|
1
|
Venkatakrishnan J, Yuan Y, Zhang J, Yu Y, Hu YC, Kao WWY. Self-complementary AAV vector therapy for treating corneal cloudiness of mucopolysaccharidosis type VII (MPS VII). Ocul Surf 2024; 32:39-47. [PMID: 38218582 DOI: 10.1016/j.jtos.2024.01.002] [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: 08/07/2023] [Revised: 11/26/2023] [Accepted: 01/06/2024] [Indexed: 01/15/2024]
Abstract
PURPOSE To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the β-Glu gene (Gusb allele). METHODS β-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas. RESULTS Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine β-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of β-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection. CONCLUSION Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.
Collapse
Affiliation(s)
- Jhuwala Venkatakrishnan
- Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA; Department of Biomedical Engineering, University of Cincinnati, OH, USA
| | - Yong Yuan
- Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA
| | - Jianhua Zhang
- Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA
| | - Yang Yu
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, OH, USA
| | - Yueh-Chiang Hu
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati, OH, USA
| | - Winston W-Y Kao
- Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, USA.
| |
Collapse
|
|
2
|
Akram F, Waheed HM, Shah FI, Haq IU, Nasir N, Akhtar MT, Farooq Gohar U. Burgeoning therapeutic strategies to curb the contemporary surging viral infections. Microb Pathog 2023; 179:106088. [PMID: 37004965 DOI: 10.1016/j.micpath.2023.106088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2022] [Revised: 03/23/2023] [Accepted: 03/24/2023] [Indexed: 04/03/2023]
Abstract
Significant efforts and initiatives were already made in the health care systems, however in the last few years; our world is facing emergences of viral infections which potentially leading to considerable challenges in terms of higher morbidity, mortality, increased and considerable financial loads on the affected populations. Over ten major epidemics or pandemics have been recorded in the twenty-first century, the ongoing coronavirus pandemic being one of them. Viruses being distinct obligate pathogens largely dependent on living beings are considered as one of the prominent causes of death globally. Although effective vaccines and antivirals have led to the eradication of imperative viral pathogens, the emergences of new viral infections as well as novel drug-resistant strains have necessitated the implementation of ingenious and efficient therapeutic approaches to treat viral outbreaks in the future. Nature being a constant source of tremendous therapeutical resources has inspired us to develop multi-target antiviral drugs, overcoming the challenges and limitations faced by pharmaceutical industry. Recent breakthroughs in the understanding of the cellular and molecular mechanisms of viral reproduction have laid the groundwork for potential treatment approaches including antiviral gene therapy relying on the application of precisely engineered nucleic acids for disabling pathogen replication. The development of RNA interference and advancements in genome manipulating tools have proven to be especially significant in this regard. In this review, we discussed mode of actions and pathophysiological events associated with the viral infections; followed by distributions, and advancement made towards the detection strategies for timely diagnosis. In the later section, current approaches to cope up the viral pathogens and their key limitations have also been elaborated. Lastly, we also explored some novel and potential targets to treat such infections, where attentions were made on next generation gene editing technologies.
Collapse
|
|
3
|
Grixti JM, Ayers D, Day PJR. An Analysis of Mechanisms for Cellular Uptake of miRNAs to Enhance Drug Delivery and Efficacy in Cancer Chemoresistance. Noncoding RNA 2021; 7:27. [PMID: 33923485 PMCID: PMC8167612 DOI: 10.3390/ncrna7020027] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2021] [Revised: 04/11/2021] [Accepted: 04/13/2021] [Indexed: 12/15/2022] Open
Abstract
Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, with the most notable one being the safe and effective delivery of the miRNA mimic/antagonist safely to the target cell cytoplasm for attaining the desired clinical outcome, particularly in miRNA-based cancer therapeutics, due to the poor efficiency of neo-vascular systems revolting around the tumour site, brought about by tumour-induced angiogenesis. This acquisition of resistance to several types of anticancer drugs can be as a result of an upregulation of efflux transporters expression, which eject drugs from cells, hence lowering drug efficacy, resulting in multidrug resistance. In this article, the latest available data on human microRNAs has been reviewed, together with the most recently described mechanisms for miRNA uptake in cells, for future therapeutic enhancements against cancer chemoresistance.
Collapse
Affiliation(s)
- Justine M. Grixti
- Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK;
| | - Duncan Ayers
- Centre for Molecular Medicine and Biobanking, University of Malta, Msida MSD 2080, Malta
- Faculty of Biology, Medicine and Human Sciences, The University of Manchester, Manchester M1 7DN, UK;
| | - Philip J. R. Day
- Faculty of Biology, Medicine and Human Sciences, The University of Manchester, Manchester M1 7DN, UK;
| |
Collapse
|
|
4
|
Kang MH, Hu J, Pratt RE, Hodgkinson CP, Asokan A, Dzau VJ. Optimizing delivery for efficient cardiac reprogramming. Biochem Biophys Res Commun 2020; 533:9-16. [PMID: 32917363 DOI: 10.1016/j.bbrc.2020.08.104] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2020] [Accepted: 08/27/2020] [Indexed: 12/14/2022]
Abstract
Following heart injury, cardiomyocytes, are lost and are not regenerated. In their place, fibroblasts invade the dead tissue where they generate a scar, which reduces cardiac function. We and others have demonstrated that combinations of specific miRNAs (miR combo) or transcription factors (GMT), delivered by individual lenti-/retro-viruses in vivo, can convert fibroblasts into cardiomyocytes and improve cardiac function. However, the effects are relatively modest due to the low efficiency of delivery of miR combo or GMT. We hypothesized that efficiency would be improved by optimizing delivery. In the first instance, we developed a multicistronic system to express all four miRNAs of miR combo from a single construct. The order of each miRNA in the multicistronic construct gave rise to different levels of miRNA expression. A combination that resulted in equivalent expression levels of each of the four miRNAs of miR combo showed the highest reprogramming efficiency. Further efficiency can be achieved by directly targeting fibroblasts. Screening of several AAV serotypes indicated that AAV1 displayed tropism towards cardiac fibroblasts. Combining multicistronic expression with AAV1 delivery robustly reprogrammed cardiac fibroblasts into cardiomyocytes in vivo.
