Egypt
| | - Christian Wölk
- Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine, Leipzig University, 04317, Leipzig, Germany
| | - Michaela Schulz-Siegmund
- Pharmaceutical Technology, Institute of Pharmacy, Faculty of Medicine, Leipzig University, 04317, Leipzig, Germany
| |
Collapse
|
|
20
|
Strobel TD, Weber M, Heber N, Holzer A, Hoppe-Seyler K, Hoppe-Seyler F. Revisiting the role of endogenous STAT3 in HPV-positive cervical cancer cells. J Med Virol 2023; 95:e29230. [PMID: 38009614 DOI: 10.1002/jmv.29230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 10/23/2023] [Accepted: 10/30/2023] [Indexed: 11/29/2023]
Abstract
Novel treatment options for human papillomavirus (HPV)-induced cancers are urgently required. The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is considered to be constitutively active in HPV-positive cervical cancer cells and essential for their proliferation. Moreover, STAT3 was reported to undergo mutually stimulatory interactions with the HPV E6/E7 oncogenes. Thus, inhibiting STAT3 in HPV-positive cancer cells is under discussion to provide a powerful novel therapeutic strategy. We here show that the antifungal drug ciclopirox destabilizes the STAT3 protein by acting as an iron chelator. However, by exploring the functional consequences of STAT3 inhibition in HPV-positive cancer cells, we obtained several unexpected results. Chemical STAT3 inhibitors heterogeneously affect cervical cancer cell proliferation and those which act antiproliferative also block the growth of STAT3 knockout cells, indicating induction of off-target effects. In contrast to several chemical inhibitors, genetic inhibition of STAT3 expression by either RNA interference or the CRISPR/Cas9 method does not appreciably affect cervical cancer cell proliferation. Transcriptome analyses indicate that blocking STAT3 expression in HPV-positive cancer cells has very limited effects on putative STAT3 target genes. Although the targeted inhibition of specific growth-promoting signaling pathways leads to a feedback activation of STAT3 in cervical cancer cells via Janus kinase 1/2, this does not lead to treatment resistance. Moreover, we did not obtain experimental evidence for a STAT3-linked activation of HPV E6/E7 oncogene expression or, vice versa, an E6/E7-dependent activation of STAT3, at endogenous conditions in cervical cancer cells. Collectively, these findings question the essential role of STAT3 in cervical cancer cell proliferation and the strategy to inhibit STAT3 in these cells for therapeutic purposes.
Collapse
Affiliation(s)
- Tobias D Strobel
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Maria Weber
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Nora Heber
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Angela Holzer
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Karin Hoppe-Seyler
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Felix Hoppe-Seyler
- Molecular Therapy of Virus-Associated Cancers, German Cancer Research Center (DKFZ), Heidelberg, Germany
| |
Collapse
|
|
21
|
Kang H, Ga YJ, Kim SH, Cho YH, Kim JW, Kim C, Yeh JY. Small interfering RNA (siRNA)-based therapeutic applications against viruses: principles, potential, and challenges. J Biomed Sci 2023; 30:88. [PMID: 37845731 PMCID: PMC10577957 DOI: 10.1186/s12929-023-00981-9] [Citation(s) in RCA: 32] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 10/04/2023] [Indexed: 10/18/2023] Open
Abstract
RNA has emerged as a revolutionary and important tool in the battle against emerging infectious diseases, with roles extending beyond its applications in vaccines, in which it is used in the response to the COVID-19 pandemic. Since their development in the 1990s, RNA interference (RNAi) therapeutics have demonstrated potential in reducing the expression of disease-associated genes. Nucleic acid-based therapeutics, including RNAi therapies, that degrade viral genomes and rapidly adapt to viral mutations, have emerged as alternative treatments. RNAi is a robust technique frequently employed to selectively suppress gene expression in a sequence-specific manner. The swift adaptability of nucleic acid-based therapeutics such as RNAi therapies endows them with a significant advantage over other antiviral medications. For example, small interfering RNAs (siRNAs) are produced on the basis of sequence complementarity to target and degrade viral RNA, a novel approach to combat viral infections. The precision of siRNAs in targeting and degrading viral RNA has led to the development of siRNA-based treatments for diverse diseases. However, despite the promising therapeutic benefits of siRNAs, several problems, including impaired long-term protein expression, siRNA instability, off-target effects, immunological responses, and drug resistance, have been considerable obstacles to the use of siRNA-based antiviral therapies. This review provides an encompassing summary of the siRNA-based therapeutic approaches against viruses while also addressing the obstacles that need to be overcome for their effective application. Furthermore, we present potential solutions to mitigate major challenges.
Collapse
Affiliation(s)
- Hara Kang
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Yun Ji Ga
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Soo Hyun Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Young Hoon Cho
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Jung Won Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
- Convergence Research Center for Insect Vectors, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Chaeyeon Kim
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea
| | - Jung-Yong Yeh
- Department of Life Sciences, College of Life Sciences and Bioengineering, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- Research Institute for New Drug Development, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- Convergence Research Center for Insect Vectors, Incheon National University, Academy-Ro 119, Yeonsu-Gu, Incheon, 22012, South Korea.
- KU Center for Animal Blood Medical Science, College of Veterinary Medicine, Konkuk University, 120 Neungdong-Ro, Gwangjin-Gu, Seoul, 05029, South Korea.
| |
Collapse
|
|
22
|
Hoffmann T, Hörmann A, Corcokovic M, Zmajkovic J, Hinterndorfer M, Salkanovic J, Spreitzer F, Köferle A, Gitschtaler K, Popa A, Oberndorfer S, Andersch F, Schaefer M, Fellner M, Budano N, Ruppert JG, Chetta P, Wurm M, Zuber J, Neumüller RA. Precision RNAi using synthetic shRNAmir target sites. eLife 2023; 12:RP84792. [PMID: 37552050 PMCID: PMC10409502 DOI: 10.7554/elife.84792] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/09/2023] Open
Abstract
Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.
