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At least 19 recordsLinked to original sources
PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed2026

Developing Highly Effective Nanoparticle mRNA Therapeutic for Pediatric Acute Respiratory Distress Syndrome.

Sepsis-induced pediatric acute lung injury (ALI) and pediatric acute respiratory distress syndrome (PARDS) are life-threatening conditions with high mortality rates and no current cure. Most ALI/ARDS studies focus on adults, albeit the pediatric population has unique challenges often underrepresented. ALI/PARDS severely impacts pulmonary endothelial cells (ECs), causing endothelial dysfunction and vascular leakage. FOXF1 is a transcription factor critical for lung repair after injury, representing a viable target for ALI/PARDS. This study developed and tested a novel nanoparticle system for precise delivery of FOXF1 mRNA into lung ECs to reduce endothelial damage and improve lung function in mouse model of PARDS. Systemic inflammatory response was induced in neonatal mice after intraperitoneal administration of lipopolysaccharide (LPS). Specifically designed nanoparticles (NP) were used to intravenously deliver stabilized FOXF1 mRNA (FOXF1 NP) after LPS injury to restore FOXF1 expression in injured lung endothelial cells. FOXF1 NP selectively targeted pulmonary ECs without affecting other cell types or organs. FOXF1 NP treatment reduced vascular leakage, enhanced endothelial barrier function, and improved survival of neonatal mice after injury. FOXF1 NP decreased EC apoptosis by restoring the expression of BCL2, an anti-apoptotic FOXF1 target gene. Nanoparticle-based rescue of lung ECs has promise for future treatments of human ALI/PARDS.

endothelial cells↗
PubMed2026

Complementation of a human disease phenotype by intercellular mRNA transfer.

There is growing evidence that mRNAs undergo intercellular transfer through cytoplasmic connections called tunneling nanotubes (TNTs), but whether transferred mRNAs are translated and affect cellular changes post-transfer is unknown. Using multiple lines of evidence, we show that transferred mRNAs undergo translation and can complement the phenotype of genetic mutations in vitro. For example, the human peroxisome biogenesis disorder, Zellweger Syndrome, results from mutations in genes such as PEX5 and PEX6. We demonstrate that the co-culture of patient-derived PEX6 mutant fibroblasts or PEX5 knockout cells with wild-type cells leads to de novo peroxisome biogenesis. We provide additional examples of genetic complementation via transfer of mRNAs encoding the HSF1 transcription factor or CRE recombinase. Complementation occurs by TNT-mediated mRNA transfer and translation in acceptor cells, but not by exosomes, nor by protein or peroxisome transfer. Our study provides evidence for the physiological significance of mRNA transfer and suggests another approach for mRNA therapeutics.

CP: cell biology↗
PubMed2025

Fluorinated Ionizable Lipids for Efficient Spleen-Targeted mRNA Delivery in Cancer Immunotherapy.

Efficient and selective mRNA delivery to immune-related organs, particularly the spleen, remains a major barrier to the broader clinical translation of mRNA therapeutics. Here, leveraging the clinically approved SM-102/ALC-0315 ionizable lipid scaffold, we rationally designed a combinatorial library of fluorinated ionizable lipids (FILs) by systematically modulating hydrophobic tails and fluorine stoichiometry. Through synthesis and evaluation of 74 candidate FILs, we identify SSC6F5 lipid nanoparticles (LNPs) as a lead formulation with exceptional spleen-targeting specificity (>90%) across intravenous, intramuscular, and subcutaneous administrations. Compared to clinically approved SM102 LNPs and spleen-tropic SM102/18PA (SORT) LNPs, intravenously administered SSC6F5 LNPs achieve 10.6-fold and 63.1-fold higher splenic mRNA transfection, respectively. Proteomic analysis of protein corona on SSC6F5 LNPs reveals significant enrichment of apolipoprotein D (Apod) and reduction in apolipoprotein H (Apoh), implicating a novel endogenous recognition pathway driving enhanced spleen targeting. Functionally, SSC6F5 LNPs enable efficient genome editing in splenic macrophages, dendritic cells, T cells, and B cells in Ai9 mice, and elicit potent CD8+ T cell and humoral responses in a B16-OVA murine melanoma model, resulting in significant tumor growth inhibition. These findings establish fluorinated lipids as a mechanistically distinct and translationally versatile platform for precision spleen-targeted mRNA delivery in gene editing and cancer immunotherapy.

Animals↗
PubMed2005

[Alternative mRNA splicing, pathology and molecular therapeutics].