Collapse
Affiliation(s)
- Martin H Kang
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA
| | - Jiabiao Hu
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA
| | - Richard E Pratt
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA
| | - Conrad P Hodgkinson
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA
| | - Aravind Asokan
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA
| | - Victor J Dzau
- Mandel Center for Heart and Vascular Research, and the Duke Cardiovascular Research Center, Duke University Medical Center, Durham, NC, 27710, USA.
| |
Collapse
|
|
5
|
Zhang X, Gao X, Hu J, Xie Y, Zuo Y, Xu H, Zhu S. ADAR1p150 Forms a Complex with Dicer to Promote miRNA-222 Activity and Regulate PTEN Expression in CVB3-Induced Viral Myocarditis. Int J Mol Sci 2019; 20:ijms20020407. [PMID: 30669342 PMCID: PMC6359435 DOI: 10.3390/ijms20020407] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2018] [Revised: 01/02/2019] [Accepted: 01/04/2019] [Indexed: 02/06/2023] Open
Abstract
Adenosine deaminases acting on RNA (ADAR) are enzymes that regulate RNA metabolism through post-transcriptional mechanisms. ADAR1 is involved in a variety of pathological conditions including inflammation, cancer, and the host defense against viral infections. However, the role of ADAR1p150 in vascular disease remains unclear. In this study, we examined the expression of ADAR1p150 and its role in viral myocarditis (VMC) in a mouse model. VMC mouse cardiomyocytes showed significantly higher expression of ADAR1p150 compared to the control samples. Coimmunoprecipitation verified that ADAR1p150 forms a complex with Dicer in VMC. miRNA-222, which is involved in many cardiac diseases, is highly expressed in cardiomyocytes in VMC. In addition, the expression of miRNA-222 was promoted by ADAR1p150/Dicer. Among the target genes of miRNA-222, the expression of phosphatase-and-tensin (PTEN) protein was significantly reduced in VMC. By using a bioinformatics tool, we found a potential binding site of miRNA-222 on the PTEN gene’s 3′-UTR, suggesting that miRNA-222 might play a regulatory role. In cultured cells, miR-222 suppressed PTEN expression. Our findings suggest that ADAR1p150 plays a key role in complexing with Dicer and promoting the expression of miRNA-222, the latter of which suppresses the expression of the target gene PTEN during VMC. Our work reveals a previously unknown role of ADAR1p150 in gene expression in VMC.
Collapse
Affiliation(s)
- Xincai Zhang
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| | - Xiangting Gao
- Department of Pathology, School of Medicine, Shihezi University, Shihezi 215021, China.
| | - Jun Hu
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| | - Yuxin Xie
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| | - Yuanyi Zuo
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| | - Hongfei Xu
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| | - Shaohua Zhu
- Institute of Forensic Medicine, Soochow University, Suzhou 215021, China.
| |
Collapse
|
|
6
|
Yang X, Wang Y, Shen X, Su C, Yang J, Piao M, Jia F, Gao G, Zhang L, Lin Q. One-step synthesis of photoluminescent carbon dots with excitation-independent emission for selective bioimaging and gene delivery. J Colloid Interface Sci 2017; 492:1-7. [DOI: 10.1016/j.jcis.2016.12.057] [Citation(s) in RCA: 74] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2016] [Revised: 12/21/2016] [Accepted: 12/22/2016] [Indexed: 11/16/2022]
|
|
7
|
Bourhill T, Arbuthnot P, Ely A. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics. J Virol Methods 2016; 235:26-33. [PMID: 27181212 DOI: 10.1016/j.jviromet.2016.05.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2016] [Revised: 04/16/2016] [Accepted: 05/11/2016] [Indexed: 02/05/2023]
Abstract
Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.
Collapse
Affiliation(s)
- Tarryn Bourhill
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa
| | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Science, University of the Witwatersrand, Johannesburg, South Africa.
| |
Collapse
|
|
8
|
Tsai MM, Wang CS, Tsai CY, Huang HW, Chi HC, Lin YH, Lu PH, Lin KH. Potential Diagnostic, Prognostic and Therapeutic Targets of MicroRNAs in Human Gastric Cancer. Int J Mol Sci 2016; 17:945. [PMID: 27322246 PMCID: PMC4926478 DOI: 10.3390/ijms17060945] [Citation(s) in RCA: 102] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2016] [Revised: 06/01/2016] [Accepted: 06/07/2016] [Indexed: 12/11/2022] Open
Abstract
Human gastric cancer (GC) is characterized by a high incidence and mortality rate, largely because it is normally not identified until a relatively advanced stage owing to a lack of early diagnostic biomarkers. Gastroscopy with biopsy is the routine method for screening, and gastrectomy is the major therapeutic strategy for GC. However, in more than 30% of GC surgical patients, cancer has progressed too far for effective medical resection. Thus, useful biomarkers for early screening or detection of GC are essential for improving patients' survival rate. MicroRNAs (miRNAs) play an important role in tumorigenesis. They contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors. Because of their stability in tissues, serum/plasma and other body fluids, miRNAs have been suggested as novel tumor biomarkers with suitable clinical potential. Recently, aberrantly expressed miRNAs have been identified and tested for clinical application in the management of GC. Aberrant miRNA expression profiles determined with miRNA microarrays, quantitative reverse transcription-polymerase chain reaction and next-generation sequencing approaches could be used to establish sample specificity and to identify tumor type. Here, we provide an up-to-date summary of tissue-based GC-associated miRNAs, describing their involvement and that of their downstream targets in tumorigenic and biological processes. We examine correlations among significant clinical parameters and prognostic indicators, and discuss recurrence monitoring and therapeutic options in GC. We also review plasma/serum-based, GC-associated, circulating miRNAs and their clinical applications, focusing especially on early diagnosis. By providing insights into the mechanisms of miRNA-related tumor progression, this review will hopefully aid in the identification of novel potential therapeutic targets.
