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Nucleic Acid Therapeutics (NATs): Advances and Perspectives

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A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Methods".

Deadline for manuscript submissions: closed (20 March 2024) | Viewed by 18618

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Special Issue Editors

Dr. Aurélie Goyenvalle

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Guest Editor

UVSQ, Inserm, END-ICAP, Université Paris-Saclay, Versailles, France
Interests: nucleic acid therapeutics; antisense oligonucleotides; splice switching; exon-skipping; neuromuscular disorders; Duchenne muscular dystrophy; AAV

Dr. Simon Guiraud

E-Mail Website
Guest Editor

UEVE, Inserm, Genethon, Université Paris-Saclay, Evry, France
Interests: neuromuscular disorders; Duchenne muscular dystrophy; gene therapy; gene editing; exon-skipping; antisense oligonucleotides; nucleic acid therapeutics; AAV

Special Issue Information

Dear Colleagues,

The field of nucleic acid therapeutics (NATs) has attracted a tremendous amount of interest in the past few decades. The increasing number of approved nucleic acid drugs demonstrates the potential of these therapies to treat a variety of diseases from neuromuscular disorders to cancers. Nucleic acid therapeutics act via multiple mechanisms of action, including gene silencing, activation, modulation, replacement, or editing. Continuous advances in the chemistry and design of these cutting-edge genetic drugs have led to numerous successful clinical applications, eliciting even more interest from researchers, including both academic groups and drug development companies. Clinical translation largely depends on delivery technologies that facilitate internalization and improve stability and target affinity.

The aim of this Special Issue is to cover recent advances in the field of nucleic acid therapeutics, including antisense oligonucleotides, small interfering RNA conjugates, lipid nanoparticles, and RNA and gene-editing technologies. We would like to put emphasis on their broad therapeutic potential due to the variety of mechanisms of action and the different tissues and diseases they can target. We invite contributions in the form of original research articles and reviews.

We look forward to your contributions.

Dr. Aurélie Goyenvalle
Dr. Simon Guiraud
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

nucleic acid therapeutics
antisense oligonucleotides
siRNA
messenger RNA
RNA and gene editing
lipid nanoparticles
targeted delivery
endosomal escape
drug development

Published Papers (10 papers)

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Research

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21 pages, 2265 KiB

Open AccessArticle

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease

by Nuria Suárez-Herrera, Catherina H. Z. Li, Nico Leijsten, Dyah W. Karjosukarso, Zelia Corradi, Femke Bukkems, Lonneke Duijkers, Frans P. M. Cremers, Carel B. Hoyng, Alejandro Garanto and Rob W. J. Collin

Cells 2024, 13(7), 601; https://doi.org/10.3390/cells13070601 - 29 Mar 2024

Viewed by 567

Abstract

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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16 pages, 2874 KiB

Open AccessArticle

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy

by Marine Geoffroy, Louna Pili, Valentina Buffa, Maëlle Caroff, Anne Bigot, Evelyne Gicquel, Grégory Rouby, Isabelle Richard and Romain Fragnoud

Cells 2023, 12(20), 2444; https://doi.org/10.3390/cells12202444 - 12 Oct 2023

Viewed by 1607

Abstract

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC₅₀) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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16 pages, 4251 KiB

Open AccessArticle

Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation

by Dhanu Gupta, Sara Orehek, Janne Turunen, Liz O’Donovan, Michael J. Gait, Samir El-Andaloussi and Matthew J. A. Wood

Cells 2023, 12(18), 2285; https://doi.org/10.3390/cells12182285 - 15 Sep 2023

Viewed by 1287

Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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13 pages, 1991 KiB

Open AccessArticle

Size Exclusion Chromatography–Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization

by Di Wu, Xiaonan Zhao, Diego Antonio Jimenez and Grzegorz Piszczek

Cells 2023, 12(18), 2264; https://doi.org/10.3390/cells12182264 - 13 Sep 2023

Cited by 2 | Viewed by 1372

Abstract

Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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27 pages, 8305 KiB

Open AccessArticle

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote

by Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Trivendra Tripathi, Jin Zhang, Ramya Ramankutty Nair, Tutku Okyay, Kevin Zhu and Lina Abi-Mosleh

Cells 2023, 12(18), 2230; https://doi.org/10.3390/cells12182230 - 07 Sep 2023

Viewed by 1258

Abstract

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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15 pages, 4242 KiB

Open AccessArticle

HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics

by Giuseppina Covello, Kavitha Siva, Valentina Adami and Michela Alessandra Denti

