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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.
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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
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Dhara D, Mulard LA, Hollenstein M. Natural, modified and conjugated carbohydrates in nucleic acids. Chem Soc Rev 2025; 54:2948-2983. [PMID: 39936337 DOI: 10.1039/d4cs00799a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025]
Abstract
Storage of genetic information in DNA occurs through a unique ordering of canonical base pairs. However, this would not be possible in the absence of the sugar-phosphate backbone which is essential for duplex formation. While over a hundred nucleobase modifications have been identified (mainly in RNA), Nature is rather conservative when it comes to alterations at the level of the (deoxy)ribose sugar moiety. This trend is not reflected in synthetic analogues of nucleic acids where modifications of the sugar entity is commonplace to improve the properties of DNA and RNA. In this review article, we describe the main incentives behind sugar modifications in nucleic acids and we highlight recent progress in this field with a particular emphasis on therapeutic applications, the development of xeno-nucleic acids (XNAs), and on interrogating nucleic acid etiology. We also describe recent strategies to conjugate carbohydrates and oligosaccharides to oligonucleotides since this represents a particularly powerful strategy to improve the therapeutic index of oligonucleotide drugs. The advent of glycoRNAs combined with progress in nucleic acid and carbohydrate chemistry, protein engineering, and delivery methods will undoubtedly yield more potent sugar-modified nucleic acids for therapeutic, biotechnological, and synthetic biology applications.
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Affiliation(s)
- Debashis Dhara
- Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, Institut Pasteur, Université Paris Cité, CNRS UMR 352328, rue du Docteur Roux, 75724 Paris Cedex 15, France.
- Department of Structural Biology and Chemistry, Laboratory for Chemistry of Biomolecules, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
| | - Laurence A Mulard
- Department of Structural Biology and Chemistry, Laboratory for Chemistry of Biomolecules, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
| | - Marcel Hollenstein
- Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, Institut Pasteur, Université Paris Cité, CNRS UMR 352328, rue du Docteur Roux, 75724 Paris Cedex 15, France.
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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.
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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
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Caddeo A, Romeo S. Precision medicine and nucleotide-based therapeutics to treat steatotic liver disease. Clin Mol Hepatol 2025; 31:S76-S93. [PMID: 39103998 PMCID: PMC11925435 DOI: 10.3350/cmh.2024.0438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/08/2024] [Accepted: 08/04/2024] [Indexed: 08/07/2024] Open
Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a complex multifactorial disease and becoming the leading cause of liver-related morbidity and mortality. MASLD spans from isolated steatosis to metabolic dysfunction-associated steatohepatitis (MASH), that may progress to cirrhosis and hepatocellular carcinoma (HCC). Genetic, metabolic, and environmental factors strongly contribute to the heterogeneity of MASLD. Lifestyle intervention and weight loss represent a viable treatment for MASLD. Moreover, Resmetirom, a thyroid hormone beta receptor agonist, has recently been approved for MASLD treatment. However, most individuals treated did not respond to this therapeutic, suggesting the need for a more tailored approach to treat MASLD. Oligonucleotide-based therapies, namely small-interfering RNA (siRNA) and antisense oligonucleotide (ASO), have been recently developed to tackle MASLD by reducing the expression of genes influencing MASH progression, such as PNPLA3 and HSD17B13. Here, we review the latest progress made in the synthesis and development of oligonucleotide-based agents targeting genetic determinants of MASH.
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Affiliation(s)
- Andrea Caddeo
- Department of Biomedical Sciences, Unit of Oncology and Molecular Pathology, University of Cagliari, Cagliari, Italy
| | - Stefano Romeo
- Clinical Nutrition Unit, Department of Medical and Surgical Sciences, University Magna Graecia, Catanzaro, Italy
- Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden
- Department of Cardiology, Sahlgrenska University Hospital, Gothenburg, Sweden
- Department of Medicine, Endocrinology (H7) Karolinska Institute and Hospital, Huddinge, Stockholm, Sweden
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Zhang Z, Ma Y, He Y, Wang D, Yue K, Zhang X, Song H. Inhibition of Hepatitis B Virus Replication by a Novel GalNAc-siRNA In Vivo and In Vitro. ACS OMEGA 2025; 10:484-497. [PMID: 39829464 PMCID: PMC11740256 DOI: 10.1021/acsomega.4c06840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 12/12/2024] [Accepted: 12/24/2024] [Indexed: 01/22/2025]
Abstract
Current antiviral therapy for the chronic hepatitis B virus (HBV) has a low clinical cure rate, high administration frequency, and limited efficacy in reducing HBsAg levels, leading to poor patient compliance. Novel agents are required to achieve HBV functional cure, and reduction of HBV antigenemia may enhance the activation of effective and long-lasting host immune control. HT-101 is a siRNA currently in phase I clinical trials with promising prospects for future applications. By designing and synthesizing siRNA targeting the conserved HBV S region, we evaluated its inhibitory effect on HBV biomarkers across four different genotypes (A-D). Additionally, potential cytotoxic effects were investigated. The in vivo effects and duration of inhibition were assessed using a HBV/adeno-associated virus mouse model. The EC50 values for HBV DNA, HBsAg, HBeAg, and HBV RNA in the supernatant of HepG2.2.15 cells were determined to be 0.3348 0.1696, 4.329, and 2.831 nM, respectively, while the CC50 of HT-101 against the viability of Hep2, H1 HeLa, MRC-5, HEK293, and Huh7 cell lines all exceeded 1 μM significantly. Compared with the vehicle group from days 7 to 70 postdosing, especially in the high-dose group (9 mpk), plasma levels of HBsAg, HBeAg, and HBV DNA were significantly reduced with mean reduction values ranging from 1.72 to 3.38 log10 copy/mL due to long-lasting suppression of HBsAg below the lower limit of quantitation (LLOQ), ultimately leading to induction of anti-HBs. In summary, the preclinical data demonstrate that HT-101 represents a significant breakthrough in reducing antigens and provides a promising strategy for functional cure of HBV.
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Affiliation(s)
- Zhipeng Zhang
- School
of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214126, China
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
| | - Yanqin Ma
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
| | - Yan He
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
| | - Dong Wang
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
| | - Kun Yue
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
| | - Xiaomei Zhang
- School
of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214126, China
| | - Huaien Song
- Suzhou
Hepa Thera Biopharmaceutical Company Limited, Shanghai 200120, China
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Li Z, Ma Y, Li C, Xiao S, Liang H. Photo-Cross-linked DNA Structures Greatly Improves Their Serum Nuclease Resistances and Gene Knock-In Efficiencies. SMALL METHODS 2024:e2401346. [PMID: 39713911 DOI: 10.1002/smtd.202401346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/24/2024] [Revised: 12/10/2024] [Indexed: 12/24/2024]
Abstract
The stabilization and structural integrity of DNA architectures remain significant challenges in their biomedical applications, particularly when inserting functional units into the genome using long single-stranded DNA (lssDNA). To address these challenges, a site-specific photo-cross-linking method is employed. Single-stranded oligonucleotides, containing one or two photosensitive cyanovinylcarbazole nucleoside (CNVK) molecules, are precisely incorporated and cross-linked at the specific sites of ssDNA through base-pairing, followed by rapid UV irradiation at 365 nm. This interstrand photo-cross-linking improves the thermal stability of DNA duplexes and allows this study to afford a tetrahedral DNA nanostructure in a yield of >94%. Most importantly, the photo-cross-linked DNA architectures exhibit high resistances against serum degradation, especially prevent digestion of exonuclease III (exo III), which is common in conventional lambda-processing method. Meanwhile, this photo-cross-linking treatment can significantly improve the knock-in (KI) efficiencies of lssDNA in different cells including 293T, K562, and HepG2, approximately three to eightfold those of the uncross-linked lssDNA, and remain a low cytotoxicity. Given the significantly enhanced nuclease resistance in serum and improved KI efficiencies, this study anticipates that this photo-cross-linking method will become a valuable tool in technologically advanced biomedical applications, such as nanotechnology and nucleic acid therapy.
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Affiliation(s)
- Zhigang Li
- Hefei National Research Center for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, China
| | - Youwei Ma
- Institute of Materials, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 1015, Switzerland
| | - Chengxu Li
- Key Laboratory of Precision and Intelligent Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui, 230026, China
| | - Shiyan Xiao
- Hefei National Research Center for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, China
| | - Haojun Liang
- Hefei National Research Center for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, China
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Purewal JS, Doshi GM. RNAi in psoriasis: A melodic exploration of miRNA, shRNA, and amiRNA with a spotlight on siRNA. Eur J Pharmacol 2024; 985:177083. [PMID: 39481628 DOI: 10.1016/j.ejphar.2024.177083] [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: 06/25/2024] [Revised: 10/28/2024] [Accepted: 10/29/2024] [Indexed: 11/02/2024]
Abstract
Psoriasis (Pso) is an autoimmune inflammatory skin disease characterised by well-demarcated, red plaques covered in silver scales. It affects people of all ages and can be passed down through generations. Genetics play an important role in determining vulnerability to develop Pso. Several large-scale genome-wide association studies have identified over 80 genetic loci associated with Pso susceptibility. Gene expression can be regulated via RNA interference (RNAi). RNAi suppresses gene expression by degrading mRNA molecules. Since its discovery, RNAi has generated considerable excitement over its potential therapeutic benefits. RNAi is mediated by endogenous small RNA molecules like microRNA (miRNA) or exogenous small RNA molecules like small interfering RNA (siRNA), short hairpin RNA (shRNA), and artificial micro RNA (amiRNA). These small RNA molecules can silence a disease-related gene in a sequence-specific manner. Targeting RNAi pathways can help modify disease-related biological processes in various medical conditions, including autoimmune disorders. In Pso, RNAi can downregulate the expression of molecules involved in the pathophysiology of the disease. Significant progress has been made in the field of RNAi therapeutics. However, further research is needed to fine-tune the design and delivery of RNAi therapeutics in humans. In this review, we discuss various effectors of RNAi, some challenges related to RNAi therapeutics (emphasizing siRNA) and strategies to overcome these challenges. Furthermore, we have discussed some studies that employ RNAi therapeutics for Pso.
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8
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Torabi C, Choi SE, Pisanic TR, Paulaitis M, Hur SC. Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation. Biomed Eng Online 2024; 23:116. [PMID: 39574085 PMCID: PMC11580418 DOI: 10.1186/s12938-024-01311-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2024] [Accepted: 11/05/2024] [Indexed: 11/25/2024] Open
Abstract
BACKGROUND Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs. RESULTS In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81+ EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation. CONCLUSIONS Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81+ EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81+ EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity.