Collapse
Affiliation(s)
- Ming-Ming Tsai
- Department of Nursing, Chang-Gung University of Science and Technology, Taoyuan 333, Taiwan.
- Department of General Surgery, Chang Gung Memorial Hospital, Chiayi 613, Taiwan.
| | - Chia-Siu Wang
- Department of General Surgery, Chang Gung Memorial Hospital, Chiayi 613, Taiwan.
| | - Chung-Ying Tsai
- Department of Biochemistry, College of Medicine, Chang-Gung University, Taoyuan 333, Taiwan.
| | - Hsiang-Wei Huang
- Department of Biochemistry, College of Medicine, Chang-Gung University, Taoyuan 333, Taiwan.
| | - Hsiang-Cheng Chi
- Department of Biochemistry, College of Medicine, Chang-Gung University, Taoyuan 333, Taiwan.
| | - Yang-Hsiang Lin
- Department of Biochemistry, College of Medicine, Chang-Gung University, Taoyuan 333, Taiwan.
| | - Pei-Hsuan Lu
- Department of Dermatology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan 333, Taiwan.
| | - Kwang-Huei Lin
- Department of Biochemistry, College of Medicine, Chang-Gung University, Taoyuan 333, Taiwan.
- Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan 333, Taiwan.
| |
Collapse
|
|
9
|
Louten J, Beach M, Palermino K, Weeks M, Holenstein G. MicroRNAs Expressed during Viral Infection: Biomarker Potential and Therapeutic Considerations. Biomark Insights 2016; 10:25-52. [PMID: 26819546 PMCID: PMC4718089 DOI: 10.4137/bmi.s29512] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 10/22/2015] [Accepted: 10/24/2015] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) are short sequences of noncoding single-stranded RNAs that exhibit inhibitory effects on complementary target mRNAs. Recently, it has been discovered that certain viruses express their own miRNAs, while other viruses activate the transcription of cellular miRNAs for their own benefit. This review summarizes the viral and/or cellular miRNAs that are transcribed during infection, with a focus on the biomarker and therapeutic potential of miRNAs (or their antagomirs). Several human viruses of clinical importance are discussed, namely, herpesviruses, polyomaviruses, hepatitis B virus, hepatitis C virus, human papillomavirus, and human immunodeficiency virus.
Collapse
Affiliation(s)
- Jennifer Louten
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Michael Beach
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Kristina Palermino
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Maria Weeks
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Gabrielle Holenstein
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| |
Collapse
|
|
10
|
van den Berg FT, Rossi JJ, Arbuthnot P, Weinberg MS. Design of Effective Primary MicroRNA Mimics With Different Basal Stem Conformations. MOLECULAR THERAPY-NUCLEIC ACIDS 2016; 5:e278. [PMID: 26756196 PMCID: PMC5012551 DOI: 10.1038/mtna.2015.53] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2015] [Accepted: 11/30/2015] [Indexed: 12/03/2022]
Abstract
Primary microRNA (pri-miRNA) mimics are important mediators of effective gene silencing and are well suited for sustained therapeutic applications. Pri-miRNA mimics are processed in the endogenous miRNA biogenesis pathway, where elements of the secondary RNA structure are crucial for efficient miRNA production. Cleavage of the pri-miRNA to a precursor miRNA (pre-miRNA) by Drosha-DGCR8 typically occurs adjacent to a basal stem of ~11 bp. However, a number of pri-miRNA structures are expected to contain slightly shorter or longer basal stems, which may be further disrupted in predicted folding of the expressed pri-miRNA sequence. We investigated the function and processing of natural and exogenous RNA guides from pri-miRNAs with various basal stems (9–13 bp), where a canonical hairpin was predicted to be well or poorly maintained in predicted structures of the expressed sequence. We have shown that RNA guides can be effectively derived from pri-miRNAs with various basal stem conformations, while predicted guide region stability can explain the function of pri-miRNA mimics, in agreement with previously proposed design principles. This study provides insight for the design of effective mimics based on naturally occurring pri-miRNAs and has identified several novel scaffolds suitable for use in gene silencing applications.
Collapse
Affiliation(s)
- Fiona T van den Berg
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa
| | - John J Rossi
- Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, USA
| | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa
| | - Marc S Weinberg
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.,HIV Pathogenesis Research Unit, Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa.,Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA
| |
Collapse
|
|
11
|
|
|
12
|
Chen H. Adeno-associated virus vectors for human gene therapy. World J Med Genet 2015; 5:28-45. [DOI: 10.5496/wjmg.v5.i3.28] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Revised: 04/08/2015] [Accepted: 05/18/2015] [Indexed: 02/06/2023] Open
Abstract
Adeno-associated virus (AAV) is a small, non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms, especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation, column chromatography, or a combination, have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road, such as cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients, the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants, and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.