Collapse
Affiliation(s)
- Thomas Hoffmann
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
- Vienna BioCenter PhD Program, Doctoral School of the University at Vienna and Medical University of Vienna, Vienna BioCenter (VBC)ViennaAustria
| | - Alexandra Hörmann
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Maja Corcokovic
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Jakub Zmajkovic
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
| | | | - Jasko Salkanovic
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Fiona Spreitzer
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Anna Köferle
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Katrin Gitschtaler
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Alexandra Popa
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Sarah Oberndorfer
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Florian Andersch
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
| | - Markus Schaefer
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
| | - Michaela Fellner
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
| | - Nicole Budano
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Jan G Ruppert
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Paolo Chetta
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Melanie Wurm
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| | - Johannes Zuber
- Research Institute of Molecular Pathology (IMP), Campus-Vienna-Biocenter 1ViennaAustria
- Medical University of Vienna, Vienna BioCenterViennaAustria
| | - Ralph A Neumüller
- Boehringer Ingelheim RCV GmbH & Co KG, Doktor-Boehringer-GasseViennaAustria
| |
Collapse
|
|
23
|
Al-U’datt DGF, Tranchant CC, Al-Husein B, Hiram R, Al-Dwairi A, AlQudah M, Al-shboul O, Jaradat S, Alqbelat J, Almajwal A. Involvement and possible role of transglutaminases 1 and 2 in mediating fibrotic signalling, collagen cross-linking and cell proliferation in neonatal rat ventricular fibroblasts. PLoS One 2023; 18:e0281320. [PMID: 36848364 PMCID: PMC9970086 DOI: 10.1371/journal.pone.0281320] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Accepted: 01/19/2023] [Indexed: 03/01/2023] Open
Abstract
Transglutaminase (TG) isoforms control diverse normal and pathophysiologic processes through their capacity to cross-link extracellular matrix (ECM) proteins. Their functional and signalling roles in cardiac fibrosis remain poorly understood, despite some evidence of TG2 involvement in abnormal ECM remodelling in heart diseases. In this study, we investigated the role of TG1 and TG2 in mediating fibrotic signalling, collagen cross-linking, and cell proliferation in healthy fibroblasts by siRNA-mediated knockdown. siRNA for TG1, TG2 or negative control was transfected into cultured neonatal rat ventricular fibroblasts and cardiomyocytes. mRNA expression of TGs and profibrotic, proliferation and apoptotic markers was assessed by qPCR. Cell proliferation and soluble and insoluble collagen were determined by ELISA and LC-MS/MS, respectively. TG1 and TG2 were both expressed in neonatal rat cardiomyocytes and fibroblasts before transfection. Other TGs were not detected before and after transfection. TG2 was predominantly expressed and more effectively silenced than TG1. Knocking down TG1 or TG2 significantly modified profibrotic markers mRNA expression in fibroblasts, decreasing connective tissue growth factor (CTGF) and increasing transforming growth factor-β1 compared to the negative siRNA control. Reduced expression of collagen 3A1 was found upon TG1 knockdown, while TG2 knockdown raised α-smooth muscle actin expression. TG2 knockdown further increased fibroblast proliferation and the expression of proliferation marker cyclin D1. Lower insoluble collagen content and collagen cross-linking were evidenced upon silencing TG1 or TG2. Transcript levels of collagen 1A1, fibronectin 1, matrix metalloproteinase-2, cyclin E2, and BCL-2-associated X protein/B-cell lymphoma 2 ratio were strongly correlated with TG1 mRNA expression, whereas TG2 expression correlated strongly with CTGF mRNA abundance. These findings support a functional and signalling role for TG1 and TG2 from fibroblasts in regulating key processes underlying myocardial ECM homeostasis and dysregulation, suggesting that these isoforms could be potential and promising targets for the development of cardiac fibrosis therapies.
Collapse
Affiliation(s)
- Doa’a G. F. Al-U’datt
- Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
| | - Carole C. Tranchant
- School of Food Science, Nutrition and Family Studies, Faculty of Health Sciences and Community Services, Université de Moncton, New Brunswick, Canada
| | - Belal Al-Husein
- Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan
| | - Roddy Hiram
- Montreal Heart Institute, Université de Montréal, Montreal, Quebec, Canada
- Department of Medicine, Université de Montréal, Montreal, Quebec, Canada
| | - Ahmed Al-Dwairi
- Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
| | - Mohammad AlQudah
- Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
- Physiology Department, Arabian Gulf University, Manama, Bahrain
| | - Othman Al-shboul
- Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
| | - Saied Jaradat
- Princess Haya Biotechnology Center, Jordan University of Science and Technology, Irbid, Jordan
| | - Jenan Alqbelat
- Department of Physiology and Biochemistry, Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan
| | - Ali Almajwal
- Department of Community Health Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia
| |
Collapse
|
|
24
|
Betlej G, Błoniarz D, Lewińska A, Wnuk M. Non-targeting siRNA-mediated responses are associated with apoptosis in chemotherapy-induced senescent skin cancer cells. Chem Biol Interact 2023; 369:110254. [PMID: 36343682 DOI: 10.1016/j.cbi.2022.110254] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 10/18/2022] [Accepted: 11/02/2022] [Indexed: 11/06/2022]
Abstract
It is widely accepted that siRNA transfection can promote some off-target effects in the genome; however, little is known about how the cells can respond to the presence of non-viral dsRNA. In the present study, non-targeting control siRNA (NTC-siRNA) was used to evaluate its effects on the activity of pathogen and host-derived nucleic acid-associated signaling pathways such as cGAS-STING, RIG-I, MDA5 and NF-κB in A431 skin cancer cells and BJ fibroblasts. NTC-siRNA treatment promoted cytotoxicity in cancer cells. Furthermore, NTC-siRNA-treated doxorubicin-induced senescent cancer cells were more prone to apoptotic cell death compared to untreated doxorubicin-induced senescent cancer cells. NTC-siRNA stimulated the levels of NF-κB, APOBECs, ALY, LRP8 and phosphorylated STING that suggested the involvement of selected components of nucleic acid sensing pathways in NTC-siRNA-mediated cell death response in skin cancer cells. NTC-siRNA-mediated apoptosis in cancer cells was not associated with IFN-β-based pro-inflammatory response and TRDMT1-based adaptive response. In contrast, in NTC-siRNA-treated fibroblasts, an increase in the levels of RIG-I and IFN-β was not accompanied by affected cell viability. We propose that the use of NTC-siRNA in genetic engineering may provoke a number of unexpected effects that should be carefully monitored. In our experimental settings, NTC-siRNA promoted the elimination of doxorubicin-induced senescent cancer cells that may have implications in skin cancer therapies.