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Affiliation(s)
- Corinna Torabi
- Department of Mechanical Engineering, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA
| | - Sung-Eun Choi
- Department of Mechanical Engineering, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA
- RASyn, LLC, 700 Main Street, Cambridge, MA, 02139, USA
| | - Thomas R Pisanic
- Institute for NanoBioTechnology, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA
- Department of Oncology, Johns Hopkins University, 600 N Wolfe St, Baltimore, MD, 21287, USA
| | - Michael Paulaitis
- Center for Nanomedicine at Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Soojung Claire Hur
- Department of Mechanical Engineering, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA.
- Institute for NanoBioTechnology, Johns Hopkins University, 3400 N Charles Street, Baltimore, MD, 21218, USA.
- Department of Oncology, Johns Hopkins University, 600 N Wolfe St, Baltimore, MD, 21287, USA.
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 401 N Broadway, Baltimore, MD, 21231, USA.
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Jiao J, Qian Y, Lv Y, Wei W, Long Y, Guo X, Buerliesi A, Ye J, Han H, Li J, Zhu Y, Zhang W. Overcoming limitations and advancing the therapeutic potential of antibody-oligonucleotide conjugates (AOCs): Current status and future perspectives. Pharmacol Res 2024; 209:107469. [PMID: 39433169 DOI: 10.1016/j.phrs.2024.107469] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 10/15/2024] [Accepted: 10/15/2024] [Indexed: 10/23/2024]
Abstract
As cancer incidence rises due to an aging population, the importance of precision medicine continues to grow. Antibody-drug conjugates (ADCs) exemplify targeted therapies by delivering cytotoxic agents to specific antigens. Building on this concept, researchers have developed antibody-oligonucleotide conjugates (AOCs), which combine antibodies with oligonucleotides to regulate gene expression. This review highlights the mechanism of AOCs, emphasizing their unique ability to selectively target and modulate disease-causing proteins. It also explores the components of AOCs and their application in tumor therapy while addressing key challenges such as manufacturing complexities, endosomal escape, and immune response. The article underscores the significance of AOCs in precision oncology and discusses future directions, highlighting their potential in treating cancers driven by genetic mutations and abnormal protein expression.
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Affiliation(s)
- Jinlan Jiao
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China
| | - Yun Qian
- Dermatologic Surgery Department, Institute of Dermatology, Chinese Academy of Medical Science & Peking Union Medical College, Nanjing 210042, China
| | - Yinhua Lv
- Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210000, China
| | - Wenqian Wei
- Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210000, China
| | - Yongxuan Long
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China
| | - Xiaoling Guo
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China
| | - Anya Buerliesi
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China
| | - Jiahui Ye
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China
| | - Hao Han
- Department of Ultrasound, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China
| | - Jinbo Li
- State Key Laboratory of Analytical Chemistry for Life Sciences, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023, China.
| | - Yun Zhu
- Department of Pharmacy, Nanjing Drum Tower Hospital, Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing 210008, China.
| | - Weijie Zhang
- Division of Breast Surgery, Department of General Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing 210008, China.
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Zhang Y, Wang J. Current status and prospects of gelatin and its derivatives in oncological applications: Review. Int J Biol Macromol 2024; 274:133590. [PMID: 38996884 DOI: 10.1016/j.ijbiomac.2024.133590] [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: 02/25/2024] [Revised: 06/23/2024] [Accepted: 06/29/2024] [Indexed: 07/14/2024]
Abstract
Treating cancer remains challenging due to the substantial side effects and unfavourable pharmacokinetic characteristics of antineoplastic medications, despite the progress made in comprehending the properties and actions of tumour cells in recent years. The advancement of biomaterials, such as stents, implants, personalised drug delivery systems, tailored grafts, cell sheets, and other transplantable materials, has brought about a significant transformation in healthcare and medicine in recent years. Gelatin is a very adaptable natural polymer that finds extensive application in healthcare-related industries owing to its favourable characteristics, including biocompatibility, biodegradability, affordability, and the presence of accessible chemical groups. Gelatin is used as a biomaterial in the biomedical sector for the creation of drug delivery systems (DDSs) since it may be applied to various synthetic procedures. Gelatin nanoparticles (NPs) have been extensively employed as carriers for drugs and genes, specifically targeting diseased tissues such as cancer, tuberculosis, and HIV infection, as well as treating vasospasm and restenosis. This is mostly due to their biocompatibility and ability to degrade naturally. Gelatins possess a diverse array of potential applications that require more elucidation. This review focuses on the use of gelatin and its derivatives in the diagnosis and treatment of cancer. The advancement of biomaterials and bioreactors, coupled with the increasing understanding of emerging applications for biomaterials, has enabled progress in enhancing the efficacy of tumour treatment.
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Affiliation(s)
- Yingfeng Zhang
- University-Town Hospital of Chongqing Medical University, Chongqing 401331, China
| | - Jia Wang
- University-Town Hospital of Chongqing Medical University, Chongqing 401331, China.
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11
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Motamedi H, Ari MM, Alvandi A, Abiri R. Principle, application and challenges of development siRNA-based therapeutics against bacterial and viral infections: a comprehensive review. Front Microbiol 2024; 15:1393646. [PMID: 38939184 PMCID: PMC11208694 DOI: 10.3389/fmicb.2024.1393646] [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: 02/29/2024] [Accepted: 05/28/2024] [Indexed: 06/29/2024] Open
Abstract
While significant progress has been made in understanding and applying gene silencing mechanisms and the treatment of human diseases, there have been still several obstacles in therapeutic use. For the first time, ONPATTRO, as the first small interfering RNA (siRNA) based drug was invented in 2018 for treatment of hTTR with polyneuropathy. Additionally, four other siRNA based drugs naming Givosiran, Inclisiran, Lumasiran, and Vutrisiran have been approved by the US Food and Drug Administration and the European Medicines Agency for clinical use by hitherto. In this review, we have discussed the key and promising advances in the development of siRNA-based drugs in preclinical and clinical stages, the impact of these molecules in bacterial and viral infection diseases, delivery system issues, the impact of administration methods, limitations of siRNA application and how to overcome them and a glimpse into future developments.
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Affiliation(s)
- Hamid Motamedi
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Marzie Mahdizade Ari
- Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Microbial Biotechnology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Amirhoushang Alvandi
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Medical Technology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Ramin Abiri
- Student Research Committee, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
- Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
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Chen L, Bosmajian C, Woo S. A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular pharmacokinetics and pharmacodynamics. Biol Methods Protoc 2024; 9:bpae029. [PMID: 38783988 PMCID: PMC11112049 DOI: 10.1093/biomethods/bpae029] [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: 03/11/2024] [Revised: 04/25/2024] [Accepted: 05/02/2024] [Indexed: 05/25/2024] Open
Abstract
Small interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-bp overlapping RT primer facilitated the distinction of full-length antisense from its 3'-metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RNA-induced silencing complex (RISC)-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing the in vitro-in vivo translation of siRNA therapeutics.
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Affiliation(s)
- Lin Chen
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, United States
| | - Caroline Bosmajian
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, United States
| | - Sukyung Woo
- Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, NY 14214, United States
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13
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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.
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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.
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14
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Chen Y, Li Y, Li C, Zhang D, Liu Y, Zhang J, Guan S, Ding X, Xiao Q. The current perspective and opportunities of small nucleic acid-based therapeutics. Drug Dev Res 2024; 85:e22164. [PMID: 38411296 DOI: 10.1002/ddr.22164] [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: 08/27/2023] [Revised: 02/03/2024] [Accepted: 02/13/2024] [Indexed: 02/28/2024]
Abstract
Compared to traditional small molecule and antibody drugs, RNA-based drugs offer a simple design, short research and development cycles, high specificity, broad treatment fields, and long-term efficacy. As a result, RNA-based drugs are extensively used to treat genetic diseases, tumors, viral infections, and other illnesses, suggesting that they have the potential to become the third-largest drug class after small molecule and antibody drugs. Currently, more than 10 small nucleic acid drugs have gained regulatory approval. The commercialization successes of small nucleic acid drugs will stimulate the development of RNA-based drugs. Small nucleic acid drugs primarily target liver diseases, metabolic diseases, genetic diseases, and tumors, and there is also significant potential for expanding indications in the future. This review provides a brief overview of the advantages and development of small nucleic acid-based therapeutics and shows a focus on platform technologies such as chemical modifications and delivery systems that have enabled the clinical translation of small nucleic acid-based therapeutics. Additionally, we summarize the latest clinical progress in small nucleic acid-based therapeutics for the treatment of various diseases, including rare diseases, liver diseases, metabolic diseases, and tumors. Finally, we highlight the future prospects for this promising treatment approach.
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Affiliation(s)
- Yang Chen
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
| | - Yang Li
- Department of Pharmacy, Southwest Hospital, Third Military Medical University, Chongqing, China
| | - Chao Li
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
| | - Dandan Zhang
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
| | - Yuheng Liu
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
- Department of Pharmacology, College of Pharmacy, Chongqing Medical University, Chongqing, China
| | - Jingjing Zhang
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
| | - Shan Guan
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
| | - Xiaoyan Ding
- Department of Pediatrics, Ludwig-Maximilians University of Munich, Munich, Germany
| | - Qin Xiao
- Department of Microbiology and Biochemical Pharmacy, National Engineering Research Center of Immunological Products, Third Military Medical University, Chongqing, China
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15
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Ishibashi R, Maki R, Toyoshima F. Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a. Sci Rep 2024; 14:7615. [PMID: 38556532 PMCID: PMC10982285 DOI: 10.1038/s41598-024-57551-8] [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: 11/21/2023] [Accepted: 03/19/2024] [Indexed: 04/02/2024] Open
Abstract
The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.
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Affiliation(s)
- Riki Ishibashi
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan.
