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1.
Pharmaceutics ; 15(4)2023 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-37111658

RESUMEN

Africa bears the highest burden of infectious diseases, yet the continent is heavily reliant on First World countries for the development and supply of life-saving vaccines. The COVID-19 pandemic was a stark reminder of Africa's vaccine dependence and since then great interest has been generated in establishing mRNA vaccine manufacturing capabilities on the African continent. Herein, we explore alphavirus-based self-amplifying RNAs (saRNAs) delivered by lipid nanoparticles (LNPs) as an alternative to the conventional mRNA vaccine platform. The approach is intended to produce dose-sparing vaccines which could assist resource-constrained countries to achieve vaccine independence. Protocols to synthesize high-quality saRNAs were optimized and in vitro expression of reporter proteins encoded by saRNAs was achieved at low doses and observed for an extended period. Permanently cationic or ionizable LNPs (cLNPs and iLNPs, respectively) were successfully produced, incorporating saRNAs either exteriorly (saRNA-Ext-LNPs) or interiorly (saRNA-Int-LNPs). DOTAP and DOTMA saRNA-Ext-cLNPs performed best and were generally below 200 nm with good PDIs (<0.3). DOTAP and DDA saRNA-Int-cLNPs performed optimally, allowing for saRNA amplification. These were slightly larger, with higher PDIs as a result of the method used, which will require further optimization. In both cases, the N:P ratio and lipid molar ratio had a distinct effect on saRNA expression kinetics, and RNA was encapsulated at high percentages of >90%. These LNPs allow the delivery of saRNA with no significant toxicity. The optimization of saRNA production and identification of potential LNP candidates will facilitate saRNA vaccine and therapeutic development. The dose-sparing properties, versatility, and manufacturing simplicity of the saRNA platform will facilitate a rapid response to future pandemics.

2.
Front Immunol ; 13: 1018961, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36353641

RESUMEN

Synthetic mRNA technologies represent a versatile platform that can be used to develop advanced drug products. The remarkable speed with which vaccine development programs designed and manufactured safe and effective COVID-19 vaccines has rekindled interest in mRNA technology, particularly for future pandemic preparedness. Although recent R&D has focused largely on advancing mRNA vaccines and large-scale manufacturing capabilities, the technology has been used to develop various immunotherapies, gene editing strategies, and protein replacement therapies. Within the mRNA technologies toolbox lie several platforms, design principles, and components that can be adapted to modulate immunogenicity, stability, in situ expression, and delivery. For example, incorporating modified nucleotides into conventional mRNA transcripts can reduce innate immune responses and improve in situ translation. Alternatively, self-amplifying RNA may enhance vaccine-mediated immunity by increasing antigen expression. This review will highlight recent advances in the field of synthetic mRNA therapies and vaccines, and discuss the ongoing global efforts aimed at reducing vaccine inequity by establishing mRNA manufacturing capacity within Africa and other low- and middle-income countries.


Asunto(s)
COVID-19 , Vacunas , Humanos , ARN Mensajero/genética , Vacunas contra la COVID-19 , COVID-19/prevención & control , Tecnología
3.
Viruses ; 14(9)2022 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-36146747

RESUMEN

Despite being vaccine-preventable, hepatitis B virus (HBV) infection remains the seventh leading cause of mortality in the world. In South Africa (SA), over 1.9 million people are chronically infected with HBV, and 70% of all Black chronic carriers are infected with HBV subgenotype A1. The virus remains a significant burden on public health in SA despite the introduction of an infant immunization program implemented in 1995 and the availability of effective treatment for chronic HBV infection. In addition, the high prevalence of HIV infection amplifies HBV replication, predisposes patients to chronicity, and complicates management of the infection. HBV research has made significant progress leading to better understanding of HBV epidemiology and management challenges in the SA context. This has led to recent revision of the national HBV infection management guidelines. Research on developing new vaccines and therapies is underway and progress has been made with designing potentially curative gene therapies against HBV. This review summarizes research carried out in SA on HBV molecular biology, epidemiology, treatment, and vaccination strategies.