Collapse
Affiliation(s)
- Gabriela Betlej
- Department of Biotechnology, Institute of Biology and Biotechnology, College of Nature Sciences, University of Rzeszow, Pigonia 1, 35-310, Rzeszow, Poland
| | - Dominika Błoniarz
- Department of Biotechnology, Institute of Biology and Biotechnology, College of Nature Sciences, University of Rzeszow, Pigonia 1, 35-310, Rzeszow, Poland
| | - Anna Lewińska
- Department of Biotechnology, Institute of Biology and Biotechnology, College of Nature Sciences, University of Rzeszow, Pigonia 1, 35-310, Rzeszow, Poland.
| | - Maciej Wnuk
- Department of Biotechnology, Institute of Biology and Biotechnology, College of Nature Sciences, University of Rzeszow, Pigonia 1, 35-310, Rzeszow, Poland.
| |
Collapse
|
|
25
|
Vester K, Preußner M, Holton N, Feng S, Schultz C, Heyd F, Wahl MC. Recruitment of a splicing factor to the nuclear lamina for its inactivation. Commun Biol 2022; 5:736. [PMID: 35869234 PMCID: PMC9307855 DOI: 10.1038/s42003-022-03689-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2022] [Accepted: 07/08/2022] [Indexed: 11/10/2022] Open
Abstract
Precursor messenger RNA splicing is a highly regulated process, mediated by a complex RNA-protein machinery, the spliceosome, that encompasses several hundred proteins and five small nuclear RNAs in humans. Emerging evidence suggests that the spatial organization of splicing factors and their spatio-temporal dynamics participate in the regulation of splicing. So far, methods to manipulate the spatial distribution of splicing factors in a temporally defined manner in living cells are missing. Here, we describe such an approach that takes advantage of a reversible chemical dimerizer, and outline the requirements for efficient, reversible re-localization of splicing factors to selected sub-nuclear compartments. In a proof-of-principle study, the partial re-localization of the PRPF38A protein to the nuclear lamina in HEK293T cells induced a moderate increase in intron retention. Our approach allows fast and reversible re-localization of splicing factors, has few side effects and can be applied to many splicing factors by fusion of a protein tag through genome engineering. Apart from the systematic analysis of the spatio-temporal aspects of splicing regulation, the approach has a large potential for the fast induction and reversal of splicing switches and can reveal mechanisms of splicing regulation in native nuclear environments. Through the use of a reversible chemical dimerizer, the splicing factor PRPF38A is re-localized to the nuclear lamina, paving the way for a systematic analysis of spatio-temporal splicing regulation.
Collapse
|
|
26
|
Surface Design Options in Polymer- and Lipid-Based siRNA Nanoparticles Using Antibodies. Int J Mol Sci 2022; 23:ijms232213929. [PMID: 36430411 PMCID: PMC9692731 DOI: 10.3390/ijms232213929] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 10/28/2022] [Accepted: 11/04/2022] [Indexed: 11/16/2022] Open
Abstract
The mechanism of RNA interference (RNAi) could represent a breakthrough in the therapy of all diseases that arise from a gene defect or require the inhibition of a specific gene expression. In particular, small interfering RNA (siRNA) offers an attractive opportunity to achieve a new milestone in the therapy of human diseases. The limitations of siRNA, such as poor stability, inefficient cell uptake, and undesired immune activation, as well as the inability to specifically reach the target tissue in the body, can be overcome by further developments in the field of nanoparticulate drug delivery. Therefore, types of surface modified siRNA nanoparticles are presented and illustrate how a more efficient and safer distribution of siRNA at the target site is possible by modifying the surface properties of nanoparticles with antibodies. However, the development of such efficient and safe delivery strategies is currently still a major challenge. In consideration of that, this review article aims to demonstrate the function and targeted delivery of siRNA nanoparticles, focusing on the surface modification via antibodies, various lipid- and polymer-components, and the therapeutic effects of these delivery systems.
Collapse
|
|
27
|
Komuro H, Aminova S, Lauro K, Harada M. Advances of engineered extracellular vesicles-based therapeutics strategy. SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 2022; 23:655-681. [PMID: 36277506 PMCID: PMC9586594 DOI: 10.1080/14686996.2022.2133342] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/11/2022] [Revised: 09/26/2022] [Accepted: 09/28/2022] [Indexed: 05/09/2023]
Abstract
Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-bound vesicles which encapsulate bioactive molecules, such as nucleic acids, proteins, and lipids. They mediate intercellular communication through transporting internally packaged molecules, making them attractive therapeutics carriers. Over the last decades, a significant amount of research has implied the potential of EVs servings as drug delivery vehicles for nuclear acids, proteins, and small molecular drugs. However, several challenges remain unresolved before the clinical application of EV-based therapeutics, including lack of specificity, stability, biodistribution, storage, large-scale manufacturing, and the comprehensive analysis of EV composition. Technical development is essential to overcome these issues and enhance the pre-clinical therapeutic effects. In this review, we summarize the current advancements in EV engineering which demonstrate their therapeutic potential.
Collapse
Affiliation(s)
- Hiroaki Komuro
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI, USA
- Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA
| | - Shakhlo Aminova
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI, USA
- Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA
| | - Katherine Lauro
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI, USA
- Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA
| | - Masako Harada
- Institute for Quantitative Health Science and Engineering (IQ), Michigan State University, East Lansing, MI, USA
- Department of Biomedical Engineering, Michigan State University, East Lansing, MI, USA
| |
Collapse
|
|
28
|
Shin JW, Hong EP, Park SS, Choi DE, Zeng S, Chen RZ, Lee JM. PAM-altering SNP-based allele-specific CRISPR-Cas9 therapeutic strategies for Huntington’s disease. Mol Ther Methods Clin Dev 2022; 26:547-561. [PMID: 36092363 PMCID: PMC9450073 DOI: 10.1016/j.omtm.2022.08.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Accepted: 08/12/2022] [Indexed: 11/30/2022]
Affiliation(s)
- Jun Wan Shin
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Eun Pyo Hong
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Seri S. Park
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | - Doo Eun Choi
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
| | - Sophia Zeng
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
| | | | - Jong-Min Lee
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA
- Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
- Medical and Population Genetics Program, the Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA
- Corresponding author Jong-Min Lee, Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
| |
Collapse
|
|
29
|
Paredes-Montero JR, Arif U, Brown JK. Knockdown of ecdysteroid synthesis genes results in impaired molting and high mortality in Bactericera cockerelli (Hemiptera: Triozidae). PEST MANAGEMENT SCIENCE 2022; 78:2204-2214. [PMID: 35191190 DOI: 10.1002/ps.6848] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 02/11/2022] [Accepted: 02/22/2022] [Indexed: 06/14/2023]
Abstract
BACKGROUND RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction. RESULTS Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP. CONCLUSIONS Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.
