Enhancing Neuronal Cell Uptake of Therapeutic Nucleic Acids with Tetrahedral DNA Nanostructures.

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.

Engineering a Novel Self-Assembled Multi-siRNA Nanocaged Architecture with Controlled Enzyme-Mediated siRNA Release.

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.

Delivery of Antisense Oligonucleotides Mediated by a Hydrogel System: In Vitro and In Vivo Application in the Context of Spinal Cord Injury.

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.

Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in 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 GSK3ß. 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.

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression.

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.

Biodegradable PEG-dendritic block copolymers: synthesis and biofunctionality assessment as vectors of siRNA.

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.

A high-throughput bioimaging study to assess the impact of chitosan-based nanoparticle degradation on DNA delivery performance.

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.

Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene.

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.

Next-generation bis-locked nucleic acids with stacking linker and 2'-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes.

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.

Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based Nanoparticles.

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.

A murine model of acute myeloid leukemia with Evi1 overexpression and autocrine stimulation by an intracellular form of GM-CSF in DA-3 cells.

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.

Functionalized chitosan derivatives as nonviral vectors: physicochemical properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes.

Cationic polymers have recently attracted attention due to their proven potential for nonviral gene delivery. In this study, we report novel biocompatible nanocomplexes produced using chemically functionalized N,N,N-trimethyl chitosan (TMC) with different N-acyl chain lengths (C5-C18) associated with single-stranded oligonucleotides. The TMC derivatives were synthesized by covalent coupling reactions of quaternized chitosan with n-pentanoic (C5), n-decanoic (C10), and n-octadecanoic (C18) fatty acids, which were extensively characterized by Fourier transform-infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance ((1)H NMR). These N-acylated TMC derivatives (TMCn) were used as cationic polymeric matrices for encapsulating anionic 18-base single-stranded thiophosphorylated oligonucleotides (ssONs), leading to the formation of polyplexes further characterized by zeta potential (ZP), dynamic light scattering (DLS), binding affinity, transfection efficiency and in vitro cytotoxicity assays. The results demonstrated that the length of the grafted hydrophobic N-acyl chain and the relative amino:phosphate groups ratio (N/P ratio) between the TMC derivatives and ssON played crucial roles in determining the physicochemical properties of the obtained nanocomplexes. While none of the tested derivatives showed appreciable cytotoxicity, the type of acyl chain had a remarkable influence on the cell transfection capacity of TMC-ssON nanocomplexes with the derivatives based on stearic acid showing the best performance based on the results of in vitro assays using a model cell line expressing luciferase (HeLa/Luc705).

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form.

Hypercholesterolemia is a medical condition often characterized by high levels of low-density lipoprotein cholesterol (LDL-C) in the blood. Despite the available therapies, not all patients show sufficient responses, especially those with very high levels of LDL-C or those with familial hypercholesterolemia. Regulation of plasma cholesterol levels is very complex and several proteins are involved (both receptors and enzymes). From these, the proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising pharmacologic target. The objective of this work is to develop a new approach to inactivate PCSK9 by splice-switching oligonucleotides (SSOs), converting the normal splice form to a natural, less abundant and inactive, splice variant. For this purpose, a new RNA therapeutic approach for hypercholesterolemia based on SSOs was developed for modulation of the splice pattern of human PCSK9 pre-mRNA. Our results show an increase of the selected splice form at both the mRNA and protein level when compared to non-treated Huh7 and HepG2 cell lines, with concomitant increase of the protein level of the low-density lipoprotein receptor (LDLR) demonstrating the specificity and efficiency of the system. In vivo, full conversion to the splice form was achieved in a reporter system when mice were treated with the specific oligonucleotide, thus further indicating the therapeutic potential of the approach. In conclusion, PCSK9 activity can be modulated by splice-switching through an RNA therapeutic approach. The tuning of the natural active to non-active isoforms represents a physiological way of regulating the cholesterol metabolism, by controlling the amount of LDL receptor available and the rate of LDL-cholesterol clearance.

Nuclease resistant oligonucleotides with cell penetrating properties.

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.

Therapeutic antisense oligonucleotides against cancer: hurdling to the clinic.

Under clinical development since the early 90's and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics has not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given toward a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Repeatable, Inducible Micro-RNA-Based Technology Tightly Controls Liver Transgene Expression.

Inducible systems for gene expression emerge as a new class of artificial vectors offering temporal and spatial exogenous control of gene expression. However, most inducible systems are less efficient in vivo and lack the target-organ specificity. In the present study, we have developed and optimized an oligonucleotide-based inducible system for the in vivo control of transgenes in the liver. We generated a set of simple, inducible plasmid-vectors based on the addition of four units of liver-specific miR-122 target sites to the 3'untranslated region of the gene of interest. Once the vector was delivered into hepatocytes this modification induced a dramatic reduction of gene expression that could be restored by the infusion of an antagomir for miR-122. The efficiency of the system was tested in vivo, and displayed low background and strong increase in gene expression upon induction. Moreover, gene expression was repeatedly induced even several months after the first induction showing no toxic effect in vivo. By combining tissue-specific control elements with antagomir treatment we generated, optimized and validated a robust inducible system that could be used successfully for in vivo experimental models requiring tight and cyclic control of gene expression.

Design and application of bispecific splice-switching oligonucleotides.

Targeting of pre-mRNA by short splice-switching oligonucleotides (SSOs) is increasingly being used as a therapeutic modality, one rationale being to disrupt splicing so as to remove exons containing premature termination codons, or to restore the translation reading frame around out-of-frame deletion mutations. The aim of this study was to investigate the effect of chemically linking individual SSOs so as to ascertain equimolar cellular uptake that would provide for more defined drug formulations. In contrast to conventional bispecific SSOs generated by conjugation in solution, here we describe a protocol for synthesis of bispecific SSOs on solid phase. These SSOs comprised of either a non-cleavable hydrocarbon linker or disulfide-based cleavable linkers. To assess the efficacy of these SSOs we have utilized splice switching to bypass a disease-causing mutation in the DMD gene concurrent with disruption of the reading frame of the myostatin gene (Mstn). The premise of this approach is that disruption of myostatin expression is known to induce muscle hypertrophy and so for Duchenne muscular dystrophy (DMD) could be expected to have a better outcome than dystrophin restoration alone. All tested SSOs mediated simultaneous robust exon removal from mature Dmd and Mstn transcripts in myotubes. Our results also demonstrate that using cleavable SSOs is preferred over the non-cleavable counterparts and that these are equally efficient at inducing exon skipping as cocktails of monospecific versions. In conclusion, we have developed a protocol for solid-phase synthesis of single molecule cleavable bispecific SSOs that can be efficiently exploited for targeting of multiple RNA transcripts.

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA.

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation.

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.