Asunto(s)
Infecciones por VIH , Hepatitis B , Infecciones por VIH/complicaciones , Infecciones por VIH/epidemiología , Hepatitis B/complicaciones , Hepatitis B/epidemiología , Hepatitis B/prevención & control , Vacunas contra Hepatitis B/uso terapéutico , Virus de la Hepatitis B/genética , Humanos , Lactante , Sudáfrica/epidemiología
4.
Front Immunol ; 13: 834650, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35154157

RESUMEN

Infection with the hepatitis B virus (HBV) continues to pose a major threat to public health as approximately 292 million people worldwide are currently living with the chronic form of the disease, for which treatment is non-curative. Chronic HBV infections often progress to hepatocellular carcinoma (HCC) which is one of the world's leading causes of cancer-related deaths. Although the process of hepatocarcinogenesis is multifaceted and has yet to be fully elucidated, several studies have implicated numerous long non-coding RNAs (lncRNAs) as contributors to the development of HCC. These host-derived lncRNAs, which are often dysregulated as a consequence of viral infection, have been shown to function as signals, decoys, guides, or scaffolds, to modulate gene expression at epigenetic, transcriptional, post-transcriptional and even post-translational levels. These lncRNAs mainly function to promote HBV replication and oncogene expression or downregulate tumor suppressors. Very few lncRNAs are known to suppress tumorigenesis and these are often downregulated in HCC. In this review, we describe the mechanisms by which lncRNA dysregulation in HBV-related HCC promotes tumorigenesis and cancer progression.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Neoplasias Hepáticas/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Regulación Neoplásica de la Expresión Génica , Hepatitis B/genética , Hepatitis B/virología , Virus de la Hepatitis B/genética , Interacciones Huésped-Patógeno , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , ARN Largo no Codificante/genética
5.
Viruses ; 13(11)2021 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-34835053

RESUMEN

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals' potency.


Asunto(s)
Dependovirus/genética , Modelos Animales de Enfermedad , Virus de la Hepatitis B/fisiología , Replicación Viral , Animales , Antivirales/uso terapéutico , Línea Celular , Expresión Génica/efectos de los fármacos , Silenciador del Gen , Vectores Genéticos , Genotipo , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Humanos , Hígado/virología , Ratones , MicroARNs/genética , MicroARNs/uso terapéutico , Transfección , Replicación Viral/efectos de los fármacos
6.
Viruses ; 13(7)2021 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-34372550

RESUMEN

Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication.


Asunto(s)
Virus de la Hepatitis B/genética , Hepatitis B/tratamiento farmacológico , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Animales , Animales no Consanguíneos , Antivirales/uso terapéutico , Línea Celular , Virus ADN/genética , ADN Circular , ADN Viral/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Modelos Animales de Enfermedad , Endonucleasas/genética , Femenino , Terapia Genética/métodos , Células HEK293 , Células Hep G2 , Hepatitis B/genética , Hepatitis B/inmunología , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Humanos , Ratones , Nucleasas de los Efectores Tipo Activadores de la Transcripción/uso terapéutico , Replicación Viral/genética
7.
Expert Opin Ther Targets ; 25(6): 451-466, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33843412

RESUMEN

Introduction: Current therapy for infection with hepatitis B virus (HBV) rarely clears the virus, and viremia commonly resurges following treatment withdrawal. To prevent serious complications of the infection, research has been aimed at identifying new viral and host targets that can be exploited to inactivate HBV replication.Areas covered: This paper reviews the use of these new molecular targets to advance anti-HBV therapy. Emphasis is on appraising data from pre-clinical and early clinical studies described in journal articles published during the past 10 years and available from PubMed.Expert opinion: The wide range of viral and host factors that can be targeted to disable HBV is impressive and improved insight into HBV molecular biology continues to provide the basis for new drug design. In addition to candidate therapies that have direct or indirect actions on HBV covalently closed circular DNA (cccDNA), compounds that inhibit HBsAg secretion, viral entry, destabilize viral RNA and effect enhanced immune responses to HBV show promise. Preclinical and clinical evaluation of drug candidates, as well as investigating use of treatment combinations, are encouraging. The field is poised at an interesting stage and indications are that reliably achieving functional cure from HBV infection is a tangible goal.


Asunto(s)
Virus de la Hepatitis B , Preparaciones Farmacéuticas , Antivirales/farmacología , Antivirales/uso terapéutico , ADN Circular , ADN Viral , Humanos , Replicación Viral
8.
J Hepatocell Carcinoma ; 8: 1-17, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33542907