Collapse
Affiliation(s)
- Jorge R Paredes-Montero
- School of Plant Sciences, The University of Arizona, Tucson, AZ, USA
- Facultad de Ciencias de la Vida, Escuela Superior Politécnica del Litoral, ESPOL, Guayaquil, Ecuador
| | - Usman Arif
- Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan
| | - Judith K Brown
- School of Plant Sciences, The University of Arizona, Tucson, AZ, USA
| |
Collapse
|
|
30
|
He L, Boulant S, Stanifer M, Guo C, Nießen A, Chen M, Felix K, Bergmann F, Strobel O, Schimmack S. The link between menin and pleiotrophin in the tumor biology of pancreatic neuroendocrine neoplasms. Cancer Sci 2022; 113:1575-1586. [PMID: 35179814 PMCID: PMC9128182 DOI: 10.1111/cas.15301] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Revised: 01/26/2022] [Accepted: 01/30/2022] [Indexed: 11/29/2022] Open
Abstract
MEN1, which encodes menin protein, is the most frequently mutated gene in pancreatic neuroendocrine neoplasms (pNEN). Pleiotrophin (PTN) was reported being a downstream factor of menin and to promote metastasis in different tumor entities. In this study, the effect of menin and its link to PTN were assessed on features of pNEN cells and outcome of pNEN patients. The expression of menin and PTN in pNEN patient tissues were examined by qRT-PCR and western blot and compared to their metastasis status. Functional assays, including transwell migration/invasion and scratch wound healing assays, were performed on specifically designed CRISPR/Cas9-mediated MEN1-knockout (MEN1-KO) pNEN cell lines (BON1MEN1-KO and QGP1MEN1-KO ) to study the metastasis of pNEN. Among 30 menin negative pNEN patients, 21 revealed a strong protein expression of PTN. This combination was associated with metastasis and shorter disease-free survival. Accordingly, in BON1MEN1-KO and QGP1MEN1-KO cells, PTN protein expression was positively associated with enhanced cell migration and invasion, which could be reversed by PTN silencing. PTN is a predicting factor of metastatic behavior of menin-deficient-pNEN. In vitro, menin is able to both promote and suppress the metastasis of pNEN by regulating PTN expression depending on the tumoral origin of pNEN cells.
Collapse
Affiliation(s)
- Liping He
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany.,Department of Medical Oncology, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310003, P.R. China
| | - Steeve Boulant
- Center for Integrative Infectious Disease Research, Heidelberg University, Heidelberg, Germany
| | - Megan Stanifer
- Center for Integrative Infectious Disease Research, Heidelberg University, Heidelberg, Germany
| | - Cuncai Guo
- Center for Integrative Infectious Disease Research, Heidelberg University, Heidelberg, Germany
| | - Anna Nießen
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany
| | - Mingyi Chen
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany.,Department of Breast Surgery, The First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, 310003, P.R. China
| | - Klaus Felix
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany
| | - Frank Bergmann
- Institute of Pathology, Heidelberg University, Heidelberg, Germany
| | - Oliver Strobel
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany
| | - Simon Schimmack
- Department of General, Visceral and Transplantation Surgery, Heidelberg University Hospital, Heidelberg, Germany
| |
Collapse
|
|
31
|
The siRNA Off-Target Effect Is Determined by Base-Pairing Stabilities of Two Different Regions with Opposite Effects. Genes (Basel) 2022; 13:genes13020319. [PMID: 35205363 PMCID: PMC8872465 DOI: 10.3390/genes13020319] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2022] [Revised: 02/06/2022] [Accepted: 02/07/2022] [Indexed: 02/07/2023] Open
Abstract
In RNA interference (RNAi), small interfering RNA (siRNA) suppresses the expression of its target mRNA with a perfect complementary sequence. In addition, siRNA also suppresses the expression of unintended mRNAs with partially complementary sequences mainly within the siRNA seed region (nucleotides 2–8). This mechanism is highly similar to microRNA (miRNA)-mediated RNA silencing, and known as the siRNA-mediated off-target effect. Previously, we revealed that the off-target effect is induced through stable base-pairing between the siRNA seed region and off-target mRNAs, but not induced through unstable base-pairing. However, in our recent study, we found that the siRNA seed region consists of two functionally different domains: nucleotides 2–5, essential for off-target effects, and nucleotides 6–8, involved in both RNAi and off-target effects. In this study, we investigated the most responsible region for the off-target effect by conducting a comprehensive analysis of the thermodynamic properties of all possible siRNA subregions that involved a machine learning technique using a random sampling procedure. As a result, the thermodynamic stability of nucleotides 2–5 showed the highest positive correlation with the off-target effect, and nucleotides 8–14 showed the most negative correlation. Thus, it is revealed that the siRNA off-target effect is determined by the base-pairing stabilities of two different subregions with opposite effects.
Collapse
|
|
32
|
Kobayashi Y, Fukuhara D, Akase D, Aida M, Ui-Tei K. siRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2'-OMe Modifications. ACS OMEGA 2022; 7:2398-2410. [PMID: 35071927 PMCID: PMC8771963 DOI: 10.1021/acsomega.1c06455] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 12/24/2021] [Indexed: 05/04/2023]
Abstract
In RNA interference (RNAi), small interfering RNA (siRNA) functions to suppress the expression of its target mRNA with perfect sequence complementarity. In a mechanism different from above, siRNA also suppresses unintended mRNAs with partial sequence complementarities, mainly to the siRNA seed region (nucleotides 2-8). This mechanism is largely utilized by microRNAs (miRNAs) and results in siRNA-mediated off-target effects. Thus, the siRNA seed region is considered to be involved in both RNAi and off-target effects. In this study, we revealed that the impact of 2'-O-methyl (2'-OMe) modification is different according to the nucleotide positions. The 2'-OMe modifications of nucleotides 2-5 inhibited off-target effects without affecting on-target RNAi activities. In contrast, 2'-OMe modifications of nucleotides 6-8 increased both RNAi and off-target activities. The computational simulation revealed that the structural change induced by 2'-OMe modifications interrupts base pairing between siRNA and target/off-target mRNAs at nucleotides 2-5 but enhances at nucleotides 6-8. Thus, our results suggest that siRNA seed region consists of two functionally different domains in response to 2'-OMe modifications: nucleotides 2-5 are essential for avoiding off-target effects, and nucleotides 6-8 are involved in the enhancement of both RNAi and off-target activities.
Collapse
Affiliation(s)
- Yoshiaki Kobayashi
- Department
of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
| | - Daiki Fukuhara
- Center
for Quantum Life Sciences and Department of Chemistry, Graduate School
of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
| | - Dai Akase
- Center
for Quantum Life Sciences and Department of Chemistry, Graduate School
of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
| | - Misako Aida
- Center
for Quantum Life Sciences and Department of Chemistry, Graduate School
of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan
| | - Kumiko Ui-Tei
- Department
of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
- Department
of Computational Biology and Medical Sciences, Graduate School of
Frontier Sciences, The University of Tokyo, Chiba 277-8561, Japan
- . Phone: +81-3-5841-3044. Fax: +81-3-5841-3044
| |
Collapse
|
|
33
|
Serumula W, Fernandez G, Gonzalez VM, Parboosing R. Anti-HIV Aptamers: Challenges and Prospects. Curr HIV Res 2022; 20:7-19. [PMID: 34503417 DOI: 10.2174/1570162x19666210908114825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2021] [Revised: 08/06/2021] [Accepted: 08/11/2021] [Indexed: 02/08/2023]
Abstract
Human Immunodeficiency Virus (HIV) infection continues to be a significant health burden in many countries around the world. Current HIV treatment through a combination of different antiretroviral drugs (cART) effectively suppresses viral replication, but drug resistance and crossresistance are significant challenges. This has prompted the search for novel targets and agents, such as nucleic acid aptamers. Nucleic acid aptamers are oligonucleotides that attach to the target sites with high affinity and specificity. This review provides a target-by-target account of research into anti-HIV aptamers and summarises the challenges and prospects of this therapeutic strategy, specifically in the unique context of HIV infection.