- Department of Mammalian Regulatory Networks, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
| | - Ritsuko Maki
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
| | - Fumiko Toyoshima
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
- Department of Mammalian Regulatory Networks, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan
- Department of Homeostatic Medicine, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
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16
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Sawada H, Kakisawa Y, Ueno Y. Properties and synergistic effects of a nonionic backbone and aminoalkyl modified nucleosides in RNAs. Bioorg Chem 2024; 144:107143. [PMID: 38309000 DOI: 10.1016/j.bioorg.2024.107143] [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: 10/30/2023] [Revised: 01/04/2024] [Accepted: 01/18/2024] [Indexed: 02/05/2024]
Abstract
In this study, we report the synthesis of two formacetal (FA)-linked dimer building blocks, namely 2'-O-methyluridyl-2'-O-methyluridine and 2'-O-methyluridyl-2'-O-aminoethyluridine. We utilize the former dimer in combination with (S)-5'-C-aminopropyl-2'-O-methylnucleosides (5'-APs) as a neutral trimer unit, and the latter dimer as a cationic unit. Double-stranded RNA containing the neutral trimer unit exhibits greater stability compared to the cationic unit and maintains nuclease stability in a serum-containing buffer. Furthermore, this unit appears to establish additional hydrogen bonds with complementary bases, as supported by modeling simulations and mismatch melting temperature assays. Importantly, siRNAs modified with this unit enhance RNA interference activity in cultured cells. These findings suggest that the trimer unit holds promise for therapeutic siRNAs.
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Affiliation(s)
- Hibiki Sawada
- The Graduate School of Natural Science and Technology, Gifu University, Japan
| | - Yuri Kakisawa
- Faculty of Applied Biological Science, Gifu University, Japan
| | - Yoshihito Ueno
- The Graduate School of Natural Science and Technology, Gifu University, Japan; Faculty of Applied Biological Science, Gifu University, Japan; Center for One Medicine Innovative Translational Research (COMIT), Gifu University Institute for Advanced Study, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
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17
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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.
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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
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18
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Zhang H, Vandesompele J, Braeckmans K, De Smedt SC, Remaut K. Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity. Chem Soc Rev 2024; 53:317-360. [PMID: 38073448 DOI: 10.1039/d3cs00194f] [Citation(s) in RCA: 11] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2024]
Abstract
Gene therapy is on its way to revolutionize the treatment of both inherited and acquired diseases, by transferring nucleic acids to correct a disease-causing gene in the target cells of patients. In the fight against infectious diseases, mRNA-based therapeutics have proven to be a viable strategy in the recent Covid-19 pandemic. Although a growing number of gene therapies have been approved, the success rate is limited when compared to the large number of preclinical and clinical trials that have been/are being performed. In this review, we highlight some of the hurdles which gene therapies encounter after administration into the human body, with a focus on nucleic acid degradation by nucleases that are extremely abundant in mammalian organs, biological fluids as well as in subcellular compartments. We overview the available strategies to reduce the biodegradation of gene therapeutics after administration, including chemical modifications of the nucleic acids, encapsulation into vectors and co-administration with nuclease inhibitors and discuss which strategies are applied for clinically approved nucleic acid therapeutics. In the final part, we discuss the currently available methods and techniques to qualify and quantify the integrity of nucleic acids, with their own strengths and limitations.
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Affiliation(s)
- Heyang Zhang
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.
- Leiden Academic Centre for Drug Research, Leiden University, 2333 CC Leiden, The Netherlands
| | - Jo Vandesompele
- Department of Biomolecular Medicine, Ghent University, 9000 Ghent, Belgium
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium
| | - Kevin Braeckmans
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.
- Centre for Nano- and Biophotonics, Ghent University, 9000 Ghent, Belgium
| | - Stefaan C De Smedt
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium
- Centre for Nano- and Biophotonics, Ghent University, 9000 Ghent, Belgium
| | - Katrien Remaut
- Laboratory for General Biochemistry and Physical Pharmacy, Department of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium.
- Cancer Research Institute Ghent (CRIG), Ghent, Belgium
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19
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Zhang J, Chen B, Gan C, Sun H, Zhang J, Feng L. A Comprehensive Review of Small Interfering RNAs (siRNAs): Mechanism, Therapeutic Targets, and Delivery Strategies for Cancer Therapy. Int J Nanomedicine 2023; 18:7605-7635. [PMID: 38106451 PMCID: PMC10725753 DOI: 10.2147/ijn.s436038] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2023] [Accepted: 11/29/2023] [Indexed: 12/19/2023] Open
Abstract
Small interfering RNA (siRNA) delivery by nanocarriers has been identified as a promising strategy in the study and treatment of cancer. Short nucleotide sequences are synthesized exogenously to create siRNA, which triggers RNA interference (RNAi) in cells and silences target gene expression in a sequence-specific way. As a nucleic acid-based medicine that has gained popularity recently, siRNA exhibits novel potential for the treatment of cancer. However, there are still many obstacles to overcome before clinical siRNA delivery devices can be developed. In this review, we discuss prospective targets for siRNA drug design, explain siRNA drug properties and benefits, and give an overview of the current clinical siRNA therapeutics for the treatment of cancer. Additionally, we introduce the siRNA chemical modifications and delivery systems that are clinically sophisticated and classify bioresponsive materials for siRNA release in a methodical manner. This review will serve as a reference for researchers in developing more precise and efficient targeted delivery systems, promoting ongoing advances in clinical applications.
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Affiliation(s)
- Jiaying Zhang
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100191, People’s Republic of China
| | - Bo Chen
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100191, People’s Republic of China
| | - Chunyuan Gan
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100191, People’s Republic of China
| | - Hongyan Sun
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100191, People’s Republic of China
| | - Jiaxin Zhang
- Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, People’s Republic of China
- Institute of Liver Diseases, Beijing University of Chinese Medicine, Beijing, People’s Republic of China
| | - Lin Feng
- School of Mechanical Engineering and Automation, Beihang University, Beijing, 100191, People’s Republic of China
- Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing, 100191, People’s Republic of China
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20
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Hülsmann J, Lindemann H, Wegener J, Kühne M, Godmann M, Koschella A, Coldewey SM, Heinze T, Heinzel T. Dually Modified Cellulose as a Non-Viral Vector for the Delivery and Uptake of HDAC3 siRNA. Pharmaceutics 2023; 15:2659. [PMID: 38140000 PMCID: PMC10747125 DOI: 10.3390/pharmaceutics15122659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2023] [Revised: 11/20/2023] [Accepted: 11/21/2023] [Indexed: 12/24/2023] Open
Abstract
RNA interference can be applied to different target genes for treating a variety of diseases, but an appropriate delivery system is necessary to ensure the transport of intact siRNAs to the site of action. In this study, cellulose was dually modified to create a non-viral vector for HDAC3 short interfering RNA (siRNA) transfer into cells. A guanidinium group introduced positive charges into the cellulose to allow complexation of negatively charged genetic material. Furthermore, a biotin group fixed by a polyethylene glycol (PEG) spacer was attached to the polymer to allow, if required, the binding of targeting ligands. The resulting polyplexes with HDAC3 siRNA had a size below 200 nm and a positive zeta potential of up to 15 mV. For N/P ratio 2 and higher, the polymer could efficiently complex siRNA. Nanoparticles, based on this dually modified derivative, revealed a low cytotoxicity. Only minor effects on the endothelial barrier integrity and a transfection efficiency in HEK293 cells higher than Lipofectamine 2000TM were found. The uptake and release of the polyplexes were confirmed by immunofluorescence imaging. This study indicates that the modified biopolymer is an auspicious biocompatible non-viral vector with biotin as a promising moiety.
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Affiliation(s)
- Juliana Hülsmann
- Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Hans-Knöll-Straße 2, 07745 Jena, Germany; (J.H.); (M.K.); (M.G.)
| | - Henry Lindemann
- Institute for Organic Chemistry and Macromolecular Chemistry, Center of Excellence for Polysaccharide Research, Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany; (H.L.); (A.K.); (T.H.)
| | - Jamila Wegener
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany; (J.W.); (S.M.C.)
- Septomics Research Center, Jena University Hospital, Albert-Einstein-Straße 10, 07745 Jena, Germany
| | - Marie Kühne
- Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Hans-Knöll-Straße 2, 07745 Jena, Germany; (J.H.); (M.K.); (M.G.)
| | - Maren Godmann
- Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Hans-Knöll-Straße 2, 07745 Jena, Germany; (J.H.); (M.K.); (M.G.)
| | - Andreas Koschella
- Institute for Organic Chemistry and Macromolecular Chemistry, Center of Excellence for Polysaccharide Research, Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany; (H.L.); (A.K.); (T.H.)
| | - Sina M. Coldewey
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany; (J.W.); (S.M.C.)
- Septomics Research Center, Jena University Hospital, Albert-Einstein-Straße 10, 07745 Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07747 Jena, Germany
| | - Thomas Heinze
- Institute for Organic Chemistry and Macromolecular Chemistry, Center of Excellence for Polysaccharide Research, Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany; (H.L.); (A.K.); (T.H.)
- Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
| | - Thorsten Heinzel
- Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Hans-Knöll-Straße 2, 07745 Jena, Germany; (J.H.); (M.K.); (M.G.)
- Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany
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21
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Leitão ADG, Ahammad RU, Spencer B, Wu C, Masliah E, Rissman RA. Novel systemic delivery of a peptide-conjugated antisense oligonucleotide to reduce α-synuclein in a mouse model of Alzheimer's disease. Neurobiol Dis 2023; 186:106285. [PMID: 37690676 PMCID: PMC10584037 DOI: 10.1016/j.nbd.2023.106285] [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: 04/18/2023] [Revised: 08/30/2023] [Accepted: 09/05/2023] [Indexed: 09/12/2023] Open
Abstract
Neurodegenerative disorders of aging are characterized by the progressive accumulation of proteins such as α-synuclein (α-syn) and amyloid beta (Aβ). Misfolded and aggregated α-syn has been implicated in neurological disorders such as Parkinson's disease, and Dementia with Lewy Bodies, but less so in Alzheimer's Disease (AD), despite the fact that accumulation of α-syn has been confirmed in over 50% of postmortem brains neuropathologically diagnosed with AD. To date, no therapeutic strategy has effectively or consistently downregulated α-syn in AD. Here we tested the hypothesis that by using a systemically-delivered peptide (ApoB11) bound to a modified antisense oligonucleotide against α-syn (ASO-α-syn), we can downregulate α-syn expression in an AD mouse model and improve behavioral and neuropathologic phenotypes. Our results demonstrate that monthly systemic treatment with of ApoB11:ASO α-syn beginning at 6 months of age reduces expression of α-synuclein in the brains of 9-month-old AD mice. Downregulation of α-syn led to reduction in Aβ plaque burden, prevented neuronal loss and astrogliosis. Furthermore, we found that AD mice treated with ApoB11:ASO α-syn had greatly improved hippocampal and spatial memory function in comparison to their control counterparts. Collectively, our data supports the reduction of α-syn through use of systemically-delivered ApoB11:ASO α-syn as a promising future disease-modifying therapeutic for AD.
