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Results for “self-amplifying RNA”

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At least 19 recordsLinked to original sources

PubMed2026

Transgene sequence codon optimization and composition determines replication competence of self-amplifying RNA.

Self-amplifying RNA (saRNA) is an emerging RNA therapeutic modality that can facilitate higher magnitude and more durable protein expression at substantially lower doses than nonreplicating mRNA. Unlike conventional messenger RNA (mRNA), alphavirus-derived saRNA must support a replicase-driven RNA amplification step in addition to translation, raising the possibility that transgene coding sequences impose sequence-level constraints on replication. Here, saRNA replication was found to be dependent on the codon composition of the transgene; multiple therapeutic transgenes were replication defective despite an intact Venezuelan Equine Encephalitis Virus (VEEV)-derived saRNA backbone. Replication defects were rescued by synonymous codon re-optimization of the same transgenes, indicating that nucleotide-level features of the coding sequence, rather than the encoded protein, govern replication competence. Comparative compositional analyses identified a distinct signature associated with productive replication, characterized by elevated GC (>53%) and GC3 (>63%) content, higher codon adaptation to human (>0.75), and reduced UpA (<43/kb) and UpU (<41/kb) dinucleotide density. Moreover, deliberate compositional perturbation of an otherwise replication-competent transgene shifted these features and abolished replication, supporting a causal and combinatorial role for sequence composition in defining saRNA replication outcome. These findings define an underappreciated constraint in saRNA therapeutics and motivate saRNA-specific payload design frameworks that incorporate alphavirus-associated compositional biases during transgene sequence optimization.

Codon↗

PubMed2003

Two ways to induce innate immune responses in human PBMCs: paracrine stimulation of IFN-alpha responses by viral protein or dsRNA.

In order to study mechanisms of induction of IFN-alpha by Newcastle disease virus (NDV), we used two replicon systems which are based respectively on DNA and RNA of the Semliki forest virus (SFV) and transfected these into baby hamster kidney cells (BHK) which do not produce interferon-alpha. Co-incubation of BHK cells which were transfected with the two vector systems, with human PBMCs, showed that production of IFN-alpha takes place by two different ways. When using the DNA-based SFV vector, only transfectants expressing cell surface HN molecules of NDV (and not the mock-transfected cells) elicited such a response via interaction of these HN molecules with viral receptors on PBMCs. In contrast, BHK cells transfected with RNA which had been in vitro transcribed from the RNA-based SFV vector without foreign gene as insert (mock-transfected) elicited a comparable IFN-alpha response. Transfer by transfection of poly(I:C), an analogue of double stranded RNA (dsRNA), into the BHK cells induced also by itself the production of IFN-alpha. Therefore induction of "danger signals" (as double-strand RNA replicative intermediates) might be responsible for this discrepancy observed in IFN-alpha induction in PBMCs between the two studied SFV vector systems based on transfection of DNA and on RNA. These observations highlight two ways of IFN-alpha induction which additively may explain the high interferonogenic capacity of NDV as virus: i) via cell-surface expressed HN after transfection of the DNA-based SFV replicon and ii) via transfection of self-amplifying RNA.

Animals↗

PubMed2026

mRNA therapy: A novel approach for retinal neurodegenerative diseases.

Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.

administration↗

PubMed1998

Mechanisms of replication-deficient vaccinia virus/T7 RNA polymerase hybrid expression: effect of T7 RNA polymerase levels and alpha-amanitin.

Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).

Amanitins↗

PubMed1998

DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-based vector.

Previously we reported the development of a plasmid DNA expression vector system derived from Sindbis virus (T. W. Dubensky, Jr., et al., J. Virol. 70:508-519, 1996). In vitro, such vectors exhibit high-level heterologous gene expression via self-amplifying cytoplasmic RNA replication. In the present study, we demonstrated the in vivo efficacy of the Sindbis virus-based pSIN vectors as DNA vaccines. A single intramuscular immunization of BALB/c mice with pSIN vectors expressing the glycoprotein B of herpes simplex virus type 1 induced a broad spectrum of immune responses, including virus-specific antibodies, cytotoxic T cells, and protection from lethal virus challenge in two different murine models. In addition, dosing studies demonstrated that the pSIN vectors were superior to a conventional plasmid DNA vector in the induction of all immune parameters tested. In general, 100- to 1,000-fold-lower doses of pSIN were needed to induce the same level of responsiveness as that achieved with the conventional plasmid DNA vector. In some instances, significant immune responses were induced with a single dose of pSIN as low as 10 ng/mouse. These results indicate the potential usefulness of alphavirus-based vectors for DNA immunization in general and more specifically as a herpes simplex virus vaccine.

Animals↗

PubMed2003

Significance in replication of the terminal nucleotides of the flavivirus genome.

