Tetrahedral DNA nanostructures (TDNs) are emerging as next-generation platforms for delivering therapeutic oligonucleotides. This study introduces a novel strategy embedding antisense oligonucleotide (ASO) sequences directly within the structural framework of TDNs, not requiring external extensions as hybridization handles and preserving flexibility for functionalization. The integration of a gapmer-based design enables structural reconfiguration upon cellular delivery, promoting ASO accessibility and efficient target engagement. To validate this approach, we engineered a TDN-gapmer targeting microRNA-21 (miR-21), a dysregulated biomarker linked to glioblastoma. Without requiring transfection agents, TDN-gapmer demonstrated autonomous delivery capacity into glioblastoma cells, leading to robust miR-21 inhibition. Mechanistic studies revealed that the integrated gapmer can potentially recruit ribonuclease (RNase) H, facilitating RNA cleavage and enhancing target suppression. Coarse-grained modeling provided a detailed view of the predicted structural transitions and thermodynamic parameters that demonstrate that cleavage initiates toehold formation, enabling strand displacement and potential catalytic reconfiguration of the nanostructures. The TDN-gapmer demonstrated stability in serum, withstanding degradation while maintaining its therapeutic potential. The ability to integrate active sequences into the structural framework increases availability for further potential multifunctionalization. This innovative TDN design establishes a versatile and transformative platform with promising implications for precision RNA-targeting therapeutics.

The successful translation of therapeutic nucleic acids (NAs) for the treatment of neurological disorders depends on their safe and efficient delivery to neural cells, in particular neurons. DNA nanostructures can be a promising NAs delivery vehicle. Nonetheless, the potential of DNA nanostructures for neuronal cell delivery of therapeutic NAs is unexplored. Here, tetrahedral DNA nanostructures (TDN) as siRNA delivery scaffolds to neuronal cells, exploring the influence of functionalization with two different reported neuronal targeting ligands: C4-3 RNA aptamer and Tet1 peptide are investigated. Nanostructures are characterized in vitro, as well as in silico using molecular dynamic simulations to better understand the overall TDN structural stability. Enhancement of neuronal cell uptake of TDN functionalized with the C4-3 Aptamer (TDN-Apt), not only in neuronal cell lines but also in primary neuronal cell cultures is demonstrated. Additionally, TDN and TDN-Apt nanostructures carrying siRNA are shown to promote silencing in a process aided by chloroquine-induced endosomal disruption. This work presents a thorough workflow for the structural and functional characterization of the proposed TDN as a nano-scaffold for neuronal delivery of therapeutic NAs and for targeting ligands evaluation, contributing to the future development of new neuronal drug delivery systems based on DNA nanostructures. Here, tetrahedral DNA nanostructures (TDN) are evaluated as nano-scaffolds for neuronal cell delivery of siRNA, exploring the functionalization with neuronal ligands (C4-3 aptamer and Tet1 peptide). Aptamer-functionalized TDN shows enhanced uptake in neuronal cells, and mediates efficient gene silencing when in the presence of an endosomal disruption agent. image

The RNA interference (RNAi) chemical and structural design space has evolved since its original definitions. Although this has led to the development of RNAi molecules that are starting to address the issues of silencing efficiency and delivery to target organs and cells, there is an on-going interest to improve upon their properties to attain wider therapeutic applicability. Taking advantage of the flexibility given by DNA and RNA structural and chemical properties, we here investigated unconventional RNAi encoding structures, designated by caged-siRNA structures (CsiRNAs), to explore novel features that could translate into advantageous properties for cellular delivery and intracellular activity. Using the principles of controlled nucleic acid self-assembly, branched DNA-RNA hybrid intermediates were formed, ultimately leading to the assembly of a "closed" structure encompassing multiple RNAi units. The RNAi active regions are further triggered by an encoded RNAse H-mediated release mechanism, while the overall structure possesses easily addressable anchors for hybridization-based functionalization with active biological moieties. We confirmed the production of correct structures and demonstrated that the encoded RNAi sequences maintain gene silencing activity even within this novel unconventional nanoarchitecture, aided by the intracellularly triggered RNAse H release mechanism. With this design, functionalization is easily achieved with no negative effects on the silencing activity, warranting further development of these novel molecular structures as a multi-RNAi platform for therapeutic delivery.

RNA interference technology (RNAi) has recently picked up momentum with the approval of the first RNAi drug for treatment of polyneuropathy caused by hereditary transthyretin-mediated amyloidosis. With the potential for small interfering RNA (siRNA) therapeutics unleashed, other applications are currently sought that can benefit from this technology. Neurological disorders are seen as an area that could immensely benefit from siRNA therapeutics. However, its development has been hampered by the lack of reliable and nontoxic delivery methods to the central nervous system (CNS). One particularly promising option for siRNA delivery entails the use of siRNA-bioconjugate systems, which have garnered exceptionally interesting results for liver applications. While promising, its development for CNS has been lagging. Thus, envisioning new developments for CNS applications, we here discuss specific delivery issues affecting therapeutic drug and nucleic acids delivery to the CNS, as well as some of the technological advancements on nucleic acids modifications and structural alterations applied to siRNA in the context of the bioconjugate systems approach.

Biomaterials-based hydrogels are attractive drug-eluting vehicles in the context of RNA therapeutics, such as those utilizing antisense oligonucleotide or RNA interference based drugs, as they can potentially reduce systemic toxicity and enhance in vivo efficacy by increasing in situ concentrations. Here we describe the preparation of antisense oligonucleotide-loaded fibrin hydrogels exploring their applications in the context of the nervous system utilizing an organotypic dorsal root ganglion explant in vitro system and an in vivo model of spinal cord injury.