Collapse
|
|
13
|
Battistella M, Marsden PA. Advances, nuances, and potential pitfalls when exploiting the therapeutic potential of RNA interference. Clin Pharmacol Ther 2015; 97:79-87. [PMID: 25670385 DOI: 10.1002/cpt.8] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2014] [Accepted: 10/25/2014] [Indexed: 12/12/2022]
Abstract
The discovery of RNA interference (RNAi) holds the potential to alter the paradigm of medical therapeutics. With the ability to selectively silence the function of a gene, RNAi not only provides an indispensable research tool for determining the function of a gene, but also offers potential for the development of novel therapeutics that will inhibit specific genes involved in disease. New concepts in therapeutics have been uncovered through the study of RNAi. Nuances have emerged. For instance, global RNAi pathways can be affected by somatic mutations in cancer and cellular stress, such as hypoxia. Also, viral gene therapy can have unexpected effects on endogenous short noncoding RNA pathways. Therefore, it is important to understand where RNAi therapeutics enter the processing pathways. We highlight the evolving use of RNAi as a new class of therapeutics, such as for amyloidosis, and address some of the anticipated challenges associated with its clinical application.
Collapse
Affiliation(s)
- M Battistella
- University Health Network and University of Toronto, Toronto, Ontario, Canada
| | | |
Collapse
|
|
14
|
Catanese MT, Dorner M. Advances in experimental systems to study hepatitis C virus in vitro and in vivo. Virology 2015; 479-480:221-33. [PMID: 25847726 DOI: 10.1016/j.virol.2015.03.014] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 02/04/2015] [Accepted: 03/03/2015] [Indexed: 12/25/2022]
Abstract
Hepatitis C virus (HCV) represents a global health concern affecting over 185 million people worldwide. Chronic HCV infection causes liver fibrosis and cirrhosis and is the leading indication for liver transplantation. Recent advances in the field of direct-acting antiviral drugs (DAAs) promise a cure for HCV in over 90% of cases that will get access to these expensive treatments. Nevertheless, the lack of a protective vaccine and likely emergence of drug-resistant viral variants call for further studies of HCV biology. With chimpanzees being for a long time the only non-human in vivo model of HCV infection, strong efforts were put into establishing in vitro experimental systems. The initial models only enabled to study specific aspects of the HCV life cycle, such as viral replication with the subgenomic replicon and entry using HCV pseudotyped particles (HCVpp). Subsequent development of protocols to grow infectious HCV particles in cell-culture (HCVcc) ignited investigations on the full cycle of HCV infection and the virus-host interactions required for virus propagation. More recently, small animal models permissive to HCV were generated that allowed in vivo testing of novel antiviral therapies as well as vaccine candidates. This review provides an overview of the currently available in vitro and in vivo experimental systems to study HCV biology. Particular emphasis is given to how these model systems furthered our understanding of virus-host interactions, viral pathogenesis and immunological responses to HCV infection, as well as drug and vaccine development.
Collapse
Affiliation(s)
| | - Marcus Dorner
- Section of Virology, Imperial College London, London, United Kingdom; Section of Hepatology, Imperial College London, London, United Kingdom.
| |
Collapse
|
|
15
|
Jiang XH, Xie YT, Jiang B, Tang MY, Ma T, Peng H. Inhibition of expression of hepatitis C virus 1b genotype core and NS4B genes in HepG2 cells using artificial microRNAs. Mol Med Rep 2015; 12:1905-13. [PMID: 25847260 DOI: 10.3892/mmr.2015.3571] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Accepted: 02/05/2015] [Indexed: 11/06/2022] Open
Abstract
The present study aimed to evaluate the silencing effect of artificial microRNAs (amiRNAs) against the hepatitis C virus (HCV) 1b (HCV1b) genotype core (C) and non-structural protein 4B (NS4B) genes. pDsRed-monomer-Core and pDsRed-monomer-NS4B plasmids, containing the target genes were constructed. A total of eight artificial micro RNA (amiRNA)-expressing plasmids, namely, pmiRE-C-mi1 to -mi4 and pmiRE-NS4B-mi1 to -mi4, were designed and constructed to interfere with various sites of the core and NS4B genes, and the amiRNA interfering plasmid and the corresponding target gene-expressing plasmid were co-transfected into HepG2 cells. At 48 h after transfection, HCV core and NS4B gene expression levels were detected using fluorescence microscopy, flow cytometry, reverse transcription quantitative polymerase chain reaction and western blot analysis. Fluorescence microscopy revealed that the target gene-transfected cells expressed red fluorescent protein, whereas the interfering plasmid-transfected cells exhibited expression of green fluorescent protein. The percentage of red fluorescent proteins and mean fluorescence intensity, as well as protein expression levels of the core and NS4B genes within the cells, which were co-transfected by the amiRNA interfering plasmid and the target gene, were significantly decreased. The results of the present study confirmed that amiRNAs may effectively and specifically inhibit the expression of HCV1b core and NS4B genes in HepG2 cells, potentially providing a novel therapeutic strategy for the treatment of HCV.