Point mutations that resulted in a substitution of the conserved 3'-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3'-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3'-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of phi6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3'-penultimate cytidines and a 3'-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3'-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

Bacteriophage phi 6↗

PubMed1997

Self-amplifying vectors for gene delivery.

Current methods of gene transfer have a rather low efficiency, especially in vivo. Therefore, one tries to achieve the highest possible levels of expression in the few cells that do take up foreign DNA. One approach is to use self-amplifying expression vectors. These vectors are based on the (+)-strand RNA viruses (alphaviruses) Sindbis virus and Semliki Forest virus. In these vectors, the viral capsid protein coding sequences are replaced with the gene of interest. After introduction into the target cells, the viral replication proteins will replicate the recombinant genome. The increased levels of mRNA generate very high transgene expression levels. Furthermore, spread throughout large cells (muscle, neurons) is much better compared to conventional expression cassettes. Self-amplifying vectors can be introduced into target cells as RNA, DNA or virions.

Journal Article↗

PubMed2026

Repeat region engineering of Cas13a crRNA enables conformational gating-based autocatalytic CRISPR biosensing.

CrRNA engineering has emerged as a pivotal strategy for extending CRISPR-Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a-crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.

CRISPR-Associated Proteins↗

PubMed1998

In vitro evolution of molecular cooperation in CATCH, a cooperatively coupled amplification system.

BACKGROUND: One of the key issues in the investigation of evolution is how complex systems evolved from simple chemical replicators. Theoretical work proposed several models in which complex replicating systems are kinetically stabilized. The development of powerful isothermal amplification technique allows complex nucleic acid based evolving in vitro systems to be set up, which may then serve to verify experimentally current theories of evolution. Recently such a system based on the 3SR (self-sustained sequence replication) reaction has been established to investigate the evolution of cooperation: the trans-cooperatively coupled CATCH (cooperative amplification by cross hybridization). RESULTS: Over four rounds of serial transfer, the cooperatively coupled two species CATCH system evolved into a more complex cooperative four species system, which then was overgrown by CATCH-derived RNA-Z-like hairpin species. In contrast to the classical RNA-Z species, these molecules have complementary loop sequences and self-amplify using a dual mechanism that includes concentration-dependent phases of noncooperative and cooperative amplification. CONCLUSIONS: The evolution of a cooperative system, under conditions that were alternately unfavorable and favorable for cooperative amplification, led to a system showing facultative cooperation. This principle of facultative cooperation preserves the complexity of the system investigated and could have general implications for the evolution and stabilization of cooperation under oscillating reaction conditions.

Alfalfa mosaic virus↗

PubMed1995

A plasmid-based self-amplifying Sindbis virus vector.

Sindbis virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA viruses.

3T3 Cells↗

PubMed1994

Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts.

To increase the levels of exogenous or foreign gene expression in mammalian cells, this study sought to develop an 'autogene' that will self-amplify. An autogene plasmid, pT7-G1, containing the T7 phage RNA polymerase-encoding modified gene (G1) under control of its cognate T7 promoter, was only obtained when the plasmid contained the encephalomyocarditis (EMC) untranslated sequence. In vitro transcription and translation studies confirmed that both the T7 promoter and the G1 gene were completely functional in the pT7-G1 plasmid. Expression from pT7-G1 was initiated in vivo either by co-transfection with its in vitro transcript or by transfection into NIH3T3 cell lines that stably expressed T7 RNA polymerase enzyme. Use of the pT7-G1 autogene enabled an approx. 50-fold increase in foreign protein production. Northern analysis suggested that this increased expression resulted from the self-amplification of the autogene. By allowing greater expression in cell lines with low T7 RNA polymerase expression, the pT7-G1 plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.

3T3 Cells↗

PubMed1994

A sustained, cytoplasmic transgene expression system delivered by cationic liposomes.

A plasmid containing the reporter gene, chloramphenicol acetyltransferase (CAT), driven by the bacteriophage T7 promoter was co-delivered with purified T7 RNA polymerase by the DC-chol cationic liposomes into human embryonic kidney 293 cells to obtain a transient (2 days) CAT gene expression. To prolong the expression, a T7 autogene which contains the T7 RNA polymerase gene driven by the T7 promoter was included in the transfection complex as a self-amplifying regeneration mechanism for the polymerase. High level CAT gene expression was observed up to 5 days after transfection. This strong and sustained expression system should be useful in gene transfer experiments.

Cell Line↗

PubMed2002

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and in Caenorhabditis elegans can spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs.

Base Sequence↗

PubMed2001

A combined in vitro/in vivo selection for polymerases with novel promoter specificities.

BACKGROUND: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection. RESULTS AND CONCLUSIONS: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides.

Amino Acid Substitution↗

PubMed1997

Semliki Forest virus-mediated production of retroviral vector RNA in retroviral packaging cells.

