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Nucleic acid therapeutics have shown great potential for the treatment of numerous diseases, such as genetic disorders, cancer and infections. Moreover, they have been successfully used as vaccines during the COVID-19 pandemic. In order to unfold full therapeutical potential, these nano agents have to overcome several barriers. Therefore, directed transport to specific tissues and cell types remains a central challenge to receive carrier systems with enhanced efficiency and desired biodistribution profiles. Active targeting strategies include receptor-targeting, mediating cellular uptake based on ligand-receptor interactions, and chemical targeting, enabling cell-specific delivery as a consequence of chemically and structurally modified carriers. With a focus on synthetic delivery systems including polyplexes, lipid-based systems such as lipoplexes and lipid nanoparticles, and direct conjugates optimized for various types of nucleic acids (DNA, mRNA, siRNA, miRNA, oligonucleotides), we highlight recent achievements, exemplified by several nucleic acid drugs on the market, and discuss challenges for targeted delivery to different organs such as brain, eye, liver, lung, spleen and muscle in vivo.

Polyethylenimine (PEI) is a gold standard polycationic transfectant. However, the highly efficient transfecting activity of PEI and many of its derivatives is accompanied by serious cytotoxic complications and safety concerns at innate immune levels, which impedes the development of therapeutic polycationic nucleic acid carriers in general and their clinical applications. In recent years, the dilemma between transfection efficacy and adverse PEI activities has been addressed from in-depth investigations of cellular processes during transfection and elucidation of molecular mechanisms of PEI-mediated toxicity and translation of these integrated events to chemical engineering of novel PEI derivatives with an improved benefit-to-risk ratio. This review addresses these perspectives and discusses molecular events pertaining to dynamic and multifaceted PEI-mediated cytotoxicity, including membrane destabilization, mitochondrial dysfunction, and perturbations of glycolytic flux and redox homeostasis as well as chemical strategies for the generation of better tolerated polycations. We further examine the effect of PEI and its derivatives on complement activation and interaction with Toll-like receptors. These perspectives are intended to lay the foundation for an improved understanding of interlinked mechanisms controlling transfection and toxicity and their translation for improved engineering of polycation-based transfectants. [Display omitted] Polycations such as polyethylenimines (PEIs) are widely used as non-viral transfectants, but they often induce cytotoxicity and may trigger immune reactions. Here we examine and discuss molecular events pertaining to dynamic and multifaceted PEI-mediated cytotoxicity and immune system modulation and their translation for improved and safer engineering of polycation-based transfectants.

Despite the great potential of DNA vaccines for a broad range of applications, ranging from prevention of infections, over treatment of autoimmune and allergic diseases to cancer immunotherapies, the implementation of such therapies for clinical treatment is far behind the expectations up to now. The main reason is the poor immunogenicity of DNA vaccines in humans. Consequently, the improvement of the performance of DNA vaccines in vivo is required. This mini‐review provides an overview of the current state of DNA vaccines and the various strategies to enhance the immunogenic potential of DNA vaccines, including (i) the optimization of the DNA construct itself regarding size, nuclear transfer and transcriptional regulation; (ii) the use of appropriate adjuvants; and (iii) improved delivery, for example, by careful choice of the administration route, physical methods such as electroporation and nanomaterials that may allow cell type‐specific targeting. Moreover, combining nanoformulated DNA vaccines with other immunotherapies and prime‐boost strategies may help to enhance success of treatment. The design of DNA vaccines and their mode of application need to be thoroughly improved to mount strong and sustained immune responses in humans, for example, by deleting prokaryotic sequences known to exert detrimental effects from the vector backbone, or by nanoformulated delivery that allows cell type‐specific transfection and codelivery of adjuvants.

Within the last decade, the introduction of checkpoint inhibitors proposed to boost the patients’ anti-tumor immune response has proven the efficacy of immunotherapeutic approaches for tumor therapy. Furthermore, especially in the context of the development of biocompatible, cell type targeting nano-carriers, nucleic acid-based drugs aimed to initiate and to enhance anti-tumor responses have come of age. This review intends to provide a comprehensive overview of the current state of the therapeutic use of nucleic acids for cancer treatment on various levels, comprising (i) mRNA and DNA-based vaccines to be expressed by antigen presenting cells evoking sustained anti-tumor T cell responses, (ii) molecular adjuvants, (iii) strategies to inhibit/reprogram tumor-induced regulatory immune cells e.g., by RNA interference (RNAi), (iv) genetically tailored T cells and natural killer cells to directly recognize tumor antigens, and (v) killing of tumor cells, and reprograming of constituents of the tumor microenvironment by gene transfer and RNAi. Aside from further improvements of individual nucleic acid-based drugs, the major perspective for successful cancer therapy will be combination treatments employing conventional regimens as well as immunotherapeutics like checkpoint inhibitors and nucleic acid-based drugs, each acting on several levels to adequately counter-act tumor immune evasion.