Collapse
Affiliation(s)
- William Serumula
- Department of Virology, National Health Laboratory Service, University of KwaZulu-Natal, c/o Inkosi Albert Luthuli Central Hospital, 5th Floor Laboratory Building, 800 Bellair Road, Mayville, Durban 4091, South Africa
| | - Geronimo Fernandez
- Departamento de Bioquímica-Investigación, Aptus Biotech SL, Avda. Cardenal Herrera Oria, 298-28035 Madrid. Spain
| | - Victor M Gonzalez
- Departamento de Bioquímica-Investigación, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS)-Hospital Ramón y Cajal, 28034 Madrid, Spain
| | - Raveen Parboosing
- Department of Virology, National Health Laboratory Service, University of KwaZulu-Natal, c/o Inkosi Albert Luthuli Central Hospital, 5th Floor Laboratory Building, 800 Bellair Road, Mayville, Durban 4091, South Africa
| |
Collapse
|
|
34
|
Matchett-Oates L, Spangenberg GC, Cogan NOI. Manipulation of Cannabinoid Biosynthesis via Transient RNAi Expression. FRONTIERS IN PLANT SCIENCE 2021; 12:773474. [PMID: 35035388 PMCID: PMC8757041 DOI: 10.3389/fpls.2021.773474] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2021] [Accepted: 11/08/2021] [Indexed: 05/04/2023]
Abstract
Cannabis sativa L. produces unique phytocannabinoids, which are used for their pharmaceutical benefits. To date, there are no reports of in vivo engineering targeting the cannabinoid biosynthesis genes to greater elucidate the role each of these genes play in synthesis of these medically important compounds. Reported here is the first modulation of cannabinoid biosynthesis genes using RNAi via agroinfiltration. Vacuum infiltrated leaf segments of the Cannbio-2 C. sativa strain, transfected with different RNAi constructs corresponding to THCAS, CBDAS, and CBCAS gene sequences, showed significant downregulation of all cannabinoid biosynthesis genes using real-time quantitative PCR. Using RNAi, significant off-targeting occurs resulting in the downregulation of highly homologous transcripts. Significant (p < 0.05) downregulation was observed for THCAS (92%), CBDAS (97%), and CBCAS (70%) using pRNAi-GG-CBDAS-UNIVERSAL. Significant (p < 0.05) upregulation of CBCAS (76%) and non-significant upregulation of THCAS (13%) were observed when transfected with pRNAi-GG-CBCAS, suggesting the related gene's ability to synthesize multiple cannabinoids. Using this approach, increased understanding of the relationship between cannabinoid biosynthesis genes can be further elucidated. This RNAi approach enables functional genomics screens for further reverse genetic studies as well as the development of designer cannabis strains with over-expression and/or downregulation of targeted cannabinoid biosynthesis genes. Functional genomics screens, such as these, will further provide insights into gene regulation of cannabinoid biosynthesis in Cannabis.
Collapse
Affiliation(s)
- Lennon Matchett-Oates
- Agriculture Victoria, AgriBio, The Centre for AgriBioscience, Bundoora, VIC, Australia
- School of Applied Systems Biology, La Trobe University, Bundoora, VIC, Australia
| | - German C. Spangenberg
- Agriculture Victoria, AgriBio, The Centre for AgriBioscience, Bundoora, VIC, Australia
- School of Applied Systems Biology, La Trobe University, Bundoora, VIC, Australia
| | - Noel O. I. Cogan
- Agriculture Victoria, AgriBio, The Centre for AgriBioscience, Bundoora, VIC, Australia
- School of Applied Systems Biology, La Trobe University, Bundoora, VIC, Australia
| |
Collapse
|
|
35
|
The LEDGF/p75 Integrase Binding Domain Interactome Contributes to the Survival, Clonogenicity, and Tumorsphere Formation of Docetaxel-Resistant Prostate Cancer Cells. Cells 2021; 10:cells10102723. [PMID: 34685704 PMCID: PMC8534522 DOI: 10.3390/cells10102723] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Revised: 10/05/2021] [Accepted: 10/06/2021] [Indexed: 12/18/2022] Open
Abstract
Patients with prostate cancer (PCa) receiving docetaxel chemotherapy invariably develop chemoresistance. The transcription co-activator lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 and PSIP1, is upregulated in several human cancers, including PCa and promotes resistance to docetaxel and other drugs. The C-terminal region of LEDGF/p75 contains an integrase binding domain (IBD) that tethers nuclear proteins, including the HIV-1 integrase and transcription factors, to active chromatin to promote viral integration and transcription of cellular survival genes. Here, we investigated the contribution of the LEDGF/p75 IBD interactome to PCa chemoresistance. Quantitative immunoblotting revealed that LEDGF/p75 and its IBD-interacting partners are endogenously upregulated in docetaxel-resistant PCa cell lines compared to docetaxel-sensitive parental cells. Using specific human autoantibodies, we co-immunoprecipitated LEDGF/p75 with its endogenous IBD-interacting partners JPO2, menin, MLL, IWS1, ASK1, and PogZ, as well as transcription factors c-MYC and HRP2, in docetaxel-resistant cells, and confirmed their nuclear co-localization by confocal microscopy. Depletion of LEDGF/p75 and selected interacting partners robustly decreased the survival, clonogenicity, and tumorsphere formation capacity of docetaxel-resistant cells. These results implicate the LEDGF/p75 IBD interactome in PCa chemoresistance and could lead to novel therapeutic strategies targeting this protein complex for the treatment of docetaxel-resistant tumors.