Retroviral vectors are efficient tools for gene transfer studies. Their major advantage is that they can permanently integrate the transgene into the target cell's genome. However, because of the compulsory nuclear expression phase of their life cycle, it can be difficult for retroviruses to carry complex expression cassettes. In a attempt to mimic the structural features of most eukaryotic genes and obtain a potentially self-amplifying system for retrovirus production, we tested the feasibility of Semliki Forest virus (SFV) expression to mediate cytoplasmic synthesis of retrovirus vector RNA. An equivalent of a retrovirus virion RNA (retrovirus vector cassette, RVC) was cloned under the SFV 26S promoter, and full-length chimeric SFV-RVC RNA was produced in vitro. This RNA was introduced into retrovirus packaging cells, either via electroporation or transduction in SFV virions, and supernatants were analyzed for the presence of biologically active retroviruses. We demonstrate that this strategy can be used for cytoplasmic retrovirus production. The resulting viral particles are fully functional; they can transduce target cells, undergo reverse transcription, and integrate into genomic DNA. We also demonstrate that the SFV virion-based RVC delivery into packaging cells can yield high transient titers, in this case more than 10(5) G418R cfu/ml. This study shows that a simple, one-plasmid, heterologous viral RNA production system can be used to create functional retroviral RNA outside the cell nucleus.

3T3 Cells↗

PubMed2001

A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli.

Infectious clone methodology is a valuable tool of modern experimental virology. However, its use is often constrained by the instability of infectious clone constructs during propagation in E. coli. To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general. An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E. coli by insertion of short introns. To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture. The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines.

Base Sequence↗

PubMed1998

IFN-gamma primes macrophage responses to bacterial DNA.

Macrophages recognize and are activated by unmethylated CpG motifs in bacterial DNA. Here we demonstrate that production of nitric oxide (NO) from murine RAW 264 macrophages and bone marrow-derived macrophages (BMM) in response to bacterial DNA is absolutely dependent on interferon-gamma (IFN-gamma) priming. Similarly, arginine uptake and expression of the inducible nitric oxide synthase (iNOS) gene in response to bacterial DNA in BMM occurred only after IFN-gamma priming. In contrast, mRNA for the cationic amino acid transporter, CAT2, was induced by plasmid DNA alone, and priming with IFN-gamma had no effect on this response. Tumor necrosis factor-alpha (TNF-alpha) release from RAW 264 and BMM in response to bacterial DNA was augmented by IFN-gamma pretreatment. In a stably transfected HIV-1 long terminal repeat (LTR) luciferase RAW 264 cell line, IFN-gamma and bacterial DNA synergized in activation of the HIV-1 LTR. Bacterial DNA has been shown to induce IFN-gamma production in vivo as an indirect consequence of interleukin-12 (IL-12) and TNF-alpha production from macrophages. The results herein suggest the existence of a self-amplifying loop that may have implications for therapeutic applications of bacterial DNA.

Animals↗

PubMed2000

Effect of endogenous and exogenous prostaglandin E(2) on interleukin-1 beta-induced cyclooxygenase-2 expression in human airway smooth-muscle cells.

We studied the effect of endogenous and exogenous prostaglandin E(2) (PGE(2)), a metabolite of arachidonic acid through the cyclooxygenase (COX) pathway, on interleukin (IL)-1 beta-induced COX-2 expression, using primary cultures of human bronchial smooth-muscle cells (HBSMC). Treatment with exogenous PGE(2) resulted in enhanced expression of IL-1 beta-induced COX-2 protein and messenger RNA (mRNA) as compared with the effect of the cytokine per se. Inhibition of PGE(2) production with a nonselective COX inhibitor (flurbiprofen, 10 microM) resulted in a significant reduction in IL-1 beta- induced COX-2 expression, supporting a role of endogenous COX metabolites in the modulation of COX-2 expression. None of the experimental conditions used in the study affected the expression of constitutive cyclooxygenase (COX-1). Treatment with cycloheximide to inhibit translation, and with dexamethasone or actinomycin D to inhibit transcription, linked the effect of PGE(2) to the transcriptional level of COX-2 mRNA rather than to a potential effect on protein and/or mRNA stabilization. PGE(2) increased adenylate cyclase activity in a concentration dependent manner, and forskolin, a direct activator of adenylate cyclase, caused a marked increase in IL-1 beta-dependent COX-2, suggesting the existence of a causal relationship between the two events. The same results were observed with salbutamol, a bronchodilator that acts by increasing cyclic adenosine monophosphate. The effect of PGE(2) on COX-2 expression may contribute to the hypothesized antiinflammatory role of PGE(2) in human airways, providing a self-amplifying loop leading to increased biosynthesis of PGE(2) during an inflammatory event.

Adenylyl Cyclases↗