Cells 2023, 12(15), 1959; https://doi.org/10.3390/cells12151959 - 28 Jul 2023

Viewed by 1338

Abstract

Nucleic acid therapeutics have demonstrated an impressive acceleration in recent years. They work through multiple mechanisms of action, including the downregulation of gene expression and the modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in the Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method in cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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17 pages, 2310 KiB

Open AccessArticle

Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models

by Amel Saoudi, Claire Fergus, Talia Gileadi, Federica Montanaro, Jennifer E. Morgan, Vincent P. Kelly, Thomas Tensorer, Luis Garcia, Cyrille Vaillend, Francesco Muntoni and Aurélie Goyenvalle

Cells 2023, 12(6), 908; https://doi.org/10.3390/cells12060908 - 15 Mar 2023

Cited by 1 | Viewed by 2253

Abstract

Nucleic acid-based therapies have demonstrated great potential for the treatment of monogenetic diseases, including neurologic disorders. To date, regulatory approval has been received for a dozen antisense oligonucleotides (ASOs); however, these chemistries cannot readily cross the blood–brain barrier when administered systemically. Therefore, an investigation of their potential effects within the central nervous system (CNS) requires local delivery. Here, we studied the brain distribution and exon-skipping efficacy of two ASO chemistries, PMO and tcDNA, when delivered to the cerebrospinal fluid (CSF) of mice carrying a deletion in exon 52 of the dystrophin gene, a model of Duchenne muscular dystrophy (DMD). Following intracerebroventricular (ICV) delivery (unilateral, bilateral, bolus vs. slow rate, repeated via cannula or very slow via osmotic pumps), ASO levels were quantified across brain regions and exon 51 skipping was evaluated, revealing that tcDNA treatment invariably generates comparable or more skipping relative to that with PMO, even when the PMO was administered at higher doses. We also performed intra-cisterna magna (ICM) delivery as an alternative route for CSF delivery and found a biased distribution of the ASOs towards posterior brain regions, including the cerebellum, hindbrain, and the cervical part of the spinal cord. Finally, we combined both ICV and ICM injection methods to assess the potential of an additive effect of this methodology in inducing efficient exon skipping across different brain regions. Our results provide useful insights into the local delivery and associated efficacy of ASOs in the CNS in mouse models of DMD. These findings pave the way for further ASO-based therapy application to the CNS for neurological disease. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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16 pages, 2852 KiB

Open AccessArticle

Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model

by Flavien Bizot, Abdallah Fayssoil, Cécile Gastaldi, Tabitha Irawan, Xaysongkhame Phongsavanh, Arnaud Mansart, Thomas Tensorer, Elise Brisebard, Luis Garcia, Rudolph L Juliano and Aurélie Goyenvalle

Cells 2023, 12(5), 702; https://doi.org/10.3390/cells12050702 - 23 Feb 2023

Cited by 1 | Viewed by 2478

Abstract

Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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15 pages, 1797 KiB

Open AccessArticle

Correction of the Splicing Defect Caused by a Recurrent Variant in ABCA4 (c.769-784C>T) That Underlies Stargardt Disease

by Tomasz Z. Tomkiewicz, Sara E. Nieuwenhuis, Frans P. M. Cremers, Alejandro Garanto and Rob W. J. Collin

Cells 2022, 11(24), 3947; https://doi.org/10.3390/cells11243947 - 07 Dec 2022

Cited by 10 | Viewed by 1825

Abstract

Stargardt disease is an inherited retinal disease caused by biallelic mutations in the ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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Review

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38 pages, 5781 KiB

Open AccessReview

Therapeutic Oligonucleotides: An Outlook on Chemical Strategies to Improve Endosomal Trafficking

by Priyanka Mangla, Quentin Vicentini and Annabelle Biscans

Cells 2023, 12(18), 2253; https://doi.org/10.3390/cells12182253 - 11 Sep 2023

Cited by 3 | Viewed by 3507

Abstract

The potential of oligonucleotide therapeutics is undeniable as more than 15 drugs have been approved to treat various diseases in the liver, central nervous system (CNS), and muscles. However, achieving effective delivery of oligonucleotide therapeutics to specific tissues still remains a major challenge, limiting their widespread use. Chemical modifications play a crucial role to overcome biological barriers to enable efficient oligonucleotide delivery to the tissues/cells of interest. They provide oligonucleotide metabolic stability and confer favourable pharmacokinetic/pharmacodynamic properties. This review focuses on the various chemical approaches implicated in mitigating the delivery problem of oligonucleotides and their limitations. It highlights the importance of linkers in designing oligonucleotide conjugates and discusses their potential role in escaping the endosomal barrier, a bottleneck in the development of oligonucleotide therapeutics. Full article

(This article belongs to the Special Issue Nucleic Acid Therapeutics (NATs): Advances and Perspectives)

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