Pre-mRNA splicing operates towards at least 95 % of the transcript pool. It is subjected to a large number of variations, collectively regrouped under the term of alternative mRNA splicing, which occurs, on average, 6 to 8 times per pre-mRNA molecule. Consequently, many more proteins may be encoded from a single gene, which may satisfy a physiological need, or mark a pathological adaptation. The identification of mutations in sequences required for splicing, both constitutive and alternative, or for their control, has permitted to determine the causes of qualitative or quantitative variations in transcript levels associated with inherited diseases or cancer development. A number of molecular approaches have been undertaken to try to compensate for the effect of deleterious splicing mutations and to restore, at least in part, sufficient amounts of either the normal or a surrogate transcript. These include overexpression of splicing proteins, improvement of their activity by post-translational modification, splice-site increased or decreased usage, and RNA-mediated trans-splicing. Using such approaches, phenotypic improvements have been obtained in animal models, carrying new hopes for the development of therapeutic strategies aimed at correcting both inherited and acquired diseases that involve pre-mRNA splicing defects.

Alternative Splicing↗
PubMed1996

Designing of chimeric DNA/RNA hammerhead ribozymes to be targeted against AML1/MTG8 mRNA.

For therapeutic purposes, two chimeric DNA/RNA hammerhead ribozymes were synthesized to cleave AML1/MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia. One ribozyme, A/MRZ-1, recognizes the area adjacent to the fusion point between AML1 and MTG8, and cleaves six bases downstream from this point. The other, MRZ-1, recognizes the MTG8 sequence. Both ribozymes cleaved synthetic chimeric DNA/RNA substrates at theoretical sites. Neither cleaved AML1 RNA. A/MRZ-1 cleaved only AML1/MTG8 RNA, and MRZ-1 cleaved both AML1/MTG8 and MTG8 RNAs. The two ribozymes showed growth inhibition of an acute myeloid leukemia cell line carrying t(8;21), SKNO-1 cells. The same extent of growth inhibition was attained by antisense oligonucleotides against AML1/MTG8 RNA. The results suggest that the ribozyme has the potential to be developed as a useful agent for gene therapy, in particular for leukemia with t(8;21).

Antineoplastic Agents↗
PubMed2004

Genetic medicine at the RNA level: modifications of the genetic repertoire for therapeutic purposes by pre-mRNA trans-splicing.

Gene therapy is conventionally carried out by transferring genetic material to the target cell where the exogenous gene is expressed using the endogenous transcription and translation machinery in parallel with the target cell genome. This review focuses on a new paradigm of gene therapy, the use of trans-splicing to modify the genetic repertoire at the pre-mRNA level to treat genetic and acquired disorders. Therapeutic trans-splicing can be used to alter coding domains, to create novel fusion proteins, to direct gene products to various cellular compartments, and to overcome some of the limitations to vector-derived gene transfer technology, including gene therapy with large genes or with genes coding for toxic proteins. To demonstrate the potential of therapeutic trans-splicing, eukaryotic cis-splicing and trans-splicing are reviewed, followed by a discussion of strategies of therapeutic pre-mRNA trans-splicing directed by exogenous gene transfer.

Animals↗
PubMed2025

Strain-Promoted mRNA Transdermal Delivery by Lipoic Lipid Nanoparticles for Therapeutic Skin Genome Editing.

Lipid nanoparticle (LNP)-mRNA formulations have revolutionized the field of nucleic acid therapeutics, yet their broader clinical application is constrained by inflammatory side effects and oxidative stress, particularly in the context of inflammatory diseases. Herein, we report the rational design and synthesis of a lipoic acid-based ionizable lipid library to address these limitations. By leveraging the antioxidant properties and thiol-mediated uptake potential of lipoic acid, we identified LA-A2B2CD3 as an optimal candidate through a structure-activity relationship study and design of experiment (DOE) optimization. LA-A2B2CD3 LNPs exhibited superior reactive oxygen species scavenging, enhanced mRNA translation, and reduced inflammatory cytokine production in vitro and in vivo. Mechanistic studies revealed that the efficient cellular uptake and the transdermal delivery capacity of LA-A2B2CD3 heavily rely on the reducible disulfide ring of lipoic acid. Application of LA-A2B2CD3 LNPs for the localized transdermal delivery of Cas9 mRNA and CD93 sgRNA in a murine model of psoriasis resulted in effective CD93 genome editing and the inhibition of the CD93-p38 MAPK-AKT-SMAD2/3 pathway, leading to significant therapeutic improvement. This work presents a robust, biocompatible LNP platform with minimized immunogenicity and strong potential for genome-editing therapies in inflammatory conditions, offering a transformative approach for the mRNA-based treatment of skin and other inflammation-related disorders.

Animals↗
PubMedDate not supplied

In-vivo particle mediated delivery of mRNA to mammalian tissues: ballistic and biologic effects.