RESUMEN

After being overlooked for decades, circular RNAs (circRNAs) have recently generated considerable interest. circRNAs play a role in a variety of normal and pathological biological processes, including hepatocarcinogenesis. Many circRNAs contribute to hepatocarcinogenesis through sponging of microRNAs (miRs) and disruption of cellular signaling pathways that play a part in control of cell proliferation, metastasis and apoptosis. In most cases, overexpressed circRNAs sequester miRs to cause de-repressed translation of mRNAs that encode oncogenic proteins. Conversely, low expression of circRNAs has also been described in hepatocellular carcinoma (HCC) and is associated with inhibited production of tumor suppressor proteins. Other functions of circRNAs that contribute to hepatocarcinogenesis include translation of truncated proteins and acting as adapters to regulate influence of transcription factors on target gene expression. circRNAs also affect hepatocyte transformation indirectly. For example, the molecules regulate immune surveillance of cancerous cells and influence the liver fibrosis that commonly precedes HCC. Marked over- or under-expression of circRNA expression in HCC, with correlating plasma concentrations, has diagnostic utility and assays of these RNAs are being developed as biomarkers of HCC. Although knowledge in the field has recently surged, the myriad of described effects suggests that not all may be vital to hepatocarcinogenesis. Nevertheless, investigation of the role of circRNAs is providing valuable insights that are likely to contribute to improved management of a serious and highly aggressive cancer.

9.
Adv Drug Deliv Rev ; 168: 134-146, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32485207

RESUMEN

Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. The ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.


Asunto(s)
Terapia Genética/métodos , Hepatitis B Crónica/genética , Hepatitis B Crónica/terapia , ARN Mensajero/administración & dosificación , Antivirales/uso terapéutico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Relación Dosis-Respuesta a Droga , Edición Génica , Vectores Genéticos/administración & dosificación , Vacunas contra Hepatitis B/administración & dosificación , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/prevención & control , Nanopartículas/química , Ribonucleasas/administración & dosificación , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Nucleasas con Dedos de Zinc/metabolismo
10.
Viruses ; 12(8)2020 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-32759756

RESUMEN

Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation.


Asunto(s)
Silenciador del Gen , Hepatitis B/terapia , Tratamiento con ARN de Interferencia/métodos , Animales , Ensayos Clínicos como Asunto , ADN Circular , ADN Viral/genética , Vectores Genéticos , Hepatitis B/complicaciones , Humanos , Hígado/patología , Hígado/virología , Ratones , MicroARNs , Tratamiento con ARN de Interferencia/tendencias , Replicación Viral/genética
11.
Methods Mol Biol ; 2115: 185-197, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32006402

RESUMEN

RNA interference (RNAi) is a promising tool for the treatment of chronic viral infection, such as that caused by the hepatitis B virus (HBV). RNAi activators, including expressed primary microRNA (pri-miRNA) mimics, can effectively silence viral gene expression and thereby inhibit viral replication. Here we describe a protocol for the design, generation and functional assessment of cassettes encoding effective single and multimeric pri-miRNA mimics. Artificial miRNAs targeting viral genes can be identified in silico and used to design corresponding pri-miRNA mimics. A two-step generation and TA cloning protocol can be used to produce single mimics, while the strategic use of restriction sites enables concatenation of mimics in a sub-cloning protocol. Basic gene silencing function of pri-miRNA mimics in cell culture can then be assessed using a dual luciferase assay and appropriate minimal targets. The methods described here for the generation of effective pri-miRNA mimics targeting HBV can be applied in the silencing of other viral or endogenous genes.


Asunto(s)
ADN/genética , Silenciador del Gen , Genes Virales , Virus de la Hepatitis B/genética , MicroARNs/genética , Expresión Génica , Células HEK293 , Células HeLa , Hepatitis B/terapia , Hepatitis B/virología , Virus de la Hepatitis B/fisiología , Humanos , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Replicación Viral
12.
BMC Infect Dis ; 19(1): 802, 2019 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-31510934

RESUMEN

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital. METHODS: To investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters. RESULTS: Evaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification. CONCLUSIONS: Epigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.


Asunto(s)
ADN Viral/genética , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/genética , Hepatitis B/terapia , Hepatitis B/virología , Proteínas Represoras/metabolismo , Replicación Viral/genética , Animales , Línea Celular , Islas de CpG , Metilación de ADN , ADN Circular/genética , ADN Viral/biosíntesis , Epigénesis Genética , Femenino , Hígado/metabolismo , Hígado/virología , Ratones , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , Proteínas Represoras/genética
13.
Mol Ther Methods Clin Dev ; 14: 100-112, 2019 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-31334303

RESUMEN

HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable. To overcome these obstacles, adeno-associated virus (AAV) vectors were used to provide long-term expression of antibodies targeting the V2 region of the HIV-1 envelope protein, including the potent CAP256-VRC26.25 bNAb, as well as non-neutralizing CAP228 antibodies that resemble those elicited by vaccination. AAVs mediated effective antibody expression in cell culture and immunocompetent mice. Mean concentrations of human immunoglobulin G (IgG) in mouse sera increased rapidly following a single AAV injection, reaching 8-60 µg/mL for CAP256 antibodies and 44-220 µg/mL for CAP228 antibodies over 24 weeks, but antibody concentrations varied for individual mice. Secreted antibodies collected from serum retained the expected binding and neutralizing activity. The vectors generated here are, therefore, suitable for the delivery of V2-targeting HIV antibodies, and they could be used in a vectored immunoprophylaxis (VIP) approach to sustain the level of antibody expression required to prevent HIV infection.

