Molecular Therapy. Nucleic Acids最新文献

RNA modifications play a crucial role in regulating gene expression by altering RNA structure and modulating interactions with RNA-binding proteins (RBPs). In this study, we explore the impact of specific RNA chemical modifications-N⁶-methyladenosine (m⁶A), A-to-I editing, and pseudouridine (Ψ)-on RNA secondary structure and protein-RNA interactions. Utilizing genome-wide data, including RNA secondary structure predictions and protein-RNA interaction datasets, we classify proteins into distinct categories based on their binding behaviors: modification specific and structure independent, or modification unspecific and structure dependent. For instance, m⁶A readers such as YTHDF2 exhibit modification-specific and structure-independent binding, consistently recognizing m⁶A regardless of structural changes. Conversely, proteins such as U2AF2 display modification-unspecific and structure-dependent behavior, altering their binding preferences in response to structural changes induced by different modifications. A-to-I editing, which causes significant structural changes, typically reduces protein interactions, while Ψ enhances RNA structural stability, albeit with variable effects on protein binding. To predict these interactions, we developed the catRAPID 2.2 RNA modifications algorithm, which computes the effects of RNA modifications on protein-RNA binding propensities. This algorithm enables the prediction and analysis of RNA modifications' impact on protein interactions, offering new insights into RNA biology and engineering.

The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral titers compared with wild-type BaEV, but vector production is impaired by syncytia formation and cell death of the HEK293T cells due to the high fusogenic activity of the glycoprotein. This lowers viral titers, leads to increased batch-to-batch variability, and impedes the establishment of stable packaging cell lines essential for the economical production of viral supernatants. Here, we show that knockout of the entry receptor ASCT2 in HEK293T producer cells eliminates syncytia formation, resulting in a 2-fold increase in viral titers, reduced toxicity of viral supernatants, and enables the generation of stable packaging cell lines. In successive steps, we stably integrated BaEVRLess and α-retroviral a.Gag/Pol expression cassettes and isolated clones supporting titers up to 10⁸ to 10⁹ infectious particles/mL, a 10-fold increase in concentrated viral titers. The additional overexpression of CD47 and knockout of β2-microglobulin in the packaging cell line are tailored for future use in in vivo gene therapy applications by reducing non-specific uptake by macrophages and the immunogenicity of viral particles.

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications.

Prime editors are CRISPR-based genome engineering tools with significant potential for rectifying patient mutations. However, their usage requires experimental optimization of the prime editing guide RNA (PegRNA) to achieve high editing efficiency. This paper introduces the deep transformer-based model for predicting prime editing efficiency (DTMP-Prime), a tool specifically designed to predict PegRNA activity and prime editing (PE) efficiency. DTMP-Prime facilitates the design of appropriate PegRNA and ngRNA. A transformer-based model was constructed to scrutinize a wide-ranging set of PE data, enabling the extraction of effective features of PegRNAs and target DNA sequences. The integration of these features with the proposed encoding strategy and DNABERT-based embedding has notably improved the predictive capabilities of DTMP-Prime for off-target sites. Moreover, DTMP-Prime is a promising tool for precisely predicting off-target sites in CRISPR experiments. The integration of a multi-head attention framework has additionally improved the precision and generalizability of DTMP-Prime across various PE models and cell lines. Evaluation results based on the Pearson and Spearman correlation coefficient demonstrate that DTMP-Prime outperforms other state-of-the-art models in predicting the efficiency and outcomes of PE experiments.

Immune checkpoint blockade (ICB) therapy has significantly benefited patients with several types of solid tumors and some lymphomas. However, many of the treated patients do not have a durable clinical response. It has been demonstrated that rescuing exhausted CD8⁺ T cells is required for ICB-mediated antitumor effects. We recently developed an immunostimulatory strategy based on silencing STAT3 while stimulating immune responses by CpG, a ligand for Toll-like receptor 9 (TLR9). The CpG-small interfering RNA (siRNA) conjugates efficiently enter immune cells, silencing STAT3 and activating innate immunity to enhance T cell-mediated antitumor immune responses. In the present study, we demonstrate that blocking STAT3 through locally delivered CpG-Stat3 siRNA enhances the efficacies of the systemic PD-1 and CTLA4 blockade against mouse A20 B cell lymphoma. In addition, locally delivered CpG-Stat3 siRNA combined with systemic administration of PD-1 antibody significantly augmented both local and systemic antitumor effects against mouse B16 melanoma tumors, with enhanced tumor-associated T cell activation. Furthermore, locally delivered CpG-Stat3 siRNA enhanced CD8⁺ T cell tumor infiltration and antitumor activity in a xenograft tumor model. Overall, our studies in both B cell lymphoma and melanoma mouse models demonstrate the potential of combinatory immunotherapy with CpG-Stat3 siRNA and checkpoint inhibitors as a therapeutic strategy for B cell lymphoma and melanoma.

Pronounced T cell exhaustion characterizes immunosuppressive tumors, with the tumor microenvironment (TME) employing multiple mechanisms to elicit this suppression. Traditional immunotherapies, such as immune checkpoint blockade, often fail due to their focus primarily on T cells. To overcome this, we utilized a proinflammatory cytokine, interleukin (IL)-12, that re-wires the immunosuppressive TME by inducing T cell effector function while also repolarizing immunosuppressive myeloid cells. Due to toxicities observed with systemic administration of this cytokine, we utilized lipid nanoparticles encapsulating mRNA encoding IL-12 for intratumoral injection. This strategy has been proven safe and tolerable in early clinical trials for solid malignancies. We report an unprecedented loss of exhausted T cells and the emergence of an activated phenotype in murine pancreatic ductal adenocarcinoma (PDAC) treated with stereotactic body radiation therapy (SBRT) and IL-12mRNA. Our mechanistic findings reveal that each treatment modality contributes to the T cell response differently, with SBRT expanding the T cell receptor repertoire and IL-12mRNA promoting robust T cell proliferation and effector status. This distinctive T cell signature mediated marked growth reductions and long-term survival in local and metastatic PDAC models. This is the first study of its kind combining SBRT with IL-12mRNA and provides a promising new approach for treating this aggressive malignancy.

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk _ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.

Myotonic dystrophy type 1 (DM1), the leading cause of adult-onset muscular dystrophy, is caused by a CTG repeat expansion. Expression of the repeat causes widespread alternative splicing (AS) defects and downstream pathogenesis, including significant skeletal muscle impacts. The HSA ^LR mouse model plays a significant role in therapeutic development. This mouse model features a transgene composed of approximately 220 interrupted CTG repeats, which results in skeletal muscle pathology that mirrors DM1. To better understand this model and the growing number of therapeutic approaches developed with it, we performed a meta-analysis of publicly available RNA sequencing data for AS changes across three widely examined skeletal muscles: quadriceps, gastrocnemius, and tibialis anterior. Our analysis demonstrated that transgene expression correlated with the extent of splicing dysregulation across these muscles from gastrocnemius (highest), quadriceps (medium), to tibialis anterior (lowest). We identified 95 splicing events consistently dysregulated across all examined datasets. Comparison of splicing rescue across seven therapeutic approaches showed a range of rescue across the 95 splicing events from the three muscle groups. This analysis contributes to our understanding of the HSA ^LR model and the growing number of therapeutic approaches currently in preclinical development for DM1.