Molecular Therapy. Nucleic Acids最新文献

Challenges with vaccine reactogenicity, stability, and access have highlighted the need to develop alternative strategies for formulation and delivery. We explored the incorporation of cucurbit[n]urils (CBs), as supramolecular "hosts," into nucleic acid-polymer polyplexes. CBs are small, non-toxic, barrel-shaped molecules that transiently crosslink polymers containing supramolecular "guests," thereby increasing molecular weight (MW) of the complex, a correlate of transfection efficiency. We tested whether the supramolecular interactions of CB[8] impact polyplex function. We generated a library of different CB[8] polyplexes using plasmid DNA (pDNA), varying N/P (the ratio of polymer to plasmid), the length, and guest (phenylalanine [Phe]) group frequency of the polyethylenimine (PEI) polymer backbone. We found that N/P 32 and the 20Phe1 (20kDa PEI with 1 mol% Phe) gave optimal gene expression and that incorporating CB[8] in polyplex formulations improved gene expression, both in vitro and in vivo. Despite increases in gene expression, inclusion of CB[8] in formulations with higher guest-binding capacity led to decreased immunogenicity, possibly as a result of dampened innate immune responses. Our data show that CB[8] polyplexes increase gene delivery and expression but alter inflammatory responses. These findings highlight that rational design of the CB[8] polymer system can enable nucleic acid delivery for both vaccine and therapeutic applications.

Acute myeloid leukemia (AML) is a highly aggressive blood cancer marked by impaired differentiation and uncontrolled proliferation of myeloid cells. This phenotype is often driven by dysregulated expression of the transcription factor C/EBPα (encoded by CEBPA), especially in high-risk subtypes with FLT3 mutations. We hypothesized that RNA activation (RNAa) of CEBPA could reduce the growth of FLT3-mutated AML, and synergize with currently approved FLT3 inhibitors, thereby offering an alternative treatment strategy for a deadly disease. Our study shows that MTL-CEBPA, a chemically modified small activating RNA encapsulated in NOV340 liposomes, selectively targets myeloid cells, boosts CEBPA expression, and promotes a non-proliferative, mature state in FLT3-mutated AML cells. Importantly, MTL-CEBPA enhances the efficacy of commonly prescribed FLT3 inhibitor, gilteritinib, both in vitro and in vivo. All together, these findings support RNAa of CEBPA as a potential adjuvant therapy for FLT3-mutated AML.

Current seasonal influenza vaccines offer strain-specific protection and, thus, are less effective against mismatched strains. A broadly protective influenza vaccine is desirable to provide comprehensive protection against a wide range of influenza viruses for seasonal and pandemic influenza preparedness. Here, we evaluated the vaccine candidates based on bovine adenoviral (BAd) vectors expressing nucleoprotein (NP) of influenza A (BAd-C5-NP/A) and B (BAd-C5-NP/B) viruses linked to the autophagy-inducing peptide C5 (AIP-C5 or C5) to develop a predominantly T-cell-based vaccine. Robust cellular immune responses and humoral responses were elicited in mice with a single intranasal inoculation. Mice immunized with the BAd Bivalent (BAd-C5-NP/A + BAd-C5-NP/B) vaccine formulation exhibited protective immunity, providing protection against a broad panel of homosubtypic and heterosubtypic influenza A and B viruses, as evidenced by the absence of morbidity and mortality, along with significant reductions in lung viral titers. Protective immunity against seasonal influenza viruses was observed in ferrets following the BAd Bivalent vaccine immunization. These findings support further investigation of the potential of a unique Ad vaccine platform for mucosal immunization expressing NP linked to AIP-C5 as a broadly protective influenza vaccine.

Small interfering RNA (siRNA) therapeutics are a new class of drugs that is rapidly expanding to tackle various diseases. Extrahepatic delivery of siRNAs, especially to the parenchyma of solid tumors, is challenging with multiple strategies being explored such as lipid nanoparticle based delivery and ligand conjugation strategies. Here, we report that an albumin-binding dendritic siRNA (D-siRNA) boosts blood circulation time following systemic administration, leading to improved delivery and silencing activity in a melanoma tumor model, in comparison to non-albumin binding lipophilic siRNAs. D-siRNAs increased the tumor-to-liver delivery ratio, including both immune and non-immune cell types within the tumor parenchyma. Using D-siRNAs to target JAK1 expression as an adjuvant to immune checkpoint inhibitors, we found that D-siRNAs was able to enhance PD1 antibody treatment and slow tumor progression of melanoma. Thus, this work demonstrates the utility of D-siRNAs as a systemically administered tumor delivery strategy, enabling the use of siRNAs as chemotherapeutic agents. Further mechanistic studies into the role of JAK1 in melanoma pathology and progression may expand this into additional targets as potential treatments.