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Affiliation(s)
- André D G Leitão
- Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States of America
| | - Rijwan U Ahammad
- Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States of America
| | - Brian Spencer
- Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States of America
| | - Chengbiao Wu
- Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States of America; Alzheimer's Therapeutic Research Institute, Keck School of Medicine of the University of Southern California, San Diego, CA 92121, United States of America
| | - Eliezer Masliah
- Laboratory of Neurogenetics, National Institute of Aging, National Institute of Health, Bethesda, MD 20892, United States of America
| | - Robert A Rissman
- Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, United States of America; Alzheimer's Therapeutic Research Institute, Keck School of Medicine of the University of Southern California, San Diego, CA 92121, United States of America; VA San Diego Healthcare System, San Diego, CA 92161, United States of America.
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22
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Grilli F, Hassan EM, Variola F, Zou S. Harnessing graphene oxide nanocarriers for siRNA delivery in a 3D spheroid model of lung cancer. Biomater Sci 2023; 11:6635-6649. [PMID: 37609774 DOI: 10.1039/d3bm00732d] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/24/2023]
Abstract
Gene therapy has been recently proposed as an effective strategy for cancer treatment. A significant body of literature proved the effectiveness of nanocarriers to deliver therapeutic agents to 2D tumour models, which are simple but not always representative of the in vivo reality. In this study, we analyze the efficiency of 3D spheroids combined with a minimally modified graphene oxide (GO)-based nanocarrier for siRNA delivery as a new system for cell transfection. Small interfering RNA (siRNA) targeting cluster of differentiation 47 (CD47; CD47_siRNA) was used as an anti-tumour therapeutic agent to silence the genes expressing CD47. This is a surface marker able to send a "don't eat me" signal to macrophages to prevent their phagocytosis. Also, we report the analysis of different GO formulations, in terms of size (small: about 100 nm; large: >650 nm) and functionalization (unmodified or modified with polyethylene glycol (PEG) and the dendrimer PAMAM), aiming to establish the efficiency of unmodified GO as a nanocarrier for the transfection of A549 lung cancer spheroids. Small modified GO (smGO) showed the highest transfection efficiency values (>90%) in 3D models. Interestingly, small unmodified GO (sGO) was found to be promising for transfection, with efficiency values >80% using a higher siRNA ratio (i.e., 3 : 1). These results demonstrated the higher efficiency of spheroids compared to 2D models for transfection, and the high potential of unmodified GO to carry siRNA, providing a promising new in vitro model system for the analysis of anticancer gene therapies.
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Affiliation(s)
- Francesca Grilli
- Metrology Research Centre, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
- Department of Mechanical Engineering, University of Ottawa, 800 King Edward Avenue, Ottawa, ON K1N 6N5, Canada
| | - Eman M Hassan
- Metrology Research Centre, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
| | - Fabio Variola
- Department of Mechanical Engineering, University of Ottawa, 800 King Edward Avenue, Ottawa, ON K1N 6N5, Canada
| | - Shan Zou
- Metrology Research Centre, National Research Council of Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
- Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
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23
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Monopoli KR, Korkin D, Khvorova A. Asymmetric trichotomous partitioning overcomes dataset limitations in building machine learning models for predicting siRNA efficacy. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 33:93-109. [PMID: 37456778 PMCID: PMC10338369 DOI: 10.1016/j.omtn.2023.06.010] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Accepted: 06/09/2023] [Indexed: 07/18/2023]
Abstract
Chemically modified small interfering RNAs (siRNAs) are promising therapeutics guiding sequence-specific silencing of disease genes. Identifying chemically modified siRNA sequences that effectively silence target genes remains challenging. Such determinations necessitate computational algorithms. Machine learning is a powerful predictive approach for tackling biological problems but typically requires datasets significantly larger than most available siRNA datasets. Here, we describe a framework applying machine learning to a small dataset (356 modified sequences) for siRNA efficacy prediction. To overcome noise and biological limitations in siRNA datasets, we apply a trichotomous, two-threshold, partitioning approach, producing several combinations of classification threshold pairs. We then test the effects of different thresholds on random forest machine learning model performance using a novel evaluation metric accounting for class imbalances. We identify thresholds yielding a model with high predictive power, outperforming a linear model generated from the same data, that was predictive upon experimental evaluation. Using a novel model feature extraction method, we observe target site base importances and base preferences consistent with our current understanding of the siRNA-mediated silencing mechanism, with the random forest providing higher resolution than the linear model. This framework applies to any classification challenge involving small biological datasets, providing an opportunity to develop high-performing design algorithms for oligonucleotide therapies.
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Affiliation(s)
- Kathryn R. Monopoli
- Department of Bioinformatics & Computational Biology, Worcester Polytechnic Institute, Worcester, MA 01609, USA
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA
| | - Dmitry Korkin
- Department of Bioinformatics & Computational Biology, Worcester Polytechnic Institute, Worcester, MA 01609, USA
| | - Anastasia Khvorova
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA
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24
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Mangla P, Vicentini Q, Biscans A. Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking. Cells 2023; 12:2253. [PMID: 37759475 PMCID: PMC10527716 DOI: 10.3390/cells12182253] [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: 06/21/2023] [Revised: 08/30/2023] [Accepted: 09/07/2023] [Indexed: 09/29/2023] Open
Abstract
The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics.
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Affiliation(s)
- Priyanka Mangla
- Oligonucleotide Discovery, Discovery Sciences Research and Development, AstraZeneca, 431 38 Gothenburg, Sweden; (P.M.); (Q.V.)
| | - Quentin Vicentini
- Oligonucleotide Discovery, Discovery Sciences Research and Development, AstraZeneca, 431 38 Gothenburg, Sweden; (P.M.); (Q.V.)
- Department of Laboratory Medicine, Clinical Research Centre, Karolinska Institute, 141 57 Stockholm, Sweden
| | - Annabelle Biscans
- Oligonucleotide Discovery, Discovery Sciences Research and Development, AstraZeneca, 431 38 Gothenburg, Sweden; (P.M.); (Q.V.)
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25
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Afrin H, Geetha Bai R, Kumar R, Ahmad SS, Agarwal SK, Nurunnabi M. Oral delivery of RNAi for cancer therapy. Cancer Metastasis Rev 2023; 42:699-724. [PMID: 36971908 PMCID: PMC10040933 DOI: 10.1007/s10555-023-10099-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Accepted: 03/14/2023] [Indexed: 03/29/2023]
Abstract
Cancer is a major health concern worldwide and is still in a continuous surge of seeking for effective treatments. Since the discovery of RNAi and their mechanism of action, it has shown promises in targeted therapy for various diseases including cancer. The ability of RNAi to selectively silence the carcinogenic gene makes them ideal as cancer therapeutics. Oral delivery is the ideal route of administration of drug administration because of its patients' compliance and convenience. However, orally administered RNAi, for instance, siRNA, must cross various extracellular and intracellular biological barriers before it reaches the site of action. It is very challenging and important to keep the siRNA stable until they reach to the targeted site. Harsh pH, thick mucus layer, and nuclease enzyme prevent siRNA to diffuse through the intestinal wall and thereby induce a therapeutic effect. After entering the cell, siRNA is subjected to lysosomal degradation. Over the years, various approaches have been taken into consideration to overcome these challenges for oral RNAi delivery. Therefore, understanding the challenges and recent development is crucial to offer a novel and advanced approach for oral RNAi delivery. Herein, we have summarized the delivery strategies for oral delivery RNAi and recent advancement towards the preclinical stages.
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Affiliation(s)
- Humayra Afrin
- Environmental Science & Engineering, University of Texas at El Paso, El Paso, TX, 79965, USA
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, 1101 N. Campbell St, El Paso, TX, 79902, USA
| | - Renu Geetha Bai
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, 1101 N. Campbell St, El Paso, TX, 79902, USA
- Chair of Biosystems Engineering, Institute of Forestry and Engineering, Estonian University of Life Sciences, Kreutzwaldi 56/1, 51006, Tartu, Estonia
| | - Raj Kumar
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, 1101 N. Campbell St, El Paso, TX, 79902, USA
| | - Sheikh Shafin Ahmad
- Environmental Science & Engineering, University of Texas at El Paso, El Paso, TX, 79965, USA
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, 1101 N. Campbell St, El Paso, TX, 79902, USA
- Aerospace Center (cSETR), University of Texas at El Paso, El Paso, TX, 79965, USA
| | - Sandeep K Agarwal
- Section of Immunology, Allergy and Rheumatology, Department of Medicine, Baylor College of Medicine, Houston, TX, 77030, USA
| | - Md Nurunnabi
- Environmental Science & Engineering, University of Texas at El Paso, El Paso, TX, 79965, USA.
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Texas at El Paso, 1101 N. Campbell St, El Paso, TX, 79902, USA.
- Aerospace Center (cSETR), University of Texas at El Paso, El Paso, TX, 79965, USA.
- Biomedical Engineering, College of Engineering, University of Texas at El Paso, El Paso, TX, 79965, USA.
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26
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Geisler A, Dieringer B, Elsner L, Klingel K, Klopfleisch R, Vornlocher HP, Kurreck J, Fechner H. Lipid nanoparticle-encapsulated, chemically modified anti-adenoviral siRNAs inhibit hepatic adenovirus infection in immunosuppressed Syrian hamsters. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 32:923-936. [PMID: 37346978 PMCID: PMC10280093 DOI: 10.1016/j.omtn.2023.05.016] [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: 12/12/2022] [Accepted: 05/10/2023] [Indexed: 06/23/2023]
Abstract
RNA interference has demonstrated its potential as an antiviral therapy for treatment of human adenovirus (hAd) infections. The only existing viral vector-based system for delivery of anti-adenoviral artificial microRNAs available for in vivo use, however, has proven to be inefficient in therapeutic applications. In this study, we investigated the potential of stabilized small interfering RNA (siRNA) encapsulated in lipid nanoparticles (LNPs) for treatment of hepatic hAd serotype 5 (hAd5) infection in an hAd infection model using immunosuppressed Syrian hamsters. The siRNA sipTPmod directed against the adenoviral pre-terminal protein (pTP) and containing 2'-O-methyl modifications as well as phosphorothioate linkages effectively inhibited hAd5 infection in vitro. In light of this success, sipTPmod was encapsulated in LNPs containing the cationic lipid XL-10, which enables hepatocyte-specific siRNA transfer, and injected intravenously into hAd5-infected immunosuppressed Syrian hamsters. This resulted in a significant reduction of liver hAd5 titers, a trend toward reduced liver injury and inflammation, and reduction of viral titers in the blood and spleen compared with hAd5-infected animals that received a non-silencing siRNA. These effects were demonstrated in animals infected with low and moderate doses of hAd5. These data demonstrate that hepatic hAd5 infection can be successfully treated with anti-adenoviral sipTPmod encapsulated in LNPs.
