Peptide nucleic acids (PNAs) are synthetic nucleic acid analogs with a neutral N-(2-aminoethyl) glycine backbone. PNAs possess unique physicochemical characteristics such as increased resistance to enzymatic degradation, ionic strength and stability over a wide range of temperatures and pH, and low intrinsic electrostatic repulsion against complementary target oligonucleotides. PNA has been widely used as an antisense oligonucleotide (ASO). Despite the favorable characteristics of PNA, in comparison with other ASO technologies, the use of antisense PNA for novel therapeutics has lagged. This review provides a brief overview of PNA, its antisense mechanisms of action, delivery strategies, and highlights successful applications of PNA, focusing on anti-pathogenic, anti-neurodegenerative disease, anti-cancer, and diagnostic agents. For each application, several studies are discussed focusing on the different target sites of the PNA, design of different PNAs and the therapeutic outcome in different cell lines and animal models. Thereafter, persisting limitations slowing the successful integration of antisense PNA therapeutics are discussed in order to highlight actionable next steps in the development and optimization of PNA as an ASO.

The EphA2 receptor tyrosine kinase is overexpressed in most solid tumors and acts as the major driver of tumorigenesis. In this study, we developed a novel approach for targeting the EphA2 receptor using a 2\'-fluoro-modified pyrimidine RNA aptamer termed ATOP. We identified the ATOP EphA2 aptamer using a novel bioinformatics strategy that compared aptamers enriched during a protein SELEX using recombinant human EphA2 and a cell-internalization SELEX using EphA2-expressing MDA231 tumor cells. When applied to EphA2-expressing tumor cell lines, the ATOP EphA2 aptamer attenuated tumor cell migration and clonogenicity. In a mouse model of spontaneous metastasis, the ATOP EphA2 aptamer slowed primary tumor growth and significantly reduced the number of lung metastases. The EphA2 ATOP aptamer represents a promising candidate for the development of next-generation targeted therapies that provide safer and more effective treatment of EphA2-overexpressing tumors.

Vascularized composite allotransplantation has great potential in face transplantation by supporting functional restoration following tissue grafting. However, the need for lifelong administration of immunosuppressive drugs still limits its wide use. Modified mRNA (modRNA) technology provides an efficient and safe method to directly produce protein in vivo. Nevertheless, the use of IL-10 modRNA-based protein replacement, which exhibits anti-inflammatory properties, has not been shown to prolong composite facial allograft survival. In this study, IL-10 modRNA was demonstrated to produce functional IL-10 protein in vitro, which inhibited pro-inflammatory cytokines and in vivo formation of an anti-inflammatory environments. We found that without any immunosuppression, C57BL/6J mice with fully major histocompatibility complex (MHC)-mismatched facial allografts and local injection of IL-10 modRNA had a significantly prolonged survival rate. Decreased lymphocyte infiltration and pro-inflammatory T helper 1 subsets and increased anti-inflammatory regulatory T cells (Tregs) were seen in IL-10 modRNA-treated mice. Moreover, IL-10 modRNA induced multilineage chimerism, especially the development of donor Treg chimerism, which protected allografts from destruction because of recipient alloimmunity. These results support the use of monotherapy based on immunomodulatory IL-10 cytokines encoded by modRNA, which inhibit acute rejection and prolong allograft survival through the induction of donor Treg chimerism.

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the β-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) β-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the β-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human β-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Skin injuries and chronic non-healing wounds are one of the major global burdens on the healthcare systems worldwide due to their difficult-to-treat nature, associated co-morbidities, and high health care costs. Angiogenesis has a pivotal role in the wound-healing process, which becomes impaired in many chronic non-healing wounds, leading to several healing disorders and complications. Therefore, induction or promotion of angiogenesis can be considered a promising approach for healing of chronic wounds. Gene therapy is one of the most promising upcoming strategies for the treatment of chronic wounds. It can be classified into three main approaches: gene augmentation, gene silencing, and gene editing. Despite the increasing number of encouraging results obtained using nucleic acids (NAs) as active pharmaceutical ingredients of gene therapy, efficient delivery of NAs to their site of action (cytoplasm or nucleus) remains a key challenge. Selection of the right therapeutic cargo and delivery methods is crucial for a favorable prognosis of the healing process. This article presents an overview of gene therapy and non-viral delivery methods for angiogenesis induction in chronic wounds.

Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy.

The use of messenger RNA (mRNA) enables the transient production of therapeutic proteins with stable and predictable translational kinetics and without the risk of insertional mutagenesis. Recent findings highlight the enormous potential of mRNA-based therapeutics. Here, we describe the synthesis of chemically modified thrombopoietin (TPO) mRNA through in vitro transcription and in vivo delivery via lipid nanoparticles (LNPs). After delivery of TPO mRNA in mice, compared with normal physiological values, plasma TPO protein levels increased over 1000-fold in a dose-dependent manner. Moreover, through a single intravenous dose of TPO mRNA-loaded LNPs, both reticulated and total platelet count increased significantly in mice, demonstrating that TPO protein derived from the exogenous mRNA was able to maintain normal activity. Submicrogram quantity of N1-methylpseudouridine-modified TPO mRNA showed a similar effect in promoting thrombopoiesis as that by the TPO receptor agonist romiplostim. In addition, a therapeutic value was established in anti-GPIbα (CD42b) antibody-induced thrombocytopenia mouse models that showed a fast recovery of platelet count. Our study demonstrated chemically modified in-vitro-transcribed TPO mRNA as a potentially safe therapeutic intervention to stimulate thrombopoiesis.

Traditional Chinese medicines (TCMs) have been widely used for treating ischemic heart disease (IHD), and secondary metabolites are generally regarded as their pharmacologically active components. However, the effects of nucleic acids in TCMs remain unclear. We reported for the first time that a 22-mer double-strand RNA consisting of HC83 (a tRNA-derived fragment [tRF] from the 3\' end of tRNAGln(UUG) of ginseng) and its complementary sequence significantly promoted H9c2 cell survival after hypoxia/reoxygenation (H/R) in vitro. HC83_mimic could also significantly improve cardiac function by maintaining both cytoskeleton integrity and mitochondrial function of cardiomyocytes. Further in vivo investigations revealed that HC83_mimic is more potent than metoprolol by >500-fold against myocardial ischemia/reperfusion (MI/R) injury. In-depth studies revealed that HC83 directly downregulated a lncRNA known as myocardial infarction-associated transcript (MIAT) that led to a subsequent upregulation of VEGFA expression. These findings provided the first evidence that TCM-derived tRFs can exert miRNA-like functions in mammalian systems, therefore supporting the idea that TCM-derived tRFs are promising RNA drug candidates shown to have extraordinarily potent effects. In summary, this study provides a novel strategy not only for discovering pharmacologically active tRFs from TCMs but also for efficiently exploring new therapeutic targets for various diseases.