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1.
Nucleic Acids Res ; 52(9): 4799-4817, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38613388

RESUMO

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Oligonucleotídeos Antissenso , RNA Interferente Pequeno , Ensaios Antitumorais Modelo de Xenoenxerto , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Animais , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Humanos , Camundongos , Linhagem Celular Tumoral , Neoplasias Encefálicas/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/uso terapêutico , Inativação Gênica , Camundongos Nus
2.
J Am Chem Soc ; 145(4): 2142-2151, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36651186

RESUMO

A significant barrier to biological applications of DNA structures is their instability to nucleases. UV-mediated thymine dimerization can crosslink and stabilize DNA nanostructures, but its effect on DNA strand hybridization fidelity and function is unclear. In this work, we first compare a number of methods for DNA irradiation with different wavelengths of light and different photosensitizers. We demonstrate that all approaches can achieve nuclease protection; however, the levels of DNA off-target crosslinking and damage vary. We then describe mild irradiation conditions intended to safeguard DNA against nuclease degradation. We demonstrate up to 25× increase in serum stability while minimizing off-target damage and maintaining functions such as hybridization efficiency, gene silencing, aptamer binding, and DNA nanostructure formation. Our methodology requires no complex instruments beyond a UV light source and no synthetic modification of the DNA itself, allowing for applications in numerous areas of nucleic acid therapy and nanotechnology.


Assuntos
DNA , Nanoestruturas , DNA/química , Nanoestruturas/química , Nanotecnologia/métodos , Oligonucleotídeos/química , Hibridização de Ácido Nucleico , Conformação de Ácido Nucleico
3.
Nucleic Acids Res ; 48(9): 4643-4657, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32282904

RESUMO

We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Proteínas Argonautas/metabolismo , Configuração de Carboidratos , Células HeLa , Humanos , Luciferases de Vaga-Lume/genética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/imunologia , Proteína Supressora de Tumor p53/genética
5.
Nucleic Acid Ther ; 34(2): 52-72, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38507678

RESUMO

Nucleic acid-based therapies have become the third major drug class after small molecules and antibodies. The role of nucleic acid-based therapies has been strengthened by recent regulatory approvals and tremendous clinical success. In this review, we look at the major obstacles that have hindered the field, the historical milestones that have been achieved, and what is yet to be resolved and anticipated soon. This review provides a view of the key innovations that are expanding nucleic acid capabilities, setting the stage for the future of nucleic acid therapeutics.


Assuntos
Ácidos Nucleicos , Ácidos Nucleicos/genética , Ácidos Nucleicos/uso terapêutico , Sistemas de Liberação de Medicamentos
6.
ACS Nano ; 18(5): 3996-4007, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38265027

RESUMO

Spherical nucleic acids─nanospheres with nucleic acids on their corona─have emerged as a promising class of nanocarriers, aiming to address the shortcomings of traditional nucleic therapeutics, namely, their poor stability, biodistribution, and cellular entry. By conjugating hydrophobic monomers to a growing nucleic acid strand in a sequence-defined manner, our group has developed self-assembled spherical nucleic acids (SaSNAs), for unaided, enhanced gene silencing. By virtue of their self-assembled nature, SaSNAs can disassemble under certain conditions in contrast to covalent or gold nanoparticle SNAs. Gene silencing involves multiple steps including cellular uptake, endosomal escape, and therapeutic cargo release. Whether assembly vs disassembly is advantageous to any of these steps has not been previously studied. In this work, we modify the DNA and hydrophobic portions of SaSNAs and examine their effects on stability, cellular uptake, and gene silencing. When the linkages between the hydrophobic units are changed from phosphate to phosphorothioate, we find that the SaSNAs disassemble better in endosomal conditions and exhibit more efficacious silencing, despite having cellular uptake similar to that of their phosphate counterparts. Thus, disassembly in the endolysosomal compartments is advantageous, facilitating the release of the nucleic acid cargo and the interactions between the hydrophobic units and endosomal lipids. We also find that SaSNAs partially disassemble in serum to bind albumin; the disassembled, albumin-bound strands are less efficient at cellular uptake and gene silencing than their assembled counterparts, which can engage scavenger receptors for internalization. When the DNA portion is cross-linked by G-quadruplex formation, disassembly decreases and cellular uptake significantly increases. However, this does not translate to greater gene silencing, again illustrating the need for disassembly of the SaSNAs when they are in the endosome. This work showcases the advantages of the dual nature of SaSNAs for gene silencing, requiring extracellular assembly and disassembly inside the cell compartments.


