Molecular Therapy. Nucleic Acids最新文献

Nucleic acid molecules are emerging as potential therapeutic tools, as evidenced by the transfection of small interfering RNA (siRNA) molecules in therapeutic applications and messenger RNAs in immunotherapeutic vaccination. In most cases, these nucleic acids are conditioned as lipid nanoparticles made with different lipid moieties to promote their intracellular delivery. Over the past few years, we have documented the delivery of siRNAs using a single short (15 amino acids) peptide called WRAP5, which follows an extremely simplified formulation phase that enables the formation of nanoparticles with a diameter of 60-80 nm. We indeed demonstrated the expected dose-response reduction in the levels of the targeted proteins. To apply this technology to the cellular delivery of mRNAs, we investigated the ability of the WRAP5 peptide to transfect mRNAs of different sizes and promote the expression of their proteins. These peptide-based nanoparticles, which also have diameters ranging from 60 to 80 nm, showed remarkable stability over time when simply stored at 4°C and fully retained their transfection properties in vitro for up to several months post-formulation. Interestingly, we demonstrated in vivo that these nanoparticles were able to induce an immune response against the protein synthesized from the vectorized mRNA.

CRISPR-Cas9-based genome editors can precisely target and edit genes efficiently. However, prolonged Cas9 activity poses challenges for laboratory experiments and raises safety concerns for therapeutic applications due to unintended consequences such as off-target editing, genotoxicity, immunogenicity, and undesired on-target modifications. Here, we evaluate a novel molecular glue degradation system, called Cas9-degron (Cas9-d), designed to degrade Cas9 in the presence of the US Food and Drug Administration (FDA)-approved drug, pomalidomide (POM). This system is highly biocompatible and rapidly reduces Cas9 protein levels within 4 h of induction, resulting in a 3- to 5-fold decrease in editing at on-target sites. The reduction is reversible, as Cas9 levels are restored within 24 h after POM withdrawal. Without initiating degradation, the on-target editing efficiency and accuracy of the Cas9-d system remain intact in different human cell types, including hepatic cell lines and human induced pluripotent stem cell (hiPSC)-derived GABAergic neurons. Cells edited with the Cas9-d system were healthy and functional, exhibiting minimal toxicity from using the strategy. The Cas9-d system provides a versatile approach to adjust Cas9 levels, demonstrating its potential as an experimental tool for controlling genome editing outcomes in vitro and ex vivo. With further development, it holds promise for enhancing somatic cell genome editing in vivo.

We used kidney-targeted, non-viral, transposon-mediated gene delivery to express the mouse Slc3a1 transgene in one kidney of cystinuria type A (Slc3a1 ^-/-) mice. We found a 44% reduction in urinary cystine concentration at 154 days post-gene transfer, although there was no significant effect on cystine stone formation. Our results indicate that it is possible to achieve kidney-targeted gene transfer, resulting in reduction of cystine concentration in the urine of a cystinuria type A animal model. This proof of concept lays the foundation for future studies directed at gene therapy for cystinuria and other kidney diseases.

The coronavirus disease 2019 pandemic has highlighted the critical need for thermostable vaccines to ensure equitable distribution and accessibility, particularly in regions lacking cold chain infrastructure. Here we present a thermostable, solid dose DNA vaccine (SDV) platform for subcutaneous delivery, based on a sugar-sugar alcohol-polymer formulation manufactured via lyophilization and compaction. Using luciferase-expressing plasmid as a model, we demonstrate that subcutaneous vaccination with SDV formulation of C57BL/6 mice results in efficient and durable transgene expression in vivo. In vitro stability assays confirmed that the SDV formulation maintained excellent thermostability after 30 days of storage at 4°C, 25°C, 37°C, and 42°C. We next applied the SDV platform to a Zika virus (ZIKV) NS1 DNA vaccine and immunized BALB/c mice. ZIKV-SDV vaccination elicited robust NS1-specific antibody and T cell responses, and conferred protection upon ZIKV challenge. These data establish the feasibility of lyophilized SDV DNA vaccines for needle-free thermostable delivery. By eliminating the need for reconstitution, refrigeration, and skilled administration, SDV formulation has the potential to enhance the deployment, cost effectiveness, and shelf-life of DNA vaccines in resource-limited settings.

Recent advances in gene-editing technologies offer new opportunities for drug development to treat unmet medical needs in central nervous system (CNS) disorders including neurogenerative diseases of the aging brain. The adeno-associated virus (AAV) is a promising and most widely utilized vector for gene therapy application including the CNS. AAV is characterized by high transduction efficiency in both dividing and non-dividing cells, low immunogenicity and toxicity, and exceptional tissue specificity. The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based technologies has revolutionized all aspects of modern sciences and created an innovative therapeutic toolkit with the potential to address a wide range of neurological diseases, including Alzheimer's (AD) and Parkinson's (PD) diseases. However, AAV limitations for delivering CRISPR modalities continue to impede viable therapeutic interventions targeting the brain. This review highlights challenges and strategies to deliver AAV-CRISPR-based therapeutic cargos for gene therapy applications in the CNS, with a particular focus on AD and PD preclinical studies.

Circular RNA (circRNA) offers significant advantages in stability, storage, manufacturing, and pharmacokinetics, making it an attractive option for therapeutic applications over linear RNA. However, the commonly used permuted intron-exon (PIE) method for constructing circRNA introduces an exogenous "scar" sequence during splicing initiation, potentially compromising circRNA potency and inducing immunogenicity. Through exploration of the molecular mechanism of the Anabaena group I intron splicing, we conclude the sequence characterization of splice sites and the recognition rules of IG sequence. Leveraging these principles, we successfully prepared "scarless" circRNA without sacrificing its circularization efficiency using standard in vitro transcription procedures. Immunogenicity analysis of scarless circRNAs revealed that the scar will not induce an immune response, consistent with previous findings, after complete removal of linear byproducts, that circRNAs are naturally low immunogenicity. Finally, we find that the incorporation of modified nucleotides in circRNAs disrupts not only splicing function but also internal ribosome entry site function, with a low percentage of modified nucleotides destroying translation capacity.

Therapeutic vaccines are promising for cancer immunotherapy in combination with immune checkpoint blockade (ICB). Though lipid nanoparticles (LNPs) hold great potential to deliver cancer therapeutic vaccines, LNPs delivering peptide or mRNA vaccines often induce suboptimal T cell responses. Type I interferon (IFN-I) responses can enhance antigen presentation and potentiate T cell responses. Here, we report LNP codelivery of peptide or mRNA vaccines with a cyclic GMP-AMP synthase (cGAS) agonist that can specifically induce IFN-I responses to potentiate anticancer T cell responses for robust ICB combination immunotherapy of tumors. Svg3, an oligonucleotide-based cGAS agonist, can be efficiently coloaded with antigenic peptides or antigen-encoding mRNAs into LNPs and codelivered to mouse draining lymph nodes and antigen-presenting cells (APCs). Svg3 promoted the antigen presentation and antigen-specific CD8⁺ T cell responses in mice. The combination of LNP-delivered Svg3 with peptide or mRNA encoding antigens promotes anti-tumor responses, reduces immune suppression, and enhances tumor therapeutic efficacy when combined with ICB.

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.