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Affiliation(s)
- Anja Geisler
- Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
| | - Babette Dieringer
- Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
| | - Leslie Elsner
- Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
| | - Karin Klingel
- Cardiopathology, Institute for Pathology and Neuropathology, University Hospital Tübingen, Tübingen, Germany
| | - Robert Klopfleisch
- Institute of Veterinary Pathology, Freie Universität Berlin, Robert-von-Ostertag-Straße 15, 14163 Berlin, Germany
| | | | - Jens Kurreck
- Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
| | - Henry Fechner
- Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany
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27
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Yu Y, Gao Y, He L, Fang B, Ge W, Yang P, Ju Y, Xie X, Lei L. Biomaterial-based gene therapy. MedComm (Beijing) 2023; 4:e259. [PMID: 37284583 PMCID: PMC10239531 DOI: 10.1002/mco2.259] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Revised: 03/17/2023] [Accepted: 03/21/2023] [Indexed: 06/08/2023] Open
Abstract
Gene therapy, a medical approach that involves the correction or replacement of defective and abnormal genes, plays an essential role in the treatment of complex and refractory diseases, such as hereditary diseases, cancer, and rheumatic immune diseases. Nucleic acids alone do not easily enter the target cells due to their easy degradation in vivo and the structure of the target cell membranes. The introduction of genes into biological cells is often dependent on gene delivery vectors, such as adenoviral vectors, which are commonly used in gene therapy. However, traditional viral vectors have strong immunogenicity while also presenting a potential infection risk. Recently, biomaterials have attracted attention for use as efficient gene delivery vehicles, because they can avoid the drawbacks associated with viral vectors. Biomaterials can improve the biological stability of nucleic acids and the efficiency of intracellular gene delivery. This review is focused on biomaterial-based delivery systems in gene therapy and disease treatment. Herein, we review the recent developments and modalities of gene therapy. Additionally, we discuss nucleic acid delivery strategies, with a focus on biomaterial-based gene delivery systems. Furthermore, the current applications of biomaterial-based gene therapy are summarized.
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Affiliation(s)
- Yi Yu
- Department of StomatologyThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Yijun Gao
- Department of StomatologyThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Liming He
- Department of StomatologyChangsha Stomatological HospitalChangshaChina
| | - Bairong Fang
- Department of Plastic and Aesthetic (Burn) SurgeryThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Wenhui Ge
- Department of StomatologyThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Pu Yang
- Department of Plastic and Aesthetic (Burn) SurgeryThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Yikun Ju
- Department of Plastic and Aesthetic (Burn) SurgeryThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Xiaoyan Xie
- Department of StomatologyThe Second Xiangya HospitalCentral South UniversityChangshaChina
| | - Lanjie Lei
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical EngineeringSoutheast UniversityNanjingChina
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28
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Jones JD, Grassmyer KT, Kennedy RT, Koutmou KS, Maloney TD. Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics. Anal Chem 2023; 95:4404-4411. [PMID: 36812429 DOI: 10.1021/acs.analchem.2c04902] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023]
Abstract
siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.
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Affiliation(s)
- Joshua D Jones
- Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, Michigan 48109, United States.,Synthetic Molecule Design and Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States
| | - Kathleen T Grassmyer
- Synthetic Molecule Design and Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States
| | - Robert T Kennedy
- Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, Michigan 48109, United States
| | - Kristin S Koutmou
- Department of Chemistry, University of Michigan, 930 N University, Ann Arbor, Michigan 48109, United States
| | - Todd D Maloney
- Synthetic Molecule Design and Development, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, United States
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29
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Banerjee S, Hemmat MA, Shubham S, Gosai A, Devarakonda S, Jiang N, Geekiyanage C, Dillard JA, Maury W, Shrotriya P, Lamm MH, Nilsen-Hamilton M. Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing. Int J Mol Sci 2023; 24:4627. [PMID: 36902059 PMCID: PMC10003157 DOI: 10.3390/ijms24054627] [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: 12/31/2022] [Revised: 02/20/2023] [Accepted: 02/21/2023] [Indexed: 03/04/2023] Open
Abstract
The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2'FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.
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Affiliation(s)
- Soma Banerjee
- Ames Laboratory, U.S. Department of Energy, Ames, IA 50011, USA
| | - Mahsa Askary Hemmat
- Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
| | - Shambhavi Shubham
- Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
| | - Agnivo Gosai
- Department of Mechanical Engineering, Iowa State University, Ames, IA 50011, USA
| | | | - Nianyu Jiang
- Department of Mechanical Engineering, Iowa State University, Ames, IA 50011, USA
| | | | - Jacob A. Dillard
- Department of Microbiology and Immunology, University of Iowa, Iowa City, IA 50011, USA
| | - Wendy Maury
- Department of Microbiology and Immunology, University of Iowa, Iowa City, IA 50011, USA
| | - Pranav Shrotriya
- Department of Mechanical Engineering, Iowa State University, Ames, IA 50011, USA
| | - Monica H. Lamm
- Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA
| | - Marit Nilsen-Hamilton
- Ames Laboratory, U.S. Department of Energy, Ames, IA 50011, USA
- Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
- Aptalogic Inc., Ames, IA 50014, USA
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30
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Yasser M, Ribback S, Evert K, Utpatel K, Annweiler K, Evert M, Dombrowski F, Calvisi DF. Early Subcellular Hepatocellular Alterations in Mice Post Hydrodynamic Transfection: An Explorative Study. Cancers (Basel) 2023; 15:cancers15020328. [PMID: 36672277 PMCID: PMC9857294 DOI: 10.3390/cancers15020328] [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: 12/06/2022] [Revised: 12/29/2022] [Accepted: 12/30/2022] [Indexed: 01/06/2023] Open
Abstract
Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.
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Affiliation(s)
- Mohd Yasser
- Institut fuer Pathologie, Universitaetsmedizin Greifswald, Friedrich-Loeffler-Str. 23e, 17475 Greifswald, Germany
| | - Silvia Ribback
- Institut fuer Pathologie, Universitaetsmedizin Greifswald, Friedrich-Loeffler-Str. 23e, 17475 Greifswald, Germany
- Correspondence:
| | - Katja Evert
- Institut fuer Pathologie, Universitaetsklinikum Regensburg, 93053 Regensburg, Germany
| | - Kirsten Utpatel
- Institut fuer Pathologie, Universitaetsklinikum Regensburg, 93053 Regensburg, Germany
| | - Katharina Annweiler
- Institut fuer Pathologie, Universitaetsmedizin Greifswald, Friedrich-Loeffler-Str. 23e, 17475 Greifswald, Germany
| | - Matthias Evert
- Institut fuer Pathologie, Universitaetsklinikum Regensburg, 93053 Regensburg, Germany
| | - Frank Dombrowski
- Institut fuer Pathologie, Universitaetsmedizin Greifswald, Friedrich-Loeffler-Str. 23e, 17475 Greifswald, Germany
| | - Diego F. Calvisi
- Institut fuer Pathologie, Universitaetsklinikum Regensburg, 93053 Regensburg, Germany
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31
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Samad AFA, Kamaroddin MF. Innovative approaches in transforming microRNAs into therapeutic tools. WILEY INTERDISCIPLINARY REVIEWS. RNA 2023; 14:e1768. [PMID: 36437633 DOI: 10.1002/wrna.1768] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 11/06/2022] [Accepted: 11/08/2022] [Indexed: 11/29/2022]
Abstract
MicroRNA (miRNA) is regarded as a prominent genetic regulator, as it can fine-tune an entire biological pathway by targeting multiple target genes. This characteristic makes miRNAs promising therapeutic tools to reinstate cell functions that are disrupted as a consequence of diseases. Currently, miRNA replacement by miRNA mimics and miRNA inhibition by anti-miRNA oligonucleotides are the main approaches to utilizing miRNA molecules for therapeutic purposes. Nevertheless, miRNA-based therapeutics are hampered by major issues such as off-target effects, immunogenicity, and uncertain delivery platforms. Over the past few decades, several innovative approaches have been established to minimize off-target effects, reduce immunostimulation, and provide efficient transfer to the target cells in which these molecules exert their function. Recent achievements have led to the testing of miRNA-based drugs in clinical trials, and these molecules may become next-generation therapeutics for medical intervention. Despite the achievement of exciting milestones, the dosage of miRNA administration remains unclear, and ways to address this issue are proposed. Elucidating the current status of the main factors of therapeutic miRNA would allow further developments and innovations to achieve safe therapeutic tools. This article is categorized under: RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.
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Affiliation(s)
- Abdul Fatah A Samad
- Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia
| | - Mohd Farizal Kamaroddin
- Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia
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32
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CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting. Pharm Res 2023; 40:77-105. [PMID: 36380168 DOI: 10.1007/s11095-022-03433-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Accepted: 11/03/2022] [Indexed: 11/16/2022]
Abstract
Nucleic acid-based therapeutic molecules including small interfering RNA (siRNA), microRNA(miRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), and DNA-based gene therapy have tremendous potential for treating diseases in the central nervous system (CNS). However, achieving clinically meaningful delivery to the brain and particularly to target cells and sub-cellular compartments is typically very challenging. Mediating cell-specific delivery in the CNS would be a crucial advance that mitigates off-target effects and toxicities. In this review, we describe these challenges and provide contemporary evidence of advances in cellular and sub-cellular delivery using a variety of delivery mechanisms and alternative routes of administration, including the nose-to-brain approach. Strategies to achieve subcellular localization, endosomal escape, cytosolic bioavailability, and nuclear transfer are also discussed. Ultimately, there are still many challenges to translating these experimental strategies into effective and clinically viable approaches for treating patients.