14.
Biotechniques ; 66(1): 37-39, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30730207

RESUMEN

Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3' poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100 bp that is generated using the method is approximately equal to the number of 3' A residues found in the mRNA of  mammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo.


Asunto(s)
Técnicas Genéticas , Poli A/genética , ARN Mensajero/genética , ADN Circular , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Plásmidos , Transcripción Genética
15.
Genes (Basel) ; 9(4)2018 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-29649127

RESUMEN

Chronic infection with the hepatitis B virus (HBV) is a global health concern and accounts for approximately 1 million deaths annually. Amongst other limitations of current anti-HBV treatment, failure to eliminate the viral covalently closed circular DNA (cccDNA) and emergence of resistance remain the most worrisome. Viral rebound from latent episomal cccDNA reservoirs occurs following cessation of therapy, patient non-compliance, or the development of escape mutants. Simultaneous viral co-infections, such as by HIV-1, further complicate therapeutic interventions. These challenges have prompted development of novel targeted hepatitis B therapies. Given the ease with which highly specific and potent nucleic acid therapeutics can be rationally designed, gene therapy has generated interest for antiviral application. Gene therapy strategies developed for HBV include gene silencing by harnessing RNA interference, transcriptional inhibition through epigenetic modification of target DNA, genome editing by designer nucleases, and immune modulation with cytokines. DNA-binding domains and effectors based on the zinc finger (ZF), transcription activator-like effector (TALE), and clustered regularly interspaced short palindromic repeat (CRISPR) systems are remarkably well suited to targeting episomal cccDNA. This review discusses recent developments and challenges facing the field of anti-HBV gene therapy, its potential curative significance and the progress towards clinical application.

16.
Virus Res ; 244: 311-320, 2018 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-28087399

RESUMEN

Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.


Asunto(s)
Sistemas CRISPR-Cas , ADN Circular/genética , ADN Viral/genética , Hepatitis B Crónica/terapia , Hepatitis C Crónica/terapia , ARN Guía de Kinetoplastida/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , ADN Circular/metabolismo , ADN Viral/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Hepacivirus/genética , Hepacivirus/crecimiento & desarrollo , Hepacivirus/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/virología , Hepatitis C Crónica/virología , Humanos , Terapia Molecular Dirigida/métodos , Seguridad del Paciente , ARN Guía de Kinetoplastida/metabolismo , Replicación Viral
17.
Expert Opin Biol Ther ; 18(4): 381-388, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29265946

RESUMEN

INTRODUCTION: Exposure to toxins from the portal circulation, viral infection and by-products of metabolic activity make liver tissue prone to injury. When sustained, associated inflammation leads to activation of hepatic stellate cells (HSCs), deposition of extracellular matrix (ECM) proteins and complicating hepatic fibrosis. AREAS COVERED: In this article, the authors discuss utility of therapeutic gene silencing to disable key steps of hepatic fibrogenesis. Strategies aimed at inhibiting HSC activation and silencing primary causes of fibrogenesis, such as viruses that cause chronic hepatitis, are reviewed. Both synthetic and expressed artificial intermediates of the RNAi pathway have potential to treat hepatic fibrosis, and each type of gene silencer has advantages for clinical translation. Silencing expression cassettes comprising DNA templates are compatible with efficient hepatotropic viral vectors, which may effect sustained gene silencing. By contrast, synthetic short interfering RNAs are amenable to chemical modification, incorporation into non-viral formulations, more precise dose control and large scale preparation. EXPERT OPINION: Clinical translation of RNAi-based technology for treatment of hepatic fibrosis is now a realistic goal. However, achieving this aim will require safe, efficient delivery of artificial RNAi intermediates to target cells, economic large scale production of candidate drugs and specificity of action.