The 5'Cap is a modified nucleotide structure added to the 5'end of mRNA, which plays a critical role in stability and translation of the molecule. Accurate characterization of 5'Cap impurities is crucial during development of mRNA therapeutics. Here, we utilized orthogonal liquid chromatography-mass spectrometry (LC-MS) workflows to characterize Cap-1-modified mRNA. A complex mixture of uncapped and, notably, 5'Cap chemical degradation variants, was detected. We subjected Cap-1 analogs to process-related forced degradation conditions and characterized multiple degradation routes of the 5'Cap structure, including hydrolysis and depurination. 5'Cap hydrolysis was observed in a representative in vitro transcription (IVT) reaction buffer and was driven by factors including pH, temperature, and the presence of spermidine and MgCl₂. Method-induced artifactual degradation occurred during LC-MS data acquisition, emphasizing the importance of using reference standards and optimizing methods to reduce such artifacts. We investigated the effect of 5'Cap degradation on expression and reactogenicity in vitro using model mRNAs with elevated impurity levels. mRNA 5'Cap degradation impurities significantly impacted protein expression but did not trigger innate immune response pathways. 5'Cap degradation impurities should be considered as a critical quality attribute of mRNA therapeutics and should be monitored during production, purification, formulation, and storage.

In this study, we explore the therapeutic feasibility of globotriaosylceramide (Gb3) synthase (A4GALT)-specific siRNA-loaded polyhistidine (pHis)-incorporated lipid nanoparticles (HLNPs) for Fabry disease (FD). HLNPs were developed to deliver siRNAs targeting A4GALT using a microfluidic device, with pHis aiding in endosome escape. The therapy was tested on GLA-knockout human-induced pluripotent-stem-cell-derived endothelial cells (GLA-KO-hiPSC-ECs) and podocytes (GLA-KO-hiPSC-PCs). GLA-KO-hiPSCs-ECs or -PCs, upon differentiation, were treated with A4GALT-siRNA-HLNP. Successful intracellular uptake of A4GALT-siRNA-HLNP was confirmed through fluorescence and electron microscopy in both cell types. A4GALT-siRNA-HLNP treatment confirmed both cell types' stability at 5 μg/mL. Increased Gb3 deposition and zebra body formation were detected in both cell types, but A4GALT-siRNA-HLNP treatment attenuated these FD phenotypes, demonstrating reduced expression of A4GALT through western blot analysis. RNA sequencing analysis revealed that the expression of transcripts associated with FD was restored by A4GALT-siRNA-HLNP treatment in GLA-KO-hiPSCs-ECs, whereas in GLA-KO-hiPSCs-PCs, this effect was relatively less pronounced. Suppression of A4GALT via siRNA/HLNP treatment significantly rescued FD phenotypes especially in EC, presenting a novel therapeutic approach for FD.

The filamentous phage M13 is a single-stranded DNA phage with several attractive characteristics for gene delivery, including a capsid amenable to the display of foreign peptides and a simple well-characterized genome that is easy to genetically modify. Previously, we constructed a DNA minivector based on M13 (a miniphagemid), which minimized the inflammatory bacterial and phage DNA content in the vector. In general, DNA minivectors devoid of their prokaryotic components have shown improved gene transfer and safety. We examined the miniphagemid's capacity for in vitro transgene delivery to target cells through phage display of epidermal growth factor to target its cognate receptor. The absence of the prokaryotic backbone and smaller vector size conferred by the miniphagemids were associated with improved transgene expression for purified single-stranded phagemid DNA and phagemid virion particles. We further engineered this system to enhance packaging of DNA minivectors via deletion of the packaging signal within the helper plasmid used to produce miniphagemids and observed improved phage-mediated gene expression in mammalian cells. Overall, we present a set of novel transgene delivery vectors that combine cell-targeting ligand display and vector minimization. This platform showcases the flexibility of M13 as a gene delivery tool with immense therapeutic potential.