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33
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PEGylated Strontium Sulfite Nanoparticles with Spontaneously Formed Surface-Embedded Protein Corona Restrict Off-Target Distribution and Accelerate Breast Tumour-Selective Delivery of siRNA. J Funct Biomater 2022; 13:jfb13040211. [PMID: 36412852 PMCID: PMC9680366 DOI: 10.3390/jfb13040211] [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: 09/06/2022] [Revised: 10/26/2022] [Accepted: 10/27/2022] [Indexed: 11/06/2022] Open
Abstract
As transporters of RNAi therapeutics in preclinical and clinical studies, the application of nanoparticles is often hindered by their susceptibility to opsonin-mediated clearance, poor biological stability, ineffectual targeting, and undesirable effects on healthy cells. Prolonging the blood circulation time while minimizing the off-target distribution and associated toxicity is indispensable for the establishment of a clinically viable delivery system for therapeutic small interfering RNAs (siRNAs). Herein, we report a scalable and straightforward approach to fabricate non-toxic and biodegradable pH-responsive strontium sulfite nanoparticles (SSNs) wrapped with a hydrophilic coating material, biotinylated PEG to lessen unforeseen biological interactions. Surface functionalization of SSNs with PEG led to the generation of small and uniformly distributed particles with a significant affinity towards siRNAs and augmented internalization into breast cancer cells. A triple quadrupole liquid chromatography-mass spectrometry (LC-MS) was deployed to identify the proteins entrapped onto the SSNs, with the help of SwissProt.Mus_musculus database. The results demonstrated the reduction of opsonin proteins adsorption owing to the stealth effect of PEG. The distribution of PEGylated SSNs in mice after 4 h and 24 h of intravenous administration in breast tumour-bearing mice was found to be significantly less to the organs of the reticuloendothelial system (RES) and augmented accumulation in the tumour region. The anti-EGFR siRNA-loaded PEG-SSNs exerted a significant inhibitory effect on tumour development in the murine breast cancer model without any significant toxicity to healthy tissues. Therefore, PEGylated SSNs open up a new avenue for tumour-selective efficient delivery of siRNAs in managing breast cancer.
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34
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Mollica L, Cupaioli FA, Rossetti G, Chiappori F. An overview of structural approaches to study therapeutic RNAs. Front Mol Biosci 2022; 9:1044126. [PMID: 36387283 PMCID: PMC9649582 DOI: 10.3389/fmolb.2022.1044126] [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: 09/14/2022] [Accepted: 10/18/2022] [Indexed: 11/07/2023] Open
Abstract
RNAs provide considerable opportunities as therapeutic agent to expand the plethora of classical therapeutic targets, from extracellular and surface proteins to intracellular nucleic acids and its regulators, in a wide range of diseases. RNA versatility can be exploited to recognize cell types, perform cell therapy, and develop new vaccine classes. Therapeutic RNAs (aptamers, antisense nucleotides, siRNA, miRNA, mRNA and CRISPR-Cas9) can modulate or induce protein expression, inhibit molecular interactions, achieve genome editing as well as exon-skipping. A common RNA thread, which makes it very promising for therapeutic applications, is its structure, flexibility, and binding specificity. Moreover, RNA displays peculiar structural plasticity compared to proteins as well as to DNA. Here we summarize the recent advances and applications of therapeutic RNAs, and the experimental and computational methods to analyze their structure, by biophysical techniques (liquid-state NMR, scattering, reactivity, and computational simulations), with a focus on dynamic and flexibility aspects and to binding analysis. This will provide insights on the currently available RNA therapeutic applications and on the best techniques to evaluate its dynamics and reactivity.
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Affiliation(s)
- Luca Mollica
- Department of Medical Biotechnologies and Translational Medicine, L.I.T.A/University of Milan, Milan, Italy
| | | | | | - Federica Chiappori
- National Research Council—Institute for Biomedical Technologies, Milan, Italy
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Liyanage W, Wu T, Kannan S, Kannan RM. Dendrimer-siRNA Conjugates for Targeted Intracellular Delivery in Glioblastoma Animal Models. ACS APPLIED MATERIALS & INTERFACES 2022; 14:46290-46303. [PMID: 36214413 DOI: 10.1021/acsami.2c13129] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/16/2023]
Abstract
Small interfering RNAs (siRNAs) are potent weapons for gene silencing, with an opportunity to correct defective genes and stop the production of undesirable proteins, with many applications in central nervous system (CNS) disorders. However, successful delivery of siRNAs to the brain parenchyma faces obstacles such as the blood-brain barrier (BBB), brain tissue penetration, and targeting of specific cells. In addition, siRNAs are unstable under physiological conditions and are susceptible to protein binding and enzymatic degradation, necessitating a higher dosage to remain effective. To address these issues and advance siRNA delivery, we report the development of covalently conjugated hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimer-siRNA conjugates, demonstrated with a siRNA against GFP (siGFP) conjugate (D-siGFP) utilizing glutathione-sensitive linkers. This allows for precise nucleic acid loading, protects the payload from premature degradation, delivers the siRNA cargo into cells, and achieves significant GFP knockdown in vitro (∼40%) and in vivo (∼30%). Compared to commercially available delivery systems such as RNAi Max and Lipofectamine, D-siGFP retains the potency of the siRNA in vitro. In addition, the dendrimer-siGFP conjugate significantly enhances the half-life of siRNA in the presence of plasma and endonucleases and maintains the passive targeting ability of PAMAM dendrimers to reactive microglia. When administered intratumorally to orthotopic glioblastoma multiform tumors (GBM) in CX3CR-1GFP mice, D-siGFP localizes in tumor-associated macrophages (TAMs) within the tumor parenchyma, minimizing off-target effects in other cell populations. The facile conjugation strategy for dendrimer-siRNA conjugates presented here offers a promising approach for targeted, systemic intracellular delivery of siRNA, serving as a potential bridge for the clinical translation of RNAi therapies.
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Affiliation(s)
- Wathsala Liyanage
- Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States
| | - Tony Wu
- Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States
| | - Sujatha Kannan
- Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States
- Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States
- Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, Maryland 21205, United States
- Kennedy Krieger Institute-Johns Hopkins University for Cerebral Palsy Research Excellence, Baltimore, Maryland 21218, United States
| | - Rangaramanujam M Kannan
- Center for Nanomedicine, Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States
- Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, United States
- Hugo W. Moser Research Institute at Kennedy Krieger, Inc., Baltimore, Maryland 21205, United States
- Kennedy Krieger Institute-Johns Hopkins University for Cerebral Palsy Research Excellence, Baltimore, Maryland 21218, United States
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Fjelstrup S, Dupont DM, Bus C, Enghild J, Jensen J, Birkenkamp-Demtröder K, Dyrskjøt L, Kjems J. Differential RNA aptamer affinity profiling on plasma as a potential diagnostic tool for bladder cancer. NAR Cancer 2022; 4:zcac025. [PMID: 36004048 PMCID: PMC9394167 DOI: 10.1093/narcan/zcac025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Revised: 07/08/2022] [Accepted: 08/18/2022] [Indexed: 11/16/2022] Open
Abstract
The molecular composition of blood is a signature of human health, reflected in the thousands of blood biomarkers known for human diseases. However, establishing robust disease markers is challenging due to the diversity of individual samples. New sequencing methods have simplified biomarker discovery for circulating DNA and RNA while protein profiling is still laborious and costly. To harness the power of high-throughput sequencing to profile the protein content of a biological sample, we developed a method termed APTASHAPE that uses oligonucleotide aptamers to recognize proteins in complex biofluids. We selected a large pool of 2'Fluoro protected RNA sequences to recognize proteins in human plasma and identified a set of 33 cancer-specific aptamers. Differential enrichment of these aptamers after selection against 1 μl of plasma from individual patients allowed us to differentiate between healthy controls and bladder cancer-diagnosed patients (91% accuracy) and between early non-invasive tumors and late stage tumors (83% accuracy). Affinity purification and mass spectrometry of proteins bound to the predictive aptamers showed the main target proteins to be C4b-binding protein, Complement C3, Fibrinogen, Complement factor H and IgG. The APTASHAPE method thus provides a general, automated and highly sensitive platform for discovering potential new disease biomarkers.
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Affiliation(s)
- Søren Fjelstrup
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
| | - Daniel M Dupont
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
| | - Claus Bus
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
| | - Jan J Enghild
- Department of Molecular Biology and Genetics (MBG), Aarhus University, Aarhus, Denmark
| | - Jørgen B Jensen
- Department of Urology, Aarhus University Hospital, Aarhus N, Denmark
- Department of Clinical medicine, Aarhus University, Aarhus, Denmark
| | - Karin Birkenkamp-Demtröder
- Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
- Department of Clinical medicine, Aarhus University, Aarhus, Denmark
| | - Lars Dyrskjøt
- Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark
- Department of Clinical medicine, Aarhus University, Aarhus, Denmark
| | - Jørgen Kjems
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
- Department of Molecular Biology and Genetics (MBG), Aarhus University, Aarhus, Denmark
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Jin J, Yang QQ, Zhou YL. Non-Viral Delivery of Gene Therapy to the Tendon. Polymers (Basel) 2022; 14:3338. [PMID: 36015594 PMCID: PMC9415435 DOI: 10.3390/polym14163338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 07/07/2022] [Accepted: 07/18/2022] [Indexed: 01/19/2023] Open
Abstract
The tendon, as a compact connective tissue, is difficult to treat after an acute laceration or chronic degeneration. Gene-based therapy is a highly efficient strategy for diverse diseases which has been increasingly applied in tendons in recent years. As technology improves by leaps and bounds, a wide variety of non-viral vectors have been manufactured that attempt to have high biosecurity and transfection efficiency, considered to be a promising treatment modality. In this review, we examine the unwanted biological barriers, the categories of applicable genes, and the introduction and comparison of non-viral vectors. We focus on lipid-based nanoparticles and polymer-based nanoparticles, differentiating between them based on their combination with diverse chemical modifications and scaffolds.
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Affiliation(s)
| | | | - You Lang Zhou
- Hand Surgery Research Center, Research Central of Clinical Medicine, Affiliated Hospital of Nantong University, Medical School of Nantong University, Nantong 226001, China
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Pharmacokinetic and Pharmacodynamic Modeling of siRNA Therapeutics - a Minireview. Pharm Res 2022; 39:1749-1759. [PMID: 35819583 DOI: 10.1007/s11095-022-03333-8] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2022] [Accepted: 07/04/2022] [Indexed: 10/17/2022]
Abstract
The approval of four small interfering RNA (siRNA) products in the past few years has demonstrated unequivocally the therapeutic potential of this novel modality. Three such products (givosiran, lumasiran and inclisiran) are liver-targeted, using tris N-acetylgalactosamine (GalNAc)3 as the targeting ligand. Upon subcutaneous administration, GalNAc-conjugated siRNAs rapidly distribute into the liver via asialoglycoprotein receptor (ASGPR) mediated uptake in the hepatocytes, resulting in fast elimination from the systemic circulation. Patisiran, on the other hand, has been formulated in a lipid nanoparticle for optimal delivery to the liver. While several publications have described preclinical and clinical pharmacokinetic (PK) and pharmacodynamic (PD) results, including absorption, distribution, metabolism, and elimination (ADME) profiles in selected species as well as limited modeling efforts for siRNA therapeutics, there is no systematic review of the PK and PD models developed for these agents or work summarizing the utility and application(s) of such models in drug development and regulatory review. Here, we provide a mini-review of the current state of modeling efforts for siRNA therapeutics within the early preclinical, translational, and clinical stages of siRNA development. Diverse modeling methods including simple compartmental, mechanistic and systems PK/PD, physiologically-based PK (PBPK), population PK/PD, and dose-response-time models are introduced and reviewed. The utility of such models in development and regulatory review for siRNA therapeutics is also discussed with examples. Finally, the current knowledge gaps in mechanism of action of siRNA and resulting challenges in model development are summarized. The goal of this minireview is to trigger cross-functional discussion amongst all key stakeholders to generate key experimental datasets and align on current assumptions, model structures, and approaches to facilitate development and application of robust PK/PD models for siRNA therapeutics.