Collapse
Affiliation(s)
- Xiao-Hua Jiang
- Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, Hunan 410087, P.R. China
| | - Yu-Tao Xie
- Department of Infectious Diseases, Xiangya Hospital of Central South University, Changsha, Hunan 410087, P.R. China
| | - Bo Jiang
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Meng-Ying Tang
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Tao Ma
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| | - Hua Peng
- Department of Infectious Diseases, The First Affiliated Hospital of The University of South China, Hengyang, Hunan 421001, P.R. China
| |
Collapse
|
|
16
|
Arbuthnot P. Antiviral Gene Therapy. GENE THERAPY FOR VIRAL INFECTIONS 2015. [PMCID: PMC7149688 DOI: 10.1016/b978-0-12-410518-8.00012-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Antiviral gene therapy conveniently relies on use of rationally designed nucleic acids to disable replication of the pathogens. Popular targets for this therapy have been sequences encoding viral proteins and cellular host factors on which viruses depend for their replication. Therefore, availability of technologies that inactivate genes specifically, safely and effectively is crucial. Hence, discovery of RNA interference and advances in gene editing methods have been particularly important to the field. Gene transfer to induce prophylactic and therapeutic immunostimulation is also being developed to manage viral infections. Although promising, progress and ultimate success of antiviral gene therapy is dependent on several factors. These include overcoming problems that are generic to the field and influences of elements that are particular to specific viral infections. Viral and nonviral vectors (NVVs) have been used to deliver antiviral sequences, and both have advantages and drawbacks. Viral vectors are suited to delivering DNA-based expression cassettes, including immunostimulatory and virus-targeting sequences, whereas NVVs may be better applied to carrying therapeutic RNAs such as synthetic short interfering RNAs or mRNAs. Nonviral formulations are appealing because they cause limited immunostimulation, obviate mutagenic effects of DNA, and are amenable to easier dose control and large-scale synthesis. To date, encouraging results have been obtained in preclinical and clinical assessments of gene therapy against HIV-1, hepatitis B, hepatitis C, and Ebola viruses, among others. In the long term, widespread application of antiviral gene therapy will be dependent on the technology demonstrating good efficacy and advantages over other antiviral strategies. Access to appropriate material and human resources that are required for implementation of the technologies in various settings will also be important.
Collapse
|
|
17
|
Alexander MS, Kunkel LM. Skeletal Muscle MicroRNAs: Their Diagnostic and Therapeutic Potential in Human Muscle Diseases. J Neuromuscul Dis 2015; 2:1-11. [PMID: 27547731 PMCID: PMC4988517 DOI: 10.3233/jnd-140058] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
MicroRNAs (miRNAs) are small 21-24 nucleotide RNAs that are capable of regulating multiple signaling pathways across multiple tissues. MicroRNAs are dynamically regulated and change in expression levels during periods of early development, tissue regeneration, cancer, and various other disease states. Recently, microRNAs have been isolated from whole serum and muscle biopsies to identify unique diagnostic signatures for specific neuromuscular disease states. Functional studies of microRNAs in cell lines and animal models of neuromuscular diseases have elucidated their importance in contributing to neuromuscular disease progression and pathologies. The ability of microRNAs to alter the expression of an entire signaling pathway opens up their unique ability to be used as potential therapeutic entry points for the treatment of disease. Here, we will review the recent findings of key microRNAs and their dysregulation in various neuromuscular diseases. Additionally, we will highlight the current strategies being used to regulate the expression of key microRNAs as they have become important players in the clinical treatment of some of the neuromuscular diseases.
Collapse
Affiliation(s)
- Matthew S Alexander
- Division of Genetics and Genomics at Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics and Genetics at Harvard Medical School, Boston, MA, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA, USA
| | - Louis M Kunkel
- Division of Genetics and Genomics at Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics and Genetics at Harvard Medical School, Boston, MA, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA, USA; The Manton Center for Orphan Disease Research at Boston Children's Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA
| |
Collapse
|
|
18
|
Lee CH, Kim JH, Lee SW. Prospects for nucleic acid-based therapeutics against hepatitis C virus. World J Gastroenterol 2013; 19:8949-8962. [PMID: 24379620 PMCID: PMC3870548 DOI: 10.3748/wjg.v19.i47.8949] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2013] [Revised: 11/10/2013] [Accepted: 11/30/2013] [Indexed: 02/06/2023] Open
Abstract
In this review, we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus (HCV) infection. Because the HCV genome is present exclusively in RNA form during replication, various nucleic acid-based therapeutic approaches targeting the HCV genome, such as ribozymes, aptamers, siRNAs, and antisense oligonucleotides, have been suggested as potential tools against HCV. Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics. These limitations have hampered the clinical development of nucleic acid-based therapeutics. However, despite these limitations, nucleic acid-based therapeutics has clinical value due to their great specificity, easy and large-scale synthesis with chemical methods, and pharmaceutical flexibility. Moreover, nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle, and therefore they may prove to be more effective than existing therapeutics, such as interferon-α and ribavirin combination therapy. This review focuses on the current status and future prospects of ribozymes, aptamers, siRNAs, and antisense oligonucleotides as therapeutic reagents against HCV.
Collapse
|
|
19
|
Tao Y, Li Z, Ju E, Ren J, Qu X. Polycations-functionalized water-soluble gold nanoclusters: a potential platform for simultaneous enhanced gene delivery and cell imaging. NANOSCALE 2013; 5:6154-60. [PMID: 23727891 DOI: 10.1039/c3nr01326j] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
Noble metal nanoclusters have emerged as a fascinating area of widespread interest in nanomaterials. Herein, we report the synthesis of the PEI-templated gold nanoclusters (PEI-AuNCs) as an efficient carrier for gene delivery. The PEI-AuNCs integrate the advantages of PEI and AuNCs: the presence of AuNCs can effectively decrease the cytotoxicity of PEI, making it possible to apply them in biological systems, while the cationic polymer layer PEI with positive charges is essential for enhanced gene transfection efficiency. In addition, with excellent photoluminescent properties, the AuNCs also endow our system with the versatility of fluorescent imaging, indicating a great potential as an ideal fluorescent probe to track the transfection behavior. Our studies provide strong evidence that the PEI-AuNCs can be utilized as efficient gene delivery agents.