Asunto(s)
Cirrosis Hepática/tratamiento farmacológico , ARN Interferente Pequeño/uso terapéutico , Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo
18.
Sci Rep ; 7(1): 7401, 2017 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-28785016

RESUMEN

Management of infection with hepatitis B virus (HBV) remains a global health problem. Persistence of stable covalently closed circular DNA (cccDNA) during HBV replication is responsible for modest curative efficacy of currently licensed drugs. Novel gene editing technologies, such as those based on CRISPR/Cas9, provide the means for permanently disabling cccDNA. However, efficient delivery of antiviral sequences to infected hepatocytes is challenging. A limiting factor is the large size of sequences encoding Cas9 from Streptococcus pyogenes, and resultant incompatibility with the popular single stranded adeno-associated viral vectors (ssAAVs). We thus explored the utility of ssAAVs for delivery of engineered CRISPR/Cas9 of Staphylococcus aureus (Sa), which is encoded by shorter DNA sequences. Short guide RNAs (sgRNAs) were designed with cognates in the S open reading frame of HBV and incorporated into AAVs that also encoded SaCas9. Intended targeted mutation of HBV DNA was observed after transduction of cells with the all-in-one vectors. Efficacy against HBV-infected hNTCP-HepG2 cells indicated that inactivation of cccDNA was successful. Analysis of likely off-target mutagenesis revealed no unintended sequence changes. Use of ssAAVs to deliver all components required to disable cccDNA by SaCas9 is novel and the technology has curative potential for HBV infection.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Dependovirus/genética , Virus de la Hepatitis B/efectos de los fármacos , ARN Guía de Kinetoplastida/genética , Proteína 9 Asociada a CRISPR/metabolismo , Edición Génica , Vectores Genéticos/farmacología , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , ARN Guía de Kinetoplastida/síntesis química , Staphylococcus aureus/metabolismo , Replicación Viral/efectos de los fármacos
19.
Mol Ther Nucleic Acids ; 7: 190-199, 2017 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-28624194

RESUMEN

Chronic infection with hepatitis B virus (HBV) remains a problem of global significance and improving available treatment is important to prevent life-threatening complications arising in persistently infected individuals. HBV is susceptible to silencing by exogenous artificial intermediates of the RNA interference (RNAi) pathway. However, toxicity of Pol III cassettes and short duration of silencing by effectors of the RNAi pathway may limit anti-HBV therapeutic utility. To advance RNAi-based HBV gene silencing, mono- and trimeric artificial primary microRNAs (pri-miRs) derived from pri-miR-31 were placed under control of the liver-specific modified murine transthyretin promoter. The sequences, which target the X sequence of HBV, were incorporated into recombinant hepatotropic self-complementary adeno-associated viruses (scAAVs). Systemic intravenous injection of the vectors into HBV transgenic mice at a dose of 1 × 1011 per animal effected significant suppression of markers of HBV replication for at least 32 weeks. The pri-miRs were processed according to the intended design, and intrahepatic antiviral guide sequences were detectable for 40 weeks after the injection. There was no evidence of toxicity, and innate immunostimulation was not detectable following the injections. This efficacy is an improvement on previously reported RNAi-based inhibition of HBV replication and is important to clinical translation of the technology.

20.
Methods Mol Biol ; 1540: 85-95, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27975310

RESUMEN

Gene editing using designer nucleases is now widely used in many fields of molecular biology. The technology is being developed for the treatment of viral infections such as persistant hepatitis B virus (HBV). The replication intermediate of HBV comprising covalently closed circular DNA (cccDNA) is stable and resistant to available licensed antiviral agents. Advancing gene editing as a means of introducing targeted mutations into cccDNA thus potentially offers the means to cure infection by the virus. Essentially, targeted mutations are initiated by intracellular DNA cleavage, then error-prone nonhomologous end joining results in insertions and deletions (indels) at intended sites. Characterization of these mutations is crucial to confirm activity of potentially therapeutic nucleases. A convenient tool for evaluation of the efficiency of target cleavage is the single strand-specific endonuclease, T7EI. Assays employing this enzyme entail initial amplification of DNA encompassing the targeted region. Thereafter the amplicons are denatured and reannealed to allow hybridization between indel-containing and wild-type sequences. Heteroduplexes that contain mismatched regions are susceptible to action by T7EI and cleavage of the hybrid amplicons may be used as an indicator of efficiency of designer nucleases. The protocol described here provides a method of isolating cccDNA from transfected HepG2.2.15 cells and evaluation of the efficiency of mutation by a transcription activator-like effector nuclease that targets the surface open reading frame of HBV.


Asunto(s)
ADN Circular , ADN Viral , Desoxirribonucleasa I/metabolismo , Virus de la Hepatitis B/genética , Mutación , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Expresión Génica , Células Hep G2 , Humanos , Mutación INDEL , Plásmidos/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transfección
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