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Paul A, Muralidharan A, Biswas A, Venkatesh Kamath B, Joseph A, Alex AT. siRNA Therapeutics and its Challenges: Recent Advances in Effective Delivery for Cancer Therapy. OPENNANO 2022. [DOI: 10.1016/j.onano.2022.100063] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/16/2022]
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40
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Fan Y, Yang Z. Inhaled siRNA Formulations for Respiratory Diseases: From Basic Research to Clinical Application. Pharmaceutics 2022; 14:1193. [PMID: 35745766 PMCID: PMC9227582 DOI: 10.3390/pharmaceutics14061193] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Revised: 05/27/2022] [Accepted: 05/30/2022] [Indexed: 12/10/2022] Open
Abstract
The development of siRNA technology has provided new opportunities for gene-specific inhibition and knockdown, as well as new ideas for the treatment of disease. Four siRNA drugs have already been approved for marketing. However, the instability of siRNA in vivo makes systemic delivery ineffective. Inhaled siRNA formulations can deliver drugs directly to the lung, showing great potential for treating respiratory diseases. The clinical applications of inhaled siRNA formulations still face challenges because effective delivery of siRNA to the lung requires overcoming the pulmonary and cellular barriers. This paper reviews the research progress for siRNA inhalation formulations for the treatment of various respiratory diseases and summarizes the chemical structural modifications and the various delivery systems for siRNA. Finally, we conclude the latest clinical application research for inhaled siRNA formulations and discuss the potential difficulty in efficient clinical application.
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Affiliation(s)
| | - Zhijun Yang
- School of Chinese Medicine, Hong Kong Baptist University, 224 Waterloo Rd., Kowloon Tong, Hong Kong, China;
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41
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Peng YY, Hu H, Diaz-Dussan D, Zhao J, Hao X, Narain R. Glycopolymer-Cell-Penetrating Peptide (CPP) Conjugates for Efficient Epidermal Growth Factor Receptor (EGFR) Silencing. ACS Macro Lett 2022; 11:580-587. [PMID: 35575337 DOI: 10.1021/acsmacrolett.2c00046] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Overexpression of epidermal growth factor receptor (EGFR) is observed in multiple cancers such as colorectal, lung, and cervical solid tumors. Regulating the EGFR expression is an efficient strategy to manage these malignancies, and it can be achieved by using short interfering RNA (siRNA). Cell-penetrating peptides (CPPs) demonstrated an excellent capability to enhance the cellular uptake of siRNA, but high knockdown efficiencies have not been achieved due to endosomal entrapment. In this work, Schiff's base reaction was used to modify a block {P[LAEMA(2-lactobionamidoethyl methacrylamide)37]-b-P[FPMA(4-formyl phenyl methacrylate)2-st-DMA(N,N-dimethylacrylamide)2], P2} and two statistical [P(LAEMA23-st-FPMA3) (P3) and P(LAEMA25-st-FPMA2-st-DMA2) (P4)] aldehyde-based and galactose-based polymers, prepared via reversible addition-fragmentation chain-transfer (RAFT) polymerization. An arginine-rich peptide (ARP, KRRKRRRRRK) was used as a cell-penetrating peptide (CPP) and conjugated to the polymers via a Schiff base reaction. The resulting glycopolymer-peptide conjugates were utilized to condense the siRNA to prepare polyplexes with multivalent CPPs (MCPPs, a nanoparticle with multiple copies of the CPP) to enhance the endosomal escape. The polyplexes have different surface properties as determined by the architecture of polymers and the insertion of dimethyl amide moieties. The enhancement of cellular internalization of ARP was observed by labeling the polyplexes with fluorescein isothiocyanate (FITC)-siRNA showing a localization of polyplexes in the cytoplasm of a HeLa (cervical cancer) cell line. In the in vitro EFGR silencing study, the statistical glycopolymer-peptide (P3-P) polyplexes had superior EGFR silencing efficiency in comparison with the other polymers that were studied. Furthermore, P3-P polyplexes led to less off-targeting silencing than lipofectamine 3000. These encouraging results confirmed the potency of decorating galactose-based polymers with CPP, like ARP for their application in siRNA delivery and management of cervical carcinomas.
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Affiliation(s)
- Yi-Yang Peng
- Department of Chemical & Materials Engineering, University of Alberta, Edmonton T6G 1H9, Alberta Canada
| | - Haimei Hu
- The Commonwealth Scientific and Industrial Research Organization, Clayton, Victoria 3168, Australia
- School of Biosciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 51006, China
| | - Diana Diaz-Dussan
- Department of Chemical & Materials Engineering, University of Alberta, Edmonton T6G 1H9, Alberta Canada
| | - Jianyang Zhao
- The Commonwealth Scientific and Industrial Research Organization, Clayton, Victoria 3168, Australia
- Institute for Frontier Materials, Deakin University, Geelong, Victoria 3216, Australia
| | - Xiaojuan Hao
- The Commonwealth Scientific and Industrial Research Organization, Clayton, Victoria 3168, Australia
| | - Ravin Narain
- Department of Chemical & Materials Engineering, University of Alberta, Edmonton T6G 1H9, Alberta Canada
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42
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Nanoscale delivery platforms for RNA therapeutics: Challenges and the current state of the art. MED 2022; 3:167-187. [DOI: 10.1016/j.medj.2022.02.001] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2021] [Revised: 01/18/2022] [Accepted: 02/04/2022] [Indexed: 12/25/2022]
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Halloy F, Biscans A, Bujold KE, Debacker A, Hill AC, Lacroix A, Luige O, Strömberg R, Sundstrom L, Vogel J, Ghidini A. Innovative developments and emerging technologies in RNA therapeutics. RNA Biol 2022; 19:313-332. [PMID: 35188077 PMCID: PMC8865321 DOI: 10.1080/15476286.2022.2027150] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.
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Affiliation(s)
- François Halloy
- Department of Paediatrics, Medical Sciences Division, University of Oxford, Oxford, UK
| | - Annabelle Biscans
- Oligonucleotide Chemistry, Discovery Sciences, BioPharmaceuticals R&d, AstraZeneca, Gothenburg, Sweden
| | - Katherine E. Bujold
- Department of Chemistry & Chemical Biology, McMaster University, (Ontario), Canada
| | | | - Alyssa C. Hill
- Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Eth Zürich, Zürich, Switzerland
| | - Aurélie Lacroix
- Sixfold Bioscience, Translation & Innovation Hub, London, UK
| | - Olivia Luige
- Department of Biosciences and Nutrition, Karolinska Institutet, Sweden
| | - Roger Strömberg
- Department of Biosciences and Nutrition, Karolinska Institutet, Sweden
| | - Linda Sundstrom
- Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals R&d, AstraZeneca, Gothenburg, Sweden
| | - Jörg Vogel
- Helmholtz Institute for RNA-based Infection Research (Hiri), Helmholtz Center for Infection Research (Hzi), Würzburg, Germany
- RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany
| | - Alice Ghidini
- Mechanistic and Structural Biology, Discovery Sciences, BioPharmaceuticals R&d, AstraZeneca, Gothenburg, Sweden
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Xue C, Huang H, Wang L, Liao W, Jiang H, Wu ZS. Swelling of Serum-Stable DNA Nanoparticles upon Target-Induced Conformational Rearrangement of Sensing Probes for the Signal-On Detection of Cancer-Related Genes. Anal Chem 2022; 94:2749-2756. [PMID: 35099191 DOI: 10.1021/acs.analchem.1c03598] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic moiety-attached DNA nanoparticle (LDN) is constructed from a cholesterol-conjugated multifunctional hairpin-type DNA probe (Chol-DP) by hydrophobicity-mediated self-assembly. The LDN holds high serum stability and displays a low false-positive signal even in a complex biological milieu. The hydrophobic cholesterol moiety enables the hydrophobicity-mediated assembly, while hydrophilic DNA sequence serves as a recognition element and a polymerization template. The initiator-activated strand displacement amplification (SDA) reaction can convert the hairpin-shaped probe into rigid double-stranded DNA (dsDNA), causing the conformational rearrangement-based LDN swelling that can be used to reliably and fluorescently signal the cancer-related p53 gene. The size increase and structural reconfiguration are confirmed by dynamic light scattering (DLS) analysis and confocal microscopy imaging, respectively. Target p53 is specifically detected down to 10 pM. The whole assay process involved only several simple mixing steps. Recovery test and blind test further confirm the feasibility of the use of the LDN for the detection of target DNA in a complex biological milieu, indicating a promising nanotool for biomedical applications.
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Affiliation(s)
- Chang Xue
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Hong Huang
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Lei Wang
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China.,Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
| | - Wenqiang Liao
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Hao Jiang
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Zai-Sheng Wu
- Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China
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Liang S, Xu H, Ye BC. Membrane-Decorated Exosomes for Combination Drug Delivery and Improved Glioma Therapy. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2022; 38:299-308. [PMID: 34936368 DOI: 10.1021/acs.langmuir.1c02500] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/09/2023]
Abstract
Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system in adults. The standard therapy of GBM fails to eradicate it due to the drug resistance of glioblastoma stem cells (GSCs) and the presence of the blood-brain-barrier (BBB). Temozolomide (TMZ) is the first-line anti-GBM drug after surgery. However, the high activity of O6-alkylguanine-DNA alkyltransferase (AGT) limits the therapeutic effect of TMZ. Herein, we reported dual-receptor-specific exosomes as vehicles loaded with TMZ and O6-benzylguanine (BG) for eradicating TMZ-resistant GBM. Exosomes pose great promise as nanocarriers due to their intrinsic low immunogenicity, strong cargo-protective capacity, ideal size range, and natural penetration ability of the blood-brain-barrier (BBB). The target ligands angiopep-2 and CD133 RNA aptamers were conjugated on exosomes via an amphiphilic molecule bridge, which was induced to express on donor cells. The resulting nanocarriers exhibited efficient uptake by U87MG and GSCs, excellent BBB penetration ability, and perfect GBM accumulation due to An2 and CD133 aptamer functionalization. Such superior properties of the two dual-receptor-specific exosomes resulted in excellent in vitro proliferation inhibition of U87MG and GSCs and extension of the median survival time of U87MG-bearing mice, without causing adverse effects. The formed exosome nanocomposites can serve as powerful nanomedicine for GBM therapy and provide a promising avenue for targeted therapy against other diseases of the central nervous system.