Collapse
|
|
36
|
In vivo evaluation of biodistribution and toxicity of pH-responsive strontium nanoparticles for gene delivery. JOURNAL OF PHARMACEUTICAL INVESTIGATION 2021. [DOI: 10.1007/s40005-021-00547-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
|
|
37
|
Shin JW, Kim SH, Yoon JY. PTEN downregulation induces apoptosis and cell cycle arrest in uterine cervical cancer cells. Exp Ther Med 2021; 22:1100. [PMID: 34504554 PMCID: PMC8383748 DOI: 10.3892/etm.2021.10534] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Accepted: 06/18/2021] [Indexed: 11/06/2022] Open
Abstract
The tumor suppressors PTEN and p53 are often downregulated in various human cancer types, which has been associated with a poor prognosis. Recent evidence implies that PTEN downregulation may induce growth arrest of kidney cells and cancer cells. In the present study, the role of PTEN in the proliferation and survival of cervical cancer cells was investigated. It was found that PTEN silencing promoted apoptosis and cell-cycle arrest, accompanied by a significant decrease in the proportion of cells in the S1 phase of the cell cycle. Moreover, PTEN silencing in cervical cancer cells increased levels of p53, p27, p21, phospho-ERK and cleaved caspase-3, and decreased levels of cyclin A2 and cyclin D1. Furthermore, PTEN knockdown significantly impacted the viability of cervical cancer cells. P53 silencing did not affect the ability of PTEN knockdown to induce apoptosis in cervical cancer cells. Taken together, the present study results imply that PTEN silencing induces apoptosis and decreases proliferation in cervical cancer cells; hence, PTEN inhibition may represent a promising strategy for the treatment of cervical cancer.
Collapse
Affiliation(s)
- Jin Woo Shin
- Department of Obstetrics and Gynecology, Gil Medical Center, College of Medicine, Gachon University, Incheon 21565, Republic of Korea
| | - Se-Hee Kim
- Gachon Medical Research Institute, Gil Medical Center, Gachon University, Incheon 21565, Republic of Korea
| | - Jin Young Yoon
- Gachon Medical Research Institute, Gil Medical Center, Gachon University, Incheon 21565, Republic of Korea
| |
Collapse
|
|
38
|
Danielsen MB, Wengel J. Cationic oligonucleotide derivatives and conjugates: A favorable approach for enhanced DNA and RNA targeting oligonucleotides. Beilstein J Org Chem 2021; 17:1828-1848. [PMID: 34386102 PMCID: PMC8329367 DOI: 10.3762/bjoc.17.125] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Accepted: 07/14/2021] [Indexed: 12/20/2022] Open
Abstract
Antisense oligonucleotides (ASOs) have the ability of binding to endogenous nucleic acid targets, thereby inhibiting the gene expression. Although ASOs have great potential in the treatment of many diseases, the search for favorable toxicity profiles and distribution has been challenging and consequently impeded the widespread use of ASOs as conventional medicine. One strategy that has been employed to optimize the delivery profile of ASOs, is the functionalization of ASOs with cationic amine groups, either by direct conjugation onto the sugar, nucleobase or internucleotide linkage. The introduction of these positively charged groups has improved properties like nuclease resistance, increased binding to the nucleic acid target and improved cell uptake for oligonucleotides (ONs) and ASOs. The modifications highlighted in this review are some of the most prevalent cationic amine groups which have been attached as single modifications onto ONs/ASOs. The review has been separated into three sections, nucleobase, sugar and backbone modifications, highlighting what impact the cationic amine groups have on the ONs/ASOs physiochemical and biological properties. Finally, a concluding section has been added, summarizing the important knowledge from the three chapters, and examining the future design for ASOs.
Collapse
Affiliation(s)
- Mathias B Danielsen
- Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
| | - Jesper Wengel
- Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
| |
Collapse
|
|
39
|
Roles of Nicotinamide N-Methyltransferase in Obesity and Type 2 Diabetes. BIOMED RESEARCH INTERNATIONAL 2021; 2021:9924314. [PMID: 34368359 PMCID: PMC8337113 DOI: 10.1155/2021/9924314] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Accepted: 07/20/2021] [Indexed: 11/28/2022]
Abstract
Type 2 diabetes (T2D) is thought to be a complication of metabolic syndrome caused by disorders of energy utilization and storage and characterized by insulin resistance or deficiency of insulin secretion. Though the mechanism linking obesity to the development of T2D is complex and unintelligible, it is known that abnormal lipid metabolism and adipose tissue accumulation possibly play important roles in this process. Recently, nicotinamide N-methyltransferase (NNMT) has been emerging as a new mechanism-of-action target in treating obesity and associated T2D. Evidence has shown that NNMT is associated with obesity and T2D. NNMT inhibition or NNMT knockdown significantly increases energy expenditure, reduces body weight and white adipose mass, improves insulin sensitivity, and normalizes glucose tolerance and fasting blood glucose levels. Additionally, trials of oligonucleotide therapeutics and experiments with some small-molecule NNMT inhibitors in vitro and in preclinical animal models have validated NNMT as a promising therapeutic target to prevent or treat obesity and associated T2D. However, the exact mechanisms underlying these phenomena are not yet fully understood and clinical trials targeting NNMT have not been reported until now. Therefore, more researches are necessary to reveal the acting mechanism of NNMT in obesity and T2D and to develop therapeutics targeting NNMT.
Collapse
|
|
40
|
Tomela K, Karolak JA, Ginter-Matuszewska B, Kabza M, Gajecka M. Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF, TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines. Curr Issues Mol Biol 2021; 43:276-285. [PMID: 34204856 PMCID: PMC8928948 DOI: 10.3390/cimb43010023] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Revised: 05/29/2021] [Accepted: 05/29/2021] [Indexed: 12/19/2022] Open
Abstract
Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.
Collapse
Affiliation(s)
- Katarzyna Tomela
- Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (K.T.); (J.A.K.); (B.G.-M.); (M.K.)
- Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland
| | - Justyna A. Karolak
- Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (K.T.); (J.A.K.); (B.G.-M.); (M.K.)
- Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland
| | - Barbara Ginter-Matuszewska
- Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (K.T.); (J.A.K.); (B.G.-M.); (M.K.)
| | - Michal Kabza
- Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (K.T.); (J.A.K.); (B.G.-M.); (M.K.)
| | - Marzena Gajecka
- Chair and Department of Genetics and Pharmaceutical Microbiology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (K.T.); (J.A.K.); (B.G.-M.); (M.K.)
- Institute of Human Genetics, Polish Academy of Sciences, 60-479 Poznan, Poland
- Correspondence: ; Tel.: +48-61-854-6721
| |
Collapse
|
|
41
|
Vicencio J, Cerón J. A Living Organism in your CRISPR Toolbox: Caenorhabditis elegans Is a Rapid and Efficient Model for Developing CRISPR-Cas Technologies. CRISPR J 2021; 4:32-42. [PMID: 33538637 DOI: 10.1089/crispr.2020.0103] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The Cas9 nuclease from Streptococcus pyogenes (SpCas9) is the most popular enzyme for CRISPR technologies. However, considering the wide diversity of microorganisms (discovered and still unknown), a massive number of CRISPR effectors are being and will be identified and characterized in the search of optimal Cas variants for each of the many applications of CRISPR. In this context, a versatile and efficient multicellular system for CRISPR editing such as Caenorhabditis elegans would be of great help in the development of these effectors. Here, we highlight the benefits of using C. elegans for the rapid evaluation of new CRISPR effectors, and for optimizing CRISPR efficiency in animals in several ways such as by modulating the balance between repair pathways, modifying chromatin accessibility, or controlling the expression and activity of nucleases and guide RNAs.