In nature, genomes have been optimized by the evolution of their nucleic acid sequences. The design of peptide-like carriers as synthetic sequences provides a strategy for optimizing multifunctional targeted nucleic acid delivery in an iterative process. The optimization of sequence-defined nanocarriers differs for different nucleic acid cargos as well as their specific applications. Supramolecular self-assembly enriched the development of a virus-inspired non-viral nucleic acid delivery system. Incorporation of DNA barcodes presents a complementary approach of applying sequences for nanocarrier optimization. This strategy may greatly help to identify nucleic acid carriers that can overcome pharmacological barriers and facilitate targeted delivery in vivo. Barcode sequences enable simultaneous evaluation of multiple nucleic acid nanocarriers in a single test organism for in vivo biodistribution as well as in vivo bioactivity.

Acquired resistance to classical chemotherapeutics is a major obstacle in cancer treatment. Doxorubicin is frequently used in breast cancer therapy either as single-agent or in combination with other drugs like docetaxel and cyclophosphamide. All these chemotherapies have in common that they are administered sequentially and often result in chemoresistance. Here, we mimicked this pulse therapy of breast cancer patients in an in vitro cell culture model, where the epithelial breast cancer cell line BT474 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype with decreased levels of miR-200c. To investigate the involvement of miR-200c in resistance formation, we inhibited and overexpressed miR-200c in different cell lines. Thereby, the cells were rendered more resistant or susceptible to doxorubicin treatment. Moreover, the receptor tyrosine kinase TrkB and the transcriptional repressor Bmi1 were identified as miR-200c targets mediating the drug resistance. Hence, we provide a mechanism of acquired resistance to doxorubicin that is caused by the loss of miR-200c. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance highlighting the challenges in cancer therapy and the importance of novel microRNA-modulating anticancer agents.

mRNA therapeutics have emerged as powerful tools for cancer immunotherapy in accordance with their superiority in expressing all sequence‐known proteins in vivo. In particular, with a small dosage of delivered mRNA, antigen‐presenting cells (APCs) can synthesize mutant neo‐antigens and multi‐antigens and present epitopes to T lymphocytes to elicit antitumor effects. In addition, expressing receptors like chimeric antigen receptor (CAR), T‐cell receptor (TCR), CD134, and immune‐modulating factors including cytokines, interferons, and antibodies in specific cells can enhance immunological response against tumors. With the maturation of in vitro transcription (IVT) technology, large‐scale and pure mRNA encoding specific proteins can be synthesized quickly. However, the clinical translation of mRNA‐based anticancer strategies is restricted by delivering mRNA into target organs or cells and the inadequate endosomal escape efficiency of mRNA. Recently, there have been some advances in mRNA‐based cancer immunotherapy, which can be roughly classified as modifications of the mRNA structure and the development of delivery systems, especially the lipid nanoparticle platforms. In this review, the latest strategies for overcoming the limitations of mRNA‐based cancer immunotherapies and the recent advances in delivering mRNA into specific organs and cells are summarized. Challenges and opportunities for clinical applications of mRNA‐based cancer immunotherapy are also discussed. Systemic or topical mRNA delivery for immune function modulation is emerging as a promising option for cancer immunotherapy. In this review, strategies for enhancing mRNA‐based cancer immunotherapy from the perspective of mRNA structure design and delivery systems are first summarized. Advances of delivering mRNA into specific organs or cells for cancer treatment and opportunities in clinical translation are discussed.

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing.

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of > 94 % w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of Z r 6 O 4 ( O H ) 4 turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the β -globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.

Cancer progression and metastases are frequently related to changes of cell motility. Amongst others, the microRNA-200c (miR-200c) was shown to maintain the epithelial state of cells and to hamper migration. Here, we describe two miR-200c inducible breast cancer cell lines, derived from miR-200c knock-out MCF7 cells as well as from the miR-200c-negative MDA-MB-231 cells and report on the emerging phenotypic effects after miR-200s induction. The induction of miR-200c expression seems to effect a rapid reduction of cell motility, as determined by 1D microlane migration assays. Sustained expression of miR200c leads to a changed morphology and reveals a novel mechanism by which miR-200c interferes with cytoskeletal components. We find that filamin A expression is attenuated by miRNA-200c induced downregulation of the transcription factors c-Jun and MRTF/SRF. This potentially novel pathway that is independent of the prominent ZEB axis could lead to a broader understanding of the role that miR200c plays in cancer metastasis.