Assuntos
Nanopartículas Metálicas , Ácidos Nucleicos , Nanopartículas Metálicas/química , Ácidos Nucleicos/química , Ouro/química , Distribuição Tecidual , Inativação Gênica , DNA/metabolismo , Albuminas/metabolismo , Fosfatos/metabolismo
7.
Mol Ther Nucleic Acids ; 35(1): 102117, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38304729

RESUMO

Identifying therapeutic oligonucleotides that are cross-reactive to experimental animal species can dramatically accelerate the process of preclinical development and clinical translation. Here, we identify fully chemically-modified small interfering RNAs (siRNAs) that are cross-reactive to Janus kinase 1 (JAK1) in humans and a large variety of other species. We validated the identified siRNAs in silencing JAK1 in cell lines and skin tissues of multiple species. JAK1 is one of the four members of the JAK family of tyrosine kinases that mediate the signaling transduction of many inflammatory cytokine pathways. Dysregulation of these pathways is often involved in the pathogenesis of various immune disorders, and modulation of JAK family enzymes is an effective strategy in the clinic. Thus, this work may open up unprecedented opportunities for evaluating the modulation of JAK1 in many animal models of human inflammatory skin diseases. Further chemical engineering of the optimized JAK1 siRNAs may expand the utility of these compounds for treating immune disorders in additional tissues.

8.
Nat Biotechnol ; 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39090305

RESUMO

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5'-C and 5'-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3' and 5' exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA-PS backbone enhances resistance to 3' exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA.

9.
Nucleic Acid Ther ; 33(4): 265-276, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37196168

RESUMO

Antisense oligonucleotides (ASOs) can predictably alter RNA processing and control protein expression; however, challenges in the delivery of these therapeutics to specific tissues, poor cellular uptake, and endosomal escape have impeded progress in translating these agents into the clinic. Spherical nucleic acids (SNAs) are nanoparticles with a DNA external shell and a hydrophobic core that arise from the self-assembly of ASO strands conjugated to hydrophobic polymers. SNAs have recently shown significant promise as vehicles for improving the efficacy of ASO cellular uptake and gene silencing. However, to date, no studies have investigated the effect of the hydrophobic polymer sequence on the biological properties of SNAs. In this study, we created a library of ASO conjugates by covalently attaching polymers with linear or branched [dodecanediol phosphate] units and systematically varying polymer sequence and composition. We show that these parameters can significantly impact encapsulation efficiency, gene silencing activity, SNA stability, and cellular uptake, thus outlining optimized polymer architectures for gene silencing.


Assuntos
Nanopartículas , Ácidos Nucleicos , Inativação Gênica , Nanopartículas/química , Ácidos Nucleicos/genética , Ácidos Nucleicos/química , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Polímeros
10.
Mol Ther Nucleic Acids ; 34: 102080, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38089931

RESUMO

Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo. We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications.

11.
Nat Commun ; 14(1): 7099, 2023 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-37925520

RESUMO

Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.


Assuntos
Inibidores de Janus Quinases , Vitiligo , Camundongos , Animais , Humanos , RNA Interferente Pequeno/genética , Linfócitos T CD8-Positivos/metabolismo , Autoimunidade/genética , Vitiligo/tratamento farmacológico , Vitiligo/genética , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , RNA de Cadeia Dupla
12.
Chem Sci ; 12(8): 2993-3003, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34164068

RESUMO

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) - nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior - have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.

13.
J Control Release ; 324: 34-46, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32330572

RESUMO

Conjugation of lipid moieties to nucleic-acid therapeutics increases their interaction with cellular membranes, enhances their uptake and influences in vivo distribution. Once injected in biological fluids, such modifications trigger the binding of various serum proteins, which in turn play a major role in determining the fate of oligonucleotides. Yet, the role played by each of these proteins, more than 300 in serum, remains to be elucidated. Albumin, the most abundant circulating protein is an attractive candidate to study, as it was previously used to enhance the therapeutic effect of various drugs. Herein, we present a thorough fluorescent-based methodology to study the effect of strong and specific albumin-binding on the fate and cellular uptake of DNA oligonucleotides. We synthesized a library of molecules that exhibit non-covalent binding to albumin, with affinities ranging from high (nanomolar) to none. Our results revealed that strong albumin binding can be used as a strategy to reduce degradation of oligonucleotides in physiological conditions caused by enzymes (nucleases), to reduce uptake and degradation by immune cells (macrophages) and to prevent non-specific uptake by cells. We believe that introducing protein-binding domains in oligonucleotides can be used as a strategy to control the fate of oligonucleotides in physiological environments. While our study focuses on albumin, we believe that such systematic studies, which elucidate the role of serum proteins systematically, will ultimately provide a toolbox to engineer the next-generation of therapeutic oligonucleotides, overcoming many of the barriers encountered by these therapeutics, such as stability, immunogenicity and off-target effects.