Collapse
Affiliation(s)
- Yu Tao
- State Key laboratory of Rare Earth Resources Utilization and Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China
| | | | | | | | | |
Collapse
|
|
20
|
Gao M, Yin H, Fei ZW. Clinical application of microRNA in gastric cancer in Eastern Asian area. World J Gastroenterol 2013; 19:2019-2027. [PMID: 23599620 PMCID: PMC3623978 DOI: 10.3748/wjg.v19.i13.2019] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/12/2012] [Revised: 11/28/2012] [Accepted: 12/25/2012] [Indexed: 02/06/2023] Open
Abstract
Recent research has shown that microRNA (miRNA), which is involved in almost every step of gastric carcinogenesis, has broad prospective application in diagnosis and therapy of gastric carcinoma. Eastern Asia (South Korea, Japan and China) has the highest incidence of gastric cancer in the world. There were 988 000 new cases of gastric cancer worldwide and 736 000 deaths in 2008. Approximately 60% of the cases of gastric cancer are found in East Asia (mainly China). We herein provide a brief review of the clinical applications of miRNA, which include the following aspects: (1) miRNA may serve as a potential new generation of tumor markers; (2) a complete miRNA expression profile is highly specific, can reflect the evolutionary lineage and differentiation of tumors, and be used to carry out diversity analysis; (3) detecting specific miRNA expression in peripheral blood will become a new method for diagnosis of gastric cancer; (4) miRNA can predict prognosis of gastric cancer; (5) miRNA has predictive value in determining chemotherapy and radiotherapy resistance; and (6) miRNA could be a type of innovative drug. Finally, we focus on assessing the value of miRNA from laboratory to clinical application and the challenges it faces in East Asia.
Collapse
|
|
21
|
Lisowski L, Elazar M, Chu K, Glenn JS, Kay MA. The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA. Nucleic Acids Res 2013; 41:3688-98. [PMID: 23396439 PMCID: PMC3616702 DOI: 10.1093/nar/gkt068] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.
Collapse
Affiliation(s)
- Leszek Lisowski
- Department of Pediatrics, School of Medicine, Stanford University, 269 Pasteur Drive, Stanford, CA 94305, USA
| | | | | | | | | |
Collapse
|
|
22
|
All for one, one for all: new combinatorial RNAi therapies combat hepatitis C virus evolution. Mol Ther 2013; 20:1661-3. [PMID: 22945230 DOI: 10.1038/mt.2012.173] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
|
|
23
|
Yang X, Marcucci K, Anguela X, Couto LB. Preclinical evaluation of an anti-HCV miRNA cluster for treatment of HCV infection. Mol Ther 2013; 21:588-601. [PMID: 23295950 DOI: 10.1038/mt.2012.247] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17-92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection.
Collapse
Affiliation(s)
- Xiao Yang
- Children's Hospital of Philadelphia, Philadelphia, PA, USA
| | | | | | | |
Collapse
|
|
24
|
Tao Y, Ju E, Ren J, Qu X. Metallization of plasmid DNA for efficient gene delivery. Chem Commun (Camb) 2013; 49:9791-3. [DOI: 10.1039/c3cc45834b] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
|
|
25
|
Abstract
Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficult-to-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different effector molecule formats, promoters, and vector types, has meant that experiments can be tailored to target specific cell types and minimize cellular toxicities. Through the application of combinatorial RNAi (co-RNAi), multiple shRNA delivery strategies can improve gene knockdown, permit multiple transcripts to be targeted simultaneously, and curtail the emergence of viral escape mutants. This chapter reviews the history, cellular processing, and various applications of shRNAs in mammalian systems, including options for effector molecule design, vector and promoter types, and methods for multiple shRNA delivery.
Collapse
Affiliation(s)
- Luke S Lambeth
- Murdoch Childrens Research Institute, Royal Childrens Hospital, Melbourne, VIC, Australia.
| | | |
Collapse
|
|
26
|
Pihlmann M, Askou AL, Aagaard L, Bruun GH, Svalgaard JD, Holm-Nielsen MH, Dagnaes-Hansen F, Bek T, Mikkelsen JG, Jensen TG, Corydon TJ. Adeno-associated virus-delivered polycistronic microRNA-clusters for knockdown of vascular endothelial growth factor in vivo. J Gene Med 2012; 14:328-38. [PMID: 22438271 DOI: 10.1002/jgm.2623] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors. METHODS Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR. RESULTS Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF. CONCLUSIONS We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters.
Collapse
Affiliation(s)
- Maria Pihlmann
- Department of Biomedicine, Aarhus University, and Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
27
|
Bian Z, Xiao A, Cao M, Liu M, Liu S, Jiao Y, Yan W, Qi Z, Zheng Z. Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70. Virol J 2012; 9:275. [PMID: 23158906 PMCID: PMC3534549 DOI: 10.1186/1743-422x-9-275] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Accepted: 11/12/2012] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA. RESULTS Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells. CONCLUSIONS Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.
Collapse
Affiliation(s)
- Zhongqi Bian
- Center for Infectious Diseases, Kunming General Hospital, PLA, 212 Daguan Rd, Kunming 650032, PR China.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Lakner AM, Steuerwald NM, Walling TL, Ghosh S, Li T, McKillop IH, Russo MW, Bonkovsky HL, Schrum LW. Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis. Hepatology 2012; 56:300-10. [PMID: 22278637 PMCID: PMC3342471 DOI: 10.1002/hep.25613] [Citation(s) in RCA: 171] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/01/2011] [Accepted: 01/07/2012] [Indexed: 12/13/2022]
Abstract
UNLABELLED Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. CONCLUSION miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.