Collapse
Affiliation(s)
- Jeremy Vicencio
- Modeling human diseases in C. elegans Group, Genes, Disease and Therapy Program, Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain
| | - Julián Cerón
- Modeling human diseases in C. elegans Group, Genes, Disease and Therapy Program, Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain
| |
Collapse
|
|
42
|
Chen J, Peng Y, Zhang H, Wang K, Zhao C, Zhu G, Reddy Palli S, Han Z. Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA. RNA Biol 2021; 18:1747-1759. [PMID: 33397184 DOI: 10.1080/15476286.2020.1868680] [Citation(s) in RCA: 69] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.
Collapse
Affiliation(s)
- Jiasheng Chen
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| | - Yingchuan Peng
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.,Institute of Entomology, Jiangxi Agricultural University, Nanchang, China
| | - Hainan Zhang
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| | - Kangxu Wang
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.,Key Laboratory of Grains and Oils Quality Control and Processing, College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, China
| | - Chunqing Zhao
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| | - Guanheng Zhu
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA
| | - Subba Reddy Palli
- Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA
| | - Zhaojun Han
- The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
| |
Collapse
|
|
43
|
Kobayashi Y, Miyamoto K, Aida M, Ui-Tei K. Selection of Chemical Modifications in the siRNA Seed Region That Repress Off-Target Effect. Methods Mol Biol 2021; 2282:17-30. [PMID: 33928567 DOI: 10.1007/978-1-0716-1298-9_2] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
RNA interference mediated by small interfering RNA (siRNA) has been widely used as a procedure to knock down the expression of an intended target gene with perfect sequence complementarity. However, siRNA often exhibits off-target effects on genes with partial sequence complementarities. Such off-target effect is an undesirable adverse effect for knocking down a target gene specifically. Here we describe the powerful strategy to avoid off-target effects without affecting the RNAi activity by the introduction of DNA or 2'-O-methyl modifications in the siRNA seed region. These two types of chemical modifications repress off-target effects through different molecular mechanisms.
Collapse
Affiliation(s)
- Yoshiaki Kobayashi
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
| | - Kengo Miyamoto
- Center for Quantum Life Sciences and Department of Chemistry, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Misako Aida
- Center for Quantum Life Sciences and Department of Chemistry, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan
| | - Kumiko Ui-Tei
- Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
| |
Collapse
|
|
44
|
Goguen RP, Del Corpo O, Malard CMG, Daher A, Alpuche-Lazcano SP, Chen MJ, Scarborough RJ, Gatignol A. Efficacy, accumulation, and transcriptional profile of anti-HIV shRNAs expressed from human U6, 7SK, and H1 promoters. MOLECULAR THERAPY. NUCLEIC ACIDS 2021; 23:1020-1034. [PMID: 33614248 PMCID: PMC7868930 DOI: 10.1016/j.omtn.2020.12.022] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 12/17/2020] [Indexed: 01/02/2023]
Abstract
The expression of short hairpin RNAs (shRNAs) in cells has many potential therapeutic applications, including as a functional cure for HIV. The RNA polymerase III promoters H1, 7SK, and U6 have all been used to express shRNAs. However, there have been no direct and simultaneous comparisons of shRNA potency, expression level, and transcriptional profile between the promoters. We show that the 7SK and U6 promoters result in higher shRNA levels and potency compared to the H1 promoter but that in transduced T lymphocytes, higher expression levels can also lead to growth defects. We present evidence that Dicer cleavage of shRNAs is measured from the first base pair in the shRNA stem, rather than from the 5' end as previously shown for structurally related microRNAs. As a result, guide-strand identity was unaffected by variations in 5' transcription start sites among the different promoters, making expression levels the main determinant of shRNA potency. While all promoters generated shRNAs with variable start sites, the U6 promoter was the most accurate in using its intended +1 position. Our results have implications for the development of therapeutic small RNAs for gene therapy and for our understanding of how shRNAs are processed in cells.
Collapse
Affiliation(s)
- Ryan P Goguen
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada
| | - Olivier Del Corpo
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada.,Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, Canada
| | - Camille M G Malard
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada
| | - Aïcha Daher
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada
| | - Sergio P Alpuche-Lazcano
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, Canada
| | - Michelle J Chen
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada.,Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, Canada
| | - Robert J Scarborough
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada
| | - Anne Gatignol
- Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, QC H3T 1E2, Canada.,Department of Microbiology and Immunology, McGill University, Montréal, QC H3A 0G4, Canada.,Division of Experimental Medicine, Division of Infectious Diseases, Department of Medicine, McGill University, Montréal, QC H3A 0G4, Canada
| |
Collapse
|
|
45
|
Functional Screening Techniques to Identify Long Non-Coding RNAs as Therapeutic Targets in Cancer. Cancers (Basel) 2020; 12:cancers12123695. [PMID: 33317042 PMCID: PMC7763270 DOI: 10.3390/cancers12123695] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 12/06/2020] [Accepted: 12/07/2020] [Indexed: 12/16/2022] Open
Abstract
Simple Summary Long non-coding RNAs (lncRNAs) are a recently discovered class of molecules in the cell, with potential to be utilized as therapeutic targets in cancer. A number of lncRNAs have been described to play important roles in tumor progression and drive molecular processes involved in cell proliferation, apoptosis or invasion. However, the vast majority of lncRNAs have not been studied in the context of cancer thus far. With the advent of CRISPR/Cas genome editing, high-throughput functional screening approaches to identify lncRNAs that impact cancer growth are becoming more accessible. Here, we review currently available methods to study hundreds to thousands of lncRNAs in parallel to elucidate their role in tumorigenesis and cancer progression. Abstract Recent technological advancements such as CRISPR/Cas-based systems enable multiplexed, high-throughput screening for new therapeutic targets in cancer. While numerous functional screens have been performed on protein-coding genes to date, long non-coding RNAs (lncRNAs) represent an emerging class of potential oncogenes and tumor suppressors, with only a handful of large-scale screens performed thus far. Here, we review in detail currently available screening approaches to identify new lncRNA drivers of tumorigenesis and tumor progression. We discuss the various approaches of genomic and transcriptional targeting using CRISPR/Cas9, as well as methods to post-transcriptionally target lncRNAs via RNA interference (RNAi), antisense oligonucleotides (ASOs) and CRISPR/Cas13. We discuss potential advantages, caveats and future applications of each method to provide an overview and guide on investigating lncRNAs as new therapeutic targets in cancer.