After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels, allowing the regulation of cell survival and cell function, together with the availability of chemically modified nucleic acids with favorable biopharmaceutical properties, make AONs an attractive tool for novel SCI therapy developments. In this work, we explored the potential of locked nucleic acid (LNA)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration—RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system mediated potent RNA knockdown in vitro in a dorsal root ganglion explant culture system and in vivo at a SCI lesion site, achieving around 75% downregulation 5 days after hydrogel injection. Our results show that local implantation of a AON-gapmer-loaded hydrogel matrix mediated efficient gene silencing in the lesioned spinal cord and is an innovative platform that can potentially combine gene regulation with regenerative permissive substrates aiming at SCI therapeutics and nerve regeneration.

Biomimetic and bioinspired materials have clearly conquered a special position in the field of drug delivery. Due to their similarities with biologically relevant structures, either at the chemical, physical, or morphological level, they are expected to solve caveats of the current existing solutions and even outperform many of the currently available materials. Here we present the design and preparation of five of the most promising classes of bioinspired and biomimetic materials for application as drug-delivery systems. Particular focus was given to nucleic acid-based and dendrimer-based systems. These constitute newer classes of building blocks and their potential has been contributing to the growing interest in their use.

One important drawback of most of the currently used dendrimers for biomedical applications is their high stability under physiological conditions that can result in cytotoxicity or complications induced by the accumulation of non-degradable synthetic materials in the organism. Particularly in the gene therapy field, vector stability can further hinder the intracellular release of the nucleic acid from the dendriplex, consequently leading to low transfection efficiencies. Therefore, biodegradable cationic dendritic structures have been eagerly awaited. However, the development of these dendritic nanocarriers is challenging because of the undesired and/or premature degradation observed during their synthesis and/or application. Here, we report new hybrid-biodegradable, biocompatible, non-toxic, and water-soluble azide-terminated PEG-GATGE dendritic block copolymers, based on a gallic acid (GA) core and triethylene glycol (TG) butanoate arms, incorporating ester bonds (E) at the dendritic arms/shell. Their successful functionalization by "click'' chemistry with unprotected alkynated amines allowed complexation and delivery of siRNA. The hydrophobic character of the GATGE building unit confers to these hydrolyzable dendritic bionanomaterials a great ability to complex, protect and mediate the cellular internalization of siRNA. Moreover, the localization of the degradation points at the dendritic periphery, close to the complexed siRNA, was found to be important for nucleic acid release from the nanoparticles, rendering a significant improvement of the transfection efficiency compared to their hydrolytically stable PEG-GATG copolymer counterparts. The present study puts forward these biodegradable PEG-dendritic block copolymers not only as suitable vectors for nucleic acids, but also as new avenues for further developments exploring their use in theranostics.

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.

Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.

Splice switching oligonucleotides (SSOs) are a class of single-stranded antisense oligonucleotides (ssONs) being used as gene therapeutics and demonstrating great therapeutic potential. The availability of biodegradable and biocompatible delivery vectors that could improve delivery efficiencies, reduce dosage, and, in parallel, reduce toxicity concerns could be advantageous for clinical translation. In this work we explored the use of quaternized amphiphilic chitosan-based vectors in nanocomplex formation and delivery of splice switching oligonucleotides (SSO) into cells, while providing insights regarding cellular uptake of such complexes. Results show that the chitosan amphiphilic character is important when dealing with SSOs, greatly improving colloidal stability under serum conditions, as analyzed by dynamic light scattering, and enhancing cellular association. Nanocomplexes were found to follow an endolysosomal route with a long lysosome residence time. Conjugation of a hydrophobic moiety, stearic acid, to quaternized chitosan was a necessary condition to achieve transfection, as an unmodified quaternary chitosan was completely ineffective. We thus demonstrate that amphiphilic quaternized chitosan is a biomaterial that holds promise and warrants further development as a platform for SSO delivery strategies.

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ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs.

Achieving higher nuclear concentrations by active transport may give potent therapeutic effects at lower doses for many drugs. A method of increasing nuclear uptake is the use of naturally existing Nuclear Localization Signals (NLS) by conjugating NLS structures to the cargo. We have synthesized a set of 2,2,7-trimethylguanosine cap (m(3)G-CAP)-containing structures (and their biotin conjugates) as artificially attached analogs of a naturally found NLS. The origin of a naturally found NLS is a uridine rich, small nuclear ribonucleoprotein (U snRNP) that employs Snurportin1 as a nuclear transport protein. In this report the NLS activity of various m(3)G-CAP biotin constructs was studied. We have shown that a minimal requirement for nuclear uptake is the inclusion of a trinucleotide sequence between the m3G-CAP and the artificial linker.

By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process - NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design.STATEMENT OF SIGNIFICANCE:By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.

The poor treatment response of acute myeloid leukemia (AML) overexpressing high-risk oncogenes such as EVI1, demands specific animal models for new treatment evaluations. Evi1 is a common site of activating integrations in murine leukemia virus (MLV)-induced AML and in retroviral and lentiviral gene-modified HCS. Still, a model of overt AML induced by Evi1 has not been generated. Cell lines from MLV-induced AML are growth factor-dependent and non-transplantable. Hence, for the leukemia maintenance in the infected animals, a growth factor source such as chronic immune response has been suggested. We have investigated whether these leukemias are transplantable if provided with growth factors. We show that the Evi1(+)DA-3 cells modified to express an intracellular form of GM-CSF, acquired growth factor independence and transplantability and caused an overt leukemia in syngeneic hosts, without increasing serum GM-CSF levels. We propose this as a general approach for modeling different forms of high-risk human AML using similar cell lines.

2'-O-AECM modified oligonucleotides provide an unusual combination of remarkable properties. This includes the combination of high resistance towards enzymatic degradation and the spontaneous cellular uptake of AECM oligonucleotides.