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Affiliation(s)
- Shifu Liang
- Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China
| | - Huiying Xu
- Laboratory of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
| | - Bang-Ce Ye
- Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China
- Laboratory of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
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Darlington M, Reinders JD, Sethi A, Lu AL, Ramaseshadri P, Fischer JR, Boeckman CJ, Petrick JS, Roper JM, Narva KE, Vélez AM. RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions. INSECTS 2022; 13:57. [PMID: 35055900 PMCID: PMC8779393 DOI: 10.3390/insects13010057] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 12/17/2021] [Accepted: 12/28/2021] [Indexed: 02/06/2023]
Abstract
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.
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Affiliation(s)
- Molly Darlington
- Department of Entomology, University of Nebraska, Lincoln, NE 68583, USA; (M.D.); (J.D.R.)
| | - Jordan D. Reinders
- Department of Entomology, University of Nebraska, Lincoln, NE 68583, USA; (M.D.); (J.D.R.)
| | - Amit Sethi
- Corteva Agriscience, Johnston, IA 50131, USA; (A.S.); (A.L.L.); (C.J.B.); (J.M.R.)
| | - Albert L. Lu
- Corteva Agriscience, Johnston, IA 50131, USA; (A.S.); (A.L.L.); (C.J.B.); (J.M.R.)
| | | | - Joshua R. Fischer
- Bayer Crop Science, Chesterfield, MO 63017, USA; (P.R.); (J.R.F.); (J.S.P.)
| | - Chad J. Boeckman
- Corteva Agriscience, Johnston, IA 50131, USA; (A.S.); (A.L.L.); (C.J.B.); (J.M.R.)
| | - Jay S. Petrick
- Bayer Crop Science, Chesterfield, MO 63017, USA; (P.R.); (J.R.F.); (J.S.P.)
| | - Jason M. Roper
- Corteva Agriscience, Johnston, IA 50131, USA; (A.S.); (A.L.L.); (C.J.B.); (J.M.R.)
| | | | - Ana M. Vélez
- Department of Entomology, University of Nebraska, Lincoln, NE 68583, USA; (M.D.); (J.D.R.)
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Valero J, Civit L, Dupont DM, Selnihhin D, Reinert LS, Idorn M, Israels BA, Bednarz AM, Bus C, Asbach B, Peterhoff D, Pedersen FS, Birkedal V, Wagner R, Paludan SR, Kjems J. A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry. Proc Natl Acad Sci U S A 2021; 118:e2112942118. [PMID: 34876524 PMCID: PMC8685691 DOI: 10.1073/pnas.2112942118] [Citation(s) in RCA: 58] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/11/2021] [Indexed: 12/23/2022] Open
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.
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Affiliation(s)
- Julián Valero
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark;
- Centre for Cellular Signal Patterns (CellPAT), Aarhus University, Aarhus DK-8000, Denmark
| | - Laia Civit
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
| | - Daniel M Dupont
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
| | - Denis Selnihhin
- Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark
| | - Line S Reinert
- Department of Biomedicine, Aarhus University DK-8000 Aarhus, Denmark
| | - Manja Idorn
- Department of Biomedicine, Aarhus University DK-8000 Aarhus, Denmark
| | - Brett A Israels
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
- Department of Chemistry, Aarhus University, DK-8000, Aarhus, Denmark
| | - Aleksandra M Bednarz
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
- Department of Chemistry, Aarhus University, DK-8000, Aarhus, Denmark
| | - Claus Bus
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
| | - Benedikt Asbach
- Institute of Medical Microbiology and Hygiene/Molecular Microbiology (Virology), Regensburg University 93053 Regensburg, Germany
| | - David Peterhoff
- Institute of Medical Microbiology and Hygiene/Molecular Microbiology (Virology), Regensburg University 93053 Regensburg, Germany
| | - Finn S Pedersen
- Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark
| | - Victoria Birkedal
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark
- Department of Chemistry, Aarhus University, DK-8000, Aarhus, Denmark
| | - Ralf Wagner
- Institute of Medical Microbiology and Hygiene/Molecular Microbiology (Virology), Regensburg University 93053 Regensburg, Germany
- Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, Regensburg 93053, Germany
| | - Søren R Paludan
- Department of Biomedicine, Aarhus University DK-8000 Aarhus, Denmark
| | - Jørgen Kjems
- Interdisciplinary Nanoscience Center, Aarhus University DK-8000 Aarhus, Denmark;
- Centre for Cellular Signal Patterns (CellPAT), Aarhus University, Aarhus DK-8000, Denmark
- Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus, Denmark
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48
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Fàbrega C, Aviñó A, Eritja R. Chemical Modifications in Nucleic Acids for Therapeutic and Diagnostic Applications. CHEM REC 2021; 22:e202100270. [DOI: 10.1002/tcr.202100270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Revised: 11/26/2021] [Accepted: 11/26/2021] [Indexed: 11/08/2022]
Affiliation(s)
- Carme Fàbrega
- Department of Surfactants and Nanobiotechnology Institute for Advanced Chemistry of Catalonia (IQAC) Spanish National Research Council (CSIC) Jordi Girona 18–26 E-08034 Barcelona Spain
- Networking Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) E-08034 Barcelona Spain
| | - Anna Aviñó
- Department of Surfactants and Nanobiotechnology Institute for Advanced Chemistry of Catalonia (IQAC) Spanish National Research Council (CSIC) Jordi Girona 18–26 E-08034 Barcelona Spain
- Networking Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) E-08034 Barcelona Spain
| | - Ramon Eritja
- Department of Surfactants and Nanobiotechnology Institute for Advanced Chemistry of Catalonia (IQAC) Spanish National Research Council (CSIC) Jordi Girona 18–26 E-08034 Barcelona Spain
- Networking Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) E-08034 Barcelona Spain
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49
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Novel approaches in cancer treatment: preclinical and clinical development of small non-coding RNA therapeutics. J Exp Clin Cancer Res 2021; 40:383. [PMID: 34863235 PMCID: PMC8642961 DOI: 10.1186/s13046-021-02193-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Accepted: 11/23/2021] [Indexed: 11/20/2022] Open
Abstract
Short or small interfering RNAs (siRNAs) and microRNA (miRNAs) are molecules similar in size and function able to inhibit gene expression based on their complementarity with mRNA sequences, inducing the degradation of the transcript or the inhibition of their translation. siRNAs bind specifically to a single gene location by sequence complementarity and regulate gene expression by specifically targeting transcription units via posttranscriptional gene silencing. miRNAs can regulate the expression of different gene targets through their imperfect base pairing. This process - known as RNA interference (RNAi) - modulates transcription in order to maintain a correct physiological environment, playing a role in almost the totality of the cellular pathways. siRNAs have been evolutionary evolved for the protection of genome integrity in response to exogenous and invasive nucleic acids such as transgenes or transposons. Artificial siRNAs are widely used in molecular biology for transient silencing of genes of interest. This strategy allows to inhibit the expression of any target protein of known sequence and is currently used for the treatment of different human diseases including cancer. Modifications and rearrangements in gene regions encoding for miRNAs have been found in cancer cells, and specific miRNA expression profiles characterize the developmental lineage and the differentiation state of the tumor. miRNAs with different expression patterns in tumors have been reported as oncogenes (oncomirs) or tumor-suppressors (anti-oncomirs). RNA modulation has become important in cancer research not only for development of early and easy diagnosis tools but also as a promising novel therapeutic approach. Despite the emerging discoveries supporting the role of miRNAs in carcinogenesis and their and siRNAs possible use in therapy, a series of concerns regarding their development, delivery and side effects have arisen. In this review we report the biology of miRNAs and siRNAs in relation to cancer summarizing the recent methods described to use them as novel therapeutic drugs and methods to specifically deliver them to cancer cells and overcome the limitations in the use of these molecules.
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50
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Ayyar VS, Song D, Zheng S, Carpenter T, Heald DL. Minimal Physiologically Based Pharmacokinetic-Pharmacodynamic (mPBPK-PD) Model of N-Acetylgalactosamine-Conjugated Small Interfering RNA Disposition and Gene Silencing in Preclinical Species and Humans. J Pharmacol Exp Ther 2021; 379:134-146. [PMID: 34413198 DOI: 10.1124/jpet.121.000805] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2021] [Accepted: 08/16/2021] [Indexed: 12/20/2022] Open
Abstract
Conjugation of small interfering RNA (siRNA) to tris N-acetylgalactosamine [(GalNAc)3] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (∼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris N-acetylgalactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.
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Affiliation(s)
- Vivaswath S Ayyar
- Clinical Pharmacology & Pharmacometrics (V.S.A., D.S.) and Janssen BioTherapeutics (V.S.A., S.Z., T.C., D.L.H.), Janssen Research and Development, Spring House, Pennsylvania
| | - Dawei Song
- Clinical Pharmacology & Pharmacometrics (V.S.A., D.S.) and Janssen BioTherapeutics (V.S.A., S.Z., T.C., D.L.H.), Janssen Research and Development, Spring House, Pennsylvania
| | - Songmao Zheng
- Clinical Pharmacology & Pharmacometrics (V.S.A., D.S.) and Janssen BioTherapeutics (V.S.A., S.Z., T.C., D.L.H.), Janssen Research and Development, Spring House, Pennsylvania
| | - Thomas Carpenter
- Clinical Pharmacology & Pharmacometrics (V.S.A., D.S.) and Janssen BioTherapeutics (V.S.A., S.Z., T.C., D.L.H.), Janssen Research and Development, Spring House, Pennsylvania
| | - Donald L Heald
- Clinical Pharmacology & Pharmacometrics (V.S.A., D.S.) and Janssen BioTherapeutics (V.S.A., S.Z., T.C., D.L.H.), Janssen Research and Development, Spring House, Pennsylvania
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