Biolistic transmission of mRNA provides transient gene therapy to in vivo organs. This study documents particle mediated mRNA transmission to a solid organ and wound healing model using the mRNA of Green Fluorescent Protein to determine optimal delivery parameters. Renal function, bullet penetration, cellular injury, and Green Fluorescent Protein synthesis were quantified. Chimeric human epidermal growth factor-FLAG epitope cDNA or mRNA was transmitted to wounds in normal or steroid treated animals. Wound bursting strength, human epidermal growth factor-FLAG, and collagen synthesis were determined. Injury and bullet penetration correlated with the delivery velocity and bullet size. Optimal delivery parameters were established which provided widespread Green Fluorescent Protein synthesis. Human epidermal growth factor-FLAG treatment significantly increased collagen content and wound breaking strength in normal and steroid treated animals. FLAG protein synthesis was evident in mRNA treated fascia following treatment. We found the gene gun provides a novel method for efficient, in vivo delivery of mRNA-based therapeutic strategies to mammalian organs with minimal histologic damage allowing transient expression of protein in in vivo target tissues. Co-delivery of Green Fluorescent Protein mRNA may provide a useful positive control to determine effective transmission. Biolistic transmission of human epidermal growth factor-FLAG mRNA provides increased tissue epidermal growth factor levels and accelerates wound healing in normal and steroid exposed animals.

Animals↗
PubMed2004

Toxicology of antisense therapeutics.

Targeting unique mRNA molecules using antisense approaches, based on sequence specificity of double-stranded nucleic acid interactions should, in theory, allow for design of drugs with high specificity for intended targets. Antisense-induced degradation or inhibition of translation of a target mRNA is potentially capable of inhibiting the expression of any target protein. In fact, a large number of proteins of widely varied character have been successfully downregulated using an assortment of antisense-based approaches. The most prevalent approach has been to use antisense oligonucleotides (ASOs), which have progressed through the preclinical development stages including pharmacokinetics and toxicological studies. A small number of ASOs are currently in human clinical trials. These trials have highlighted several toxicities that are attributable to the chemical structure of the ASOs, and not to the particular ASO or target mRNA sequence. These include mild thrombocytopenia and hyperglycemia, activation of the complement and coagulation cascades, and hypotension. Dose-limiting toxicities have been related to hepatocellular degeneration leading to decreased levels of albumin and cholesterol. Despite these toxicities, which are generally mild and readily treatable with available standard medications, the clinical trials have clearly shown that ASOs can be safely administered to patients. Alternative chemistries of ASOs are also being pursued by many investigators to improve specificity and antisense efficacy and to reduce toxicity. In the design of ASOs for anticancer therapeutics in particular, the goal is often to enhance the cytotoxicity of traditional drugs toward cancer cells or to reduce the toxicity to normal cells to improve the therapeutic index of existing clinically relevant cancer chemotherapy drugs. We predict that use of antisense ASOs in combination with small molecule therapeutics against the target protein encoded by the antisense-targeted mRNA, or an alternate target in the same or a connected biological pathway, will likely be the most beneficial application of this emerging class of therapeutic agent.

Animals↗
PubMed1992

The antidepressants fluoxetine, idazoxan and phenelzine alter corticotropin-releasing hormone and tyrosine hydroxylase mRNA levels in rat brain: therapeutic implications.

Various classes of antidepressant drugs with distinct pharmacologic actions are differentially effective in the treatment of classic melancholic depression--characterized by pathological hyperarousal and atypical depression--associated with lethargy, hypersomnia, and hyperphagia. All antidepressant agents exert their therapeutic efficacy only after prolonged administration. In situ hybridization histochemistry was used to examine in rats the effects of short-term (2 weeks) and long-term (8 weeks) administration of 3 different classes of activating antidepressant drugs which tend to be preferentially effective in treating atypical depressions, on the expression of central nervous system genes thought to be dysregulated in major depression. Daily administration (5 mg/kg, i.p.) of the selective 5-hydroxytryptophan (5-HT) reuptake inhibitor fluoxetine, the selective alpha 2-adrenergic receptor antagonist idazoxan, and the nonspecific monoamine oxidase A and B inhibitor phenelzine increased tyrosine hydroxylase mRNA levels by 70-150% in the locus coeruleus after 2 weeks of drug and by 71-115% after 8 weeks. The 3 drugs decreased corticotropin-releasing hormone mRNA levels by 30-48% in the paraventricular nucleus of the hypothalamus. The decreases occurred at 8 weeks but not at 2 weeks. No consistent change in steroid hormone receptor mRNA levels was seen in the hippocampus with the 3 drugs, but fluoxetine and idazoxan increased the level of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA, respectively, after 8 weeks of drug administration. Proopiomelanocortin (POMC) mRNA levels in the anterior pituitary and plasma adrenocorticotropic-hormone (ACTH) levels were not altered after 2 or 8 weeks of drug treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed2002

The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number.

Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.

Animals↗
PubMed2005

Induction of BAALC and down regulation of RAMP3 in astrocytes treated with differentiation inducers.

Genes controlling proliferation and differentiation of astrocytes are likely to play an important role in the pathogenesis of astrocytic gliomas and could serve as therapeutic targets. mRNA differential display analysis identified two genes, viz. BAALC and RAMP3, whose expression is altered in primary astrocytes treated with differentiation inducers. BAALC, which has been reported to be expressed in neural progenitor cells, was found to be up regulated in both normal and malignant astrocytes on inhibition of proliferation. RAMP3 functions as a modulator of the receptor for adrenomedullin (AM). AM has been suggested to act as autocrine/paracrine growth factor for gliomas. Our studies show RAMP3 down regulation on staurosporine treatment of astrocytes, suggesting protein kinase C inhibition as a possible strategy for inhibiting AM activity and thereby growth of glioma cells.

Animals↗
PubMed2003

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis.

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare "readthrough" acetylcholinesterase variant AChE-R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE-R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE-R in blood and muscle yet leaves unaffected the synaptic variant AChE-S. Whereas animals treated with placebo or conventional anticholinesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA-targeted therapeutics for chronic cholinergic malfunctioning.

Acetylcholinesterase↗
PubMed2005

Targeting stathmin in prostate cancer.

Stathmin is the founding member of a family of microtubule-destabilizing proteins that regulate the dynamics of microtubule polymerization and depolymerization. Stathmin is expressed at high levels in a variety of human cancers and provides an attractive molecule to target in cancer therapies that disrupt the mitotic apparatus. We developed replication-deficient bicistronic adenoviral vectors that coexpress green fluorescent protein and ribozymes that target stathmin mRNA. The therapeutic potential of these recombinant adenoviruses was tested in an experimental androgen-independent LNCaP prostate cancer model. Adenovirus-mediated transfer of anti-stathmin ribozymes resulted in efficient transduction and marked inhibition of stathmin expression in these cells. Cells that were transduced with the anti-stathmin adenoviruses showed a dramatic dose-dependent growth inhibition. This was associated with accumulation of LNCaP cells in the G2-M phases of the cell cycle. A similar dose-dependent inhibition of clonogenic potential was also observed in cells infected with anti-stathmin adenoviruses. Morphologic and biochemical analysis of infected cells showed a marked increase in apoptosis characterized by detachment of the cells, increased chromatin condensation, activation of caspase-3, and fragmentation of internucleosomal DNA. If these findings are confirmed in vivo, it may provide an effective approach for the treatment of prostate cancer.

Adenoviridae↗
PubMed2000

The phosphoprotein Op18/stathmin is differentially expressed in ovarian cancer.

To identify potential prognostic indicators of ovarian cancer and identify targets for therapeutic strategies, mRNA differential display was used to analyze gene expression differences in normal, benign, and cancerous ovarian tissue. One cDNA isolated by this technique, Op18/stathmin, is a highly conserved gene that is reported to have many different functions within a cell, including signal transduction, control of the cell cycle, and the regulation of microtubules. Quantitative Northern blot analysis of 12 malignant ovarian samples, 8 benign ovarian tumors, and 10 normal ovarian tissue samples demonstrated overexpression of Op18/stathmin mRNA in the malignant cancers. Immunohistochemistry showed an apparent overexpression of Op18/stathmin protein level and an association with proliferating cells.

Blotting, Northern↗
PubMed1993

Interferon-gamma-induced differentiation of human neuroblastoma cells increases cellular uptake and halflife of metaiodobenzylguanidine.

Iodine labeled metaiodobenzylguanidine (MIBG) is a radiopharmaceutical employed for both diagnosis and metabolic radiotherapy of neuroblastoma (NB). Resistance to the radiotherapeutic effects of MIBG is common, due to lack of MIBG accumulation by NB cells. MIBG enters competent cells via the noradrenaline transporter; this function requires a relative cellular maturation and is missing in most NB cell lines. In vitro differentiation of NB cells can be achieved with gamma-interferon (gamma-IFN) and other agents. We have verified that gamma-IFN-induced differentiation of NB cells is specifically associated with an increase in their ability to incorporate MIBG. This phenomenon is due to enhancement of MIBG transporter activity, according to pharmacological sensitivity and semiquantitative PCR-based analysis of specific MIBG transporter mRNA. New therapeutic strategies based on both differentiation therapy and targeted radiotherapy of NB can so be devised.

3-Iodobenzylguanidine↗