Collapse
Affiliation(s)
- Ashley M. Lakner
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
| | - Nury M. Steuerwald
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
| | - Tracy L. Walling
- Department of General Surgery, Carolinas Medical Center, Charlotte, NC
| | - Sriparna Ghosh
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
| | - Ting Li
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
| | - Iain H. McKillop
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC
- Department of General Surgery, Carolinas Medical Center, Charlotte, NC
| | - Mark W. Russo
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
- Department of Internal Medicine, Center for Liver and Transplantation, Carolinas Medical Center, Charlotte, NC
| | - Herbert L. Bonkovsky
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
- Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
- Department of Medicine, University of Connecticut Health Center, Farmington, CT
- Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT
| | - Laura W. Schrum
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC
- Department of Internal Medicine, Liver-Biliary-Pancreatic Center, Carolinas Medical Center, Charlotte, NC
| |
Collapse
|
|
29
|
Abstract
The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.
Collapse
|
|
30
|
Hoffmann TW, Duverlie G, Gilles D, Bengrine A, Abderrahmane B. MicroRNAs and hepatitis C virus: toward the end of miR-122 supremacy. Virol J 2012; 9:109. [PMID: 22691570 PMCID: PMC3489824 DOI: 10.1186/1743-422x-9-109] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2012] [Accepted: 05/30/2012] [Indexed: 12/11/2022] Open
Abstract
The most common etiologic agents causing chronic hepatitis are hepatitis C and B viruses (HCV and HBV, respectively). Chronic infection caused by HCV is considered one of the major causative agents of liver cirrhosis and hepatocellular carcinoma worldwide. In combination with the increasing rate of new HCV infections, the lack of a current vaccine and/or an effective treatment for this virus continues to be a major public health challenge. The development of new treatments requires a better understanding of the virus and its interaction with the different components of the host cell. MicroRNAs (miRNAs) are small non-coding RNAs functioning as negative regulators of gene expression and represent an interesting lead to study HCV infection and to identify new therapeutic targets. Until now, microRNA-122 (miR-122) and its implication in HCV infection have been the focus of different published studies and reviews. Here we will review recent advances in the relationship between HCV infection and miRNAs, showing that some of them emerge in publications as challengers against the supremacy of miR-122.
Collapse
Affiliation(s)
- Thomas Walter Hoffmann
- EA4294 Unité de Virologie Clinique et Fondamentale, Université de Picardie Jules Verne, UFR de Médecine et de Pharmacie, 3 rue des Louvels, 80036, Amiens Cedex, France
| | | | | | | | | |
Collapse
|
|
31
|
In vitro characterization of the activity of PF-05095808, a novel biological agent for hepatitis C virus therapy. Antimicrob Agents Chemother 2011; 56:1364-75. [PMID: 22203606 DOI: 10.1128/aac.05357-11] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assess in vitro activity are well defined; we developed new assays to assess this RNA interference agent and so to understand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNA molecules required for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.
Collapse
|
|
32
|
Raj D, Davidoff AM, Nathwani AC. Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges. Expert Rev Hematol 2011; 4:539-49. [PMID: 21939421 PMCID: PMC3200187 DOI: 10.1586/ehm.11.48] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated virus has recently shown promise. This article will briefly outline a novel gene-transfer approach using self-complementary adeno-associated viral vectors using hemophilia B as a target disorder. This approach is currently being evaluated in the clinic. We will provide an overview of the development of self-complementary adeno-associated virus vectors as well as preclinical and clinical data with this vector system.
Collapse
Affiliation(s)
- Deepak Raj
- Department of Haematology, University College London Cancer Institute, London, UK
| | - Andrew M Davidoff
- Department of Surgery, St Jude Children’s Research Hospital, Memphis, TN, USA
- Departments of Surgery and Pediatrics, University of Tennessee Health Science Center, Memphis, TN, USA
| | - Amit C Nathwani
- Department of Haematology, University College London Cancer Institute, London, UK
- NHS Blood and Transplant, London, UK
| |
Collapse
|
|
33
|
Hucke F, Sieghart W, Schöniger-Hekele M, Peck-Radosavljevic M, Müller C. Clinical characteristics of patients with hepatocellular carcinoma in Austria - is there a need for a structured screening program? Wien Klin Wochenschr 2011; 123:542-51. [PMID: 21800047 DOI: 10.1007/s00508-011-0033-9] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2011] [Accepted: 07/03/2011] [Indexed: 12/16/2022]
Abstract
BACKGROUND We investigated the differences in clinical presentation of patients with hepatocellular carcinoma (HCC) at the time of diagnosis, before and after the publication of the European Association for the study of the Liver (EASL) guidelines of HCC management and screening. METHODS Between 1991 and 2009, 907 patients were diagnosed with HCC at our department of which 850 were included in this study. Data regarding demography, liver function and tumor stage at the time of diagnosis were retrospectively collected. Differences in clinical characteristics and overall survival (OS) were compared before (period 1) and after (period 2) the publication of the EASL guidelines in 2001. RESULTS In period 2, patients were more likely to be overweight (BMI: 26.1 vs. 27.5, p = 0.003), suffered more often from diabetes (25.4 vs. 37.3%, p = 0.001) and nonalcoholic steatohepatitis (NASH) (0.7 vs. 5.1%, p < 0.001). Alcoholic liver disease replaced viral hepatitis as the main etiology but not in the increasing number of patients with migration background where viral hepatitis (76.3%) remained the predominant etiology. No change in liver function and tumor stages at the time of HCC diagnosis was observed. Most patients presented with advanced incurable HCC. However, the median OS of all HCC patients increased in period 2 (7 vs. 14 months, p < 0.001) suggesting improvements of palliative therapy. CONCLUSIONS Patients with HCC are still predominantly diagnosed at incurable tumor stages, despite explicit European screening guidelines existing since 9 years. The implementation of a HCC surveillance program for cirrhotic patients in Austria seems to be warranted.