Collapse
|
|
46
|
Sundara Rajan S, Ludwig KR, Hall KL, Jones TL, Caplen NJ. Cancer biology functional genomics: From small RNAs to big dreams. Mol Carcinog 2020; 59:1343-1361. [PMID: 33043516 PMCID: PMC7702050 DOI: 10.1002/mc.23260] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Accepted: 09/23/2020] [Indexed: 12/14/2022]
Abstract
The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function-from a single gene to the whole genome-in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi-based methods to study carcinogenesis. We discuss how RNAi-based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome-wide scale efforts to identify cancer or cancer-type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer-relevant molecular targets and the implications for cancer of the first RNAi-based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.
Collapse
Affiliation(s)
- Soumya Sundara Rajan
- Functional Genetics Section, Genetics BranchCenter for Cancer Research, National Cancer Institute, NIHBethesdaMarylandUSA
| | - Katelyn R. Ludwig
- Functional Genetics Section, Genetics BranchCenter for Cancer Research, National Cancer Institute, NIHBethesdaMarylandUSA
| | - Katherine L. Hall
- Functional Genetics Section, Genetics BranchCenter for Cancer Research, National Cancer Institute, NIHBethesdaMarylandUSA
| | - Tamara L. Jones
- Functional Genetics Section, Genetics BranchCenter for Cancer Research, National Cancer Institute, NIHBethesdaMarylandUSA
| | - Natasha J. Caplen
- Functional Genetics Section, Genetics BranchCenter for Cancer Research, National Cancer Institute, NIHBethesdaMarylandUSA
| |
Collapse
|
|
47
|
Gebert M, Jaśkiewicz M, Moszyńska A, Collawn JF, Bartoszewski R. The Effects of Single Nucleotide Polymorphisms in Cancer RNAi Therapies. Cancers (Basel) 2020; 12:E3119. [PMID: 33113880 PMCID: PMC7694039 DOI: 10.3390/cancers12113119] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Revised: 10/14/2020] [Accepted: 10/23/2020] [Indexed: 12/12/2022] Open
Abstract
Tremendous progress in RNAi delivery methods and design has allowed for the effective development of siRNA-based therapeutics that are currently under clinical investigation for various cancer treatments. This approach has the potential to revolutionize cancer therapy by providing the ability to specifically downregulate or upregulate the mRNA of any protein of interest. This exquisite specificity, unfortunately, also has a downside. Genetic variations in the human population are common because of the presence of single nucleotide polymorphisms (SNPs). SNPs lead to synonymous and non-synonymous changes and they occur once in every 300 base pairs in both coding and non-coding regions in the human genome. Much less common are the somatic mosaicism variations associated with genetically distinct populations of cells within an individual that is derived from postzygotic mutations. These heterogeneities in the population can affect the RNAi's efficacy or more problematically, which can lead to unpredictable and sometimes adverse side effects. From a more positive viewpoint, both SNPs and somatic mosaicisms have also been implicated in human diseases, including cancer, and these specific changes could offer the ability to effectively and, more importantly, selectively target the cancer cells. In this review, we discuss how SNPs in the human population can influence the development and success of novel anticancer RNAi therapies and the importance of why SNPs should be carefully considered.
Collapse
Affiliation(s)
- Magdalena Gebert
- Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, 80-416 Gdańsk, Poland; (M.G.); (M.J.); (A.M.)
| | - Maciej Jaśkiewicz
- Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, 80-416 Gdańsk, Poland; (M.G.); (M.J.); (A.M.)
| | - Adrianna Moszyńska
- Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, 80-416 Gdańsk, Poland; (M.G.); (M.J.); (A.M.)
| | - James F. Collawn
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
| | - Rafał Bartoszewski
- Department of Biology and Pharmaceutical Botany, Medical University of Gdańsk, 80-416 Gdańsk, Poland; (M.G.); (M.J.); (A.M.)
| |
Collapse
|
|
48
|
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.
Collapse
|
|
49
|
Shanmugapriya, Sasidharan S. Functional analysis of down-regulated miRNA-221-5p in HeLa cell treated with polyphenol-rich Polyalthia longifolia as regulators of apoptotic HeLa cell death. 3 Biotech 2020; 10:206. [PMID: 32346497 PMCID: PMC7174487 DOI: 10.1007/s13205-020-02193-6] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2020] [Accepted: 04/06/2020] [Indexed: 02/06/2023] Open
Abstract
MicroRNAs are endogenous small non-coding-RNAs that control gene expression and cancer development. Previous studies reported that Polyalthia longifolia treatment induced apoptotic cell death in HeLa cells by down-regulation of miR-221-5p. Hence, the current study was conducted to validate the down-regulated miR-221-5p in HeLa cells. Functional analysis of miR-221-5p was conducted through the gain-of-function, and loss-of-function approach and the miRNA expression was quantified by a real-time polymerase chain reaction. The P. longifolia treatment significantly (p < 0.05) reduced miR-221-5p expression when compared to the untreated HeLa cells with a double delta Ct value of 6.32 and the expression fold change value was reduced up to 0.013. The transfection of miR-221-5p mimic significantly increased the expression of miR-221-5p with an expression fold change as high as 0.53 while anti-miR-221-5p transfected HeLa cells show the most significant decrease in miR-221-5p expression with an expression fold change of 0.011. The MTT assay results revealed that the over-expression of miR-221-5p increased the cell proliferation and viability of polyphenol-rich P. longifolia-treated HeLa cells and confirmed the role of downregulated miRNA 221-5p in HeLa cell death. The flow-cytometry analysis showed that the miR-221-5p over-expressed cells decreased the apoptosis of cells induced by polyphenol-rich P. longifolia treatment in HeLa cells, which proved the oncogenic role of miR-221-5p to inhibit apoptosis. Moreover, the depletion of caspase-3 in miR-221-5p-overexpressed HeLa cells showed the roles of downregulated miR-221-5p in the induction of apoptosis. In conclusion, these results suggest that the down-regulated miR-221-5p was involved in regulating apoptosis in HeLa cancer cells.
Collapse
Affiliation(s)
- Shanmugapriya
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang Malaysia
| | - Sreenivasan Sasidharan
- Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang Malaysia
| |
Collapse
|
|
50
|
Liu S, Jaouannet M, Dempsey DA, Imani J, Coustau C, Kogel KH. RNA-based technologies for insect control in plant production. Biotechnol Adv 2020; 39:107463. [DOI: 10.1016/j.biotechadv.2019.107463] [Citation(s) in RCA: 60] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 10/24/2019] [Accepted: 10/26/2019] [Indexed: 12/23/2022]
|