Assuntos
Albuminas , Oligonucleotídeos , Proteínas Sanguíneas , DNA , Lipídeos
14.
Methods Mol Biol ; 1974: 69-81, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31098996

RESUMO

DNA nanotechnology enables the design and assembly of DNA nanostructures with unprecedented control over their size and shape. Additionally, the programmable base-pairing alphabet of DNA allows the incorporation of responsive units within these DNA nanostructures. Here, we describe a general design strategy to construct responsive DNA prisms that can encapsulate and selectively release an encapsulated siRNA upon recognition of an oligonucleotide trigger. This prismatic DNA scaffold design is adaptable and can encapsulate oligonucleotides of any length and type. Moreover, the prism can be made to respond to an oligonucleotide trigger of interest like a messenger RNA (mRNA) or a microRNA (miRNA), thus enabling dual or synergistic therapeutic strategies. We present an overview of the design strategy used to access these DNA nanostructures, followed by the steps involved in DNA sequence generation, assembly, and validation of this construct.


Assuntos
DNA/genética , Nanotecnologia/métodos , Oligonucleotídeos/genética , RNA Interferente Pequeno/genética , Sequência de Bases/genética , DNA/química , Humanos , Nanoestruturas/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , RNA Interferente Pequeno/química
15.
ACS Appl Mater Interfaces ; 11(15): 13912-13920, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30720262

RESUMO

In this work, we report a component-minimal spherical nucleic acid (SNA) from monodisperse DNA-polymer conjugates that can load and release nucleic acid therapeutics in a stimuli-responsive manner. We show that this vehicle assembles from only four strands, and conditional release of its antisense therapeutic cargo can be induced upon recognition of specific oligonucleotide triggers via strand displacement. The latter (triggers) may be a microRNA that offers additional synergistic therapy, in addition to the previously shown ability of the SNA to load hydrophobic drugs. The SNA is easy to prepare, has dynamic character, releases its cargo only upon the presence of both triggers, and can survive biological conditions while protecting its cargo. The gene silencing potency of the cargo was tested in live cells and shown to be suppressed when loaded in the SNA, and its activity was restored only upon release with the two triggers. This vehicle has the essential characteristics of versatility, ease of synthesis, low cost, highly responsive behavior, and ability to support combination therapies, making it a promising candidate for cell-selective drug delivery and clinical transition.


Assuntos
Portadores de Fármacos/química , Ácidos Nucleicos/química , Oligonucleotídeos Antissenso/química , Polímeros/química , Carbocianinas/química , Transferência Ressonante de Energia de Fluorescência , Inativação Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia , Oligonucleotídeos Antissenso/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo
16.
J Phys Chem B ; 121(5): 1033-1040, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28072912

RESUMO

We report a simple, generally applicable, and noninvasive fluorescent method for mapping thermal fluctuations in hydrogel matrices using an unmodified commercially available digital single-lens reflex camera (DSLR). The nanothermometer is based on the complexation of short conjugated polyelectrolytes, poly(phenylene ethynylene) carboxylate, with an amphiphilic polymer, polyvinylpyrrolidone, which is in turn trapped within the porous network of a gel matrix. Changes in the temperature lead to a fluorescent ratiometric response with a maximum relative sensitivity of 2.0% and 1.9% at 45.0 °C for 0.5% agarose and agar, respectively. The response was reversible with no observed hysteresis when samples were cycled between 20 and 40 °C. As a proof of concept, the change in fluorescent signal/color was captured using a digital camera. The images were then dissected into their red-green-blue (RGB) components using a Matlab routine. A linear correlation was observed between the hydrogel temperature and the green and blue intensity channels. The reported sensor has the potential to provide a wealth of information when thermal fluctuations mapped in soft gels matrices are correlated with chemical or physical processes.

17.
Chem Sci ; 8(9): 6218-6229, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28989655

RESUMO

We report a spherical nucleic acid (SNA) system for the delivery of BKM120, an anticancer drug for treatment of chronic lymphocytic leukemia (CLL). While promising for cancer treatment, this drug crosses the blood-brain barrier causing significant side-effects in patients. The DNA nanoparticle encapsulates BKM120 in high efficiency, and is unparalleled in its monodispersity, ease of synthesis and stability in different biological media and in serum. These DNA nanostructures demonstrate efficient uptake in human cervical cancer (HeLa) cells, and increased internalization of cargo. In vitro studies show that BKM120-loaded nanoparticles promote apoptosis in primary patient CLL lymphocytes, and act as sensitizers of other antitumor drugs, without causing non-specific inflammation. Evaluation of this drug delivery system in vivo shows long circulation times up to 24 hours, full body distribution, accumulation at tumor sites and minimal leakage through the blood-brain barrier. Our results demonstrate the great potential of these delivery vehicles as a general platform for chemotherapeutic drug delivery.

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