Collapse
Affiliation(s)
- Florian Hucke
- Department of Gastroenterology and Hepatology, AKH and Medical University Vienna, Vienna, Austria
| | | | | | | | | |
Collapse
|
|
34
|
Liang GF, Zhu YL, Sun B, Hu FH, Tian T, Li SC, Xiao ZD. PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells. NANOSCALE RESEARCH LETTERS 2011; 6:447. [PMID: 21749688 PMCID: PMC3211866 DOI: 10.1186/1556-276x-6-447] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/14/2011] [Accepted: 07/12/2011] [Indexed: 05/28/2023]
Abstract
The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.
Collapse
Affiliation(s)
- Gao Feng Liang
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Yan Liang Zhu
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Bo Sun
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Fei Hu Hu
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Tian Tian
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Shu Chun Li
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| | - Zhong Dang Xiao
- State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China
| |
Collapse
|
|
35
|
Abstract
RNA interference (RNAi) is a powerful approach for reducing expression of endogenously expressed proteins. It is widely used for biological applications and is being harnessed to silence mRNAs encoding pathogenic proteins for therapy. Various methods - including delivering RNA oligonucleotides and expressing RNAi triggers from viral vectors - have been developed for successful RNAi in cell culture and in vivo. Recently, RNAi-based gene silencing approaches have been demonstrated in humans, and ongoing clinical trials hold promise for treating fatal disorders or providing alternatives to traditional small molecule therapies. Here we describe the broad range of approaches to achieve targeted gene silencing for therapy, discuss important considerations when developing RNAi triggers for use in humans, and review the current status of clinical trials.
Collapse
Affiliation(s)
- Beverly L Davidson
- Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
| | | |
Collapse
|
|
36
|
Kim MH, Na HK, Kim YK, Ryoo SR, Cho HS, Lee KE, Jeon H, Ryoo R, Min DH. Facile synthesis of monodispersed mesoporous silica nanoparticles with ultralarge pores and their application in gene delivery. ACS NANO 2011; 5:3568-3576. [PMID: 21452883 DOI: 10.1021/nn103130q] [Citation(s) in RCA: 243] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Among various nanoparticles, the silica nanoparticle (SiNP) is an attractive candidate as a gene delivery carrier due to advantages such as availability in porous forms for encapsulation of drugs and genes, large surface area to load biomacromolecules, biocompatibility, storage stability, and easy preparation in large quantity with low cost. Here, we report on a facile synthesis of monodispersed mesoporous silica nanoparticles (MMSN) possessing very large pores (>15 nm) and application of the nanoparticles to plasmid DNA delivery to human cells. The aminated MMSN with large pores provided a higher loading capacity for plasmids than those with small pores (∼2 nm), and the complex of MMSN with plasmid DNA readily entered into cells without supplementary polymers such as cationic dendrimers. Furthermore, MMSN with large pores could efficiently protect plasmids from nuclease-mediated degradation and showed much higher transfection efficiency of the plasmids encoding luciferase and green fluorescent protein (pLuc, pGFP) compared to MMSN with small pores (∼2 nm).
Collapse
Affiliation(s)
- Mi-Hee Kim
- Department of Chemistry, KAIST, Daejeon 305-701, Korea
| | | | | | | | | | | | | | | | | |
Collapse
|
|
37
|
Arbuthnot P. MicroRNA-like antivirals. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2011; 1809:746-55. [PMID: 21616187 DOI: 10.1016/j.bbagrm.2011.05.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2011] [Revised: 04/28/2011] [Accepted: 05/09/2011] [Indexed: 12/26/2022]
Abstract
Employing engineered DNA templates to express antiviral microRNA (miRNA) sequences has considerable therapeutic potential. The durable silencing that may be achieved with these RNAi activators is valuable to counter chronic viral infections, such as those caused by HIV-1, hepatitis B, hepatitis C and dengue viruses. Early use of expressed antiviral miRNAs entailed generation of cassettes containing Pol III promoters (e.g. U6 and H1) that transcribe virus-targeting short hairpin RNA mimics of precursor miRNAs. Virus escape from single gene silencing elements prompted later development of combinatorial antiviral miRNA expression cassettes that form multitargeting siRNAs from transcribed long hairpin RNA and polycistronic primary miRNA sequences. Weaker Pol III and Pol II promoters have also been employed to control production of antiviral miRNA mimics, improve dose regulation and address concerns about toxicity caused by saturation of the endogenous miRNA pathway. Efficient delivery of expressed antiviral sequences remains challenging and utilizing viral vectors, which include recombinant adenoviruses, adeno-associated viruses and lentiviruses, has been favored. Investigations using recombinant lentiviruses to transduce CD34+ hematological precursor cells with expressed HIV-1 gene silencers are at advanced stages and show promise in preclinical and clinical trials. Although the use of expressed antiviral miRNA sequences to treat viral infections is encouraging, eventual therapeutic application will be dependent on rigorously proving their safety, efficient delivery to target tissues and uncomplicated large scale preparation of vector formulations. This article is part of a special issue entitled: MicroRNAs in viral gene regulation.
Collapse
|