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Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (31 December 2021) | Viewed by 24692

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Dr. Vincenza Barresi

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Department of Biomedical and Biotechnological Sciences, Section of Medical Biochemistry, University of Catania, 95123 Catania, Italy
Interests: genomics; transcriptomics; cancer; molecular biomarkers; cancer driver genes; proteases
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Special Issue Information

Dear Colleagues,

In the last few decades, the exponential growth of genomic information has become crucial to develop new treatments based on molecular tools able to modify transcriptionally or post-transcriptionally the expression of coding and non-coding RNA. To date, antisense oligonucleotides (ASOs), apatmers and RNA Interference (RNAi), and CRISPR/Cas9 systems are the most common tools used for transcriptional therapy, and some of these tools have been approved by regulatory authorities for the therapy of diseases such as age-related macular degeneration (AMD), Duchenne muscular dystrophy (DMD), and spinal muscular atrophy (SMA). These approaches are exciting and advantageous for both monogenic and polygenic diseases. In the latter case, autoimmune and inflammatory diseases, neurological diseases, cancer, metabolic syndrome, and obesity represent the areas where transcriptional therapy and nucleic acid-based therapeutics are continuing to expand.

This Special Issue will gather a collection of papers reporting on investigations focused on concepts, interpretations, and experiments concerning nucleic acid-based therapeutics, ranging from the identification of molecular targets to their application in preclinical studies.

Dr. Vincenza Barresi
Guest Editor

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Keywords

antisense oligonucleotides
siRNA
CRISPR/Cas9
miRNA
nucleic acid-based therapeutics
transcriptional therapy
cancer therapy

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Published Papers (6 papers)

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Research

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18 pages, 2211 KiB

Open AccessArticle

Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides

by Alexandra Maria Psaras, Simonas Valiuska, Véronique Noé, Carlos J. Ciudad and Tracy A. Brooks

Int. J. Mol. Sci. 2022, 23(4), 2097; https://doi.org/10.3390/ijms23042097 - 14 Feb 2022

Cited by 5 | Viewed by 4102

Abstract

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4_mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4_mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4_mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt², qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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17 pages, 2625 KiB

Open AccessArticle

Level of Murine DDX3 RNA Helicase Determines Phenotype Changes of Hepatocytes In Vitro and In Vivo

by Olga Sergeeva, Tatiana Abakumova, Ilia Kurochkin, Renata Ialchina, Anna Kosyreva, Tatiana Prikazchikova, Varvara Varlamova, Evgeniya Shcherbinina and Timofei Zatsepin

Int. J. Mol. Sci. 2021, 22(13), 6958; https://doi.org/10.3390/ijms22136958 - 28 Jun 2021

Cited by 3 | Viewed by 2914

Abstract

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo—deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60–65%) showed discordant results in vitro and in vivo—similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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25 pages, 2673 KiB

Open AccessArticle

Composites of Nucleic Acids and Boron Clusters (C₂B₁₀H₁₂) as Functional Nanoparticles for Downregulation of EGFR Oncogene in Cancer Cells

by Damian Kaniowski, Katarzyna Ebenryter-Olbińska, Katarzyna Kulik, Justyna Suwara, Wojciech Cypryk, Agata Jakóbik-Kolon, Zbigniew Leśnikowski and Barbara Nawrot

Int. J. Mol. Sci. 2021, 22(9), 4863; https://doi.org/10.3390/ijms22094863 - 4 May 2021

Cited by 9 | Viewed by 3346

Abstract

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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16 pages, 2959 KiB

Open AccessArticle

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

by Sachiyo Fukushima, Manal Farea, Kazuhiro Maeta, Abdul Qawee Mahyoob Rani, Kazumichi Fujioka, Hisahide Nishio and Masafumi Matsuo

Int. J. Mol. Sci. 2020, 21(23), 9136; https://doi.org/10.3390/ijms21239136 - 30 Nov 2020

Cited by 3 | Viewed by 4312

Abstract

Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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16 pages, 1817 KiB

Open AccessArticle

Translational Read-Through Therapy of RPGR Nonsense Mutations

by Christine Vössing, Marta Owczarek-Lipska, Kerstin Nagel-Wolfrum, Charlotte Reiff, Christoph Jüschke and John Neidhardt

Int. J. Mol. Sci. 2020, 21(22), 8418; https://doi.org/10.3390/ijms21228418 - 10 Nov 2020

Cited by 15 | Viewed by 3254

Abstract

X-chromosomal retinitis pigmentosa (RP) frequently is caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We evaluated the potential of PTC124 (Ataluren, Translama^TM) treatment to promote ribosomal read-through of premature termination codons (PTC) in RPGR. Expression constructs in HEK293T cells showed that the efficacy of read-through reagents is higher for UGA than UAA PTCs. We identified the novel hemizygous nonsense mutation c.1154T > A, p.Leu385* (NM_000328.3) causing a UAA PTC in RPGR and generated patient-derived fibroblasts. Immunocytochemistry of serum-starved control fibroblasts showed the RPGR protein in a dot-like expression pattern along the primary cilium. In contrast, RPGR was no longer detectable at the primary cilium in patient-derived cells. Applying PTC124 restored RPGR at the cilium in approximately 8% of patient-derived cells. RT-PCR and Western blot assays verified the pathogenic mechanisms underlying the nonsense variant. Immunofluorescence stainings confirmed the successful PTC124 treatment. Our results showed for the first time that PTC124 induces read-through of PTCs in RPGR and restores the localization of the RPGR protein at the primary cilium in patient-derived cells. These results may provide a promising new treatment option for patients suffering from nonsense mutations in RPGR or other genetic diseases. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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Review

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27 pages, 2636 KiB

Open AccessReview

Transcript-Targeted Therapy Based on RNA Interference and Antisense Oligonucleotides: Current Applications and Novel Molecular Targets

by Vincenza Barresi, Camillo Musmeci, Alessandro Rinaldi and Daniele Filippo Condorelli

Int. J. Mol. Sci. 2022, 23(16), 8875; https://doi.org/10.3390/ijms23168875 - 9 Aug 2022

Cited by 24 | Viewed by 5857

Abstract

The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA (e.g., Inclisiran targets mRNA of protein PCSK9, permitting a longer half-life of LDL receptors in heterozygous familial hypercholesteremia), by arresting mRNA translation (i.e., Fomivirsen that binds to UL123-RNA and blocks the translation into IE2 protein in CMV-retinitis), or by reactivating modified functional protein (e.g., Eteplirsen able to restore a functional shorter dystrophin by skipping the exon 51 in Duchenne muscular dystrophy) or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs (e.g., Nusinersen acts on the splicing of exon 7 in SMN2 mRNA normally not expressed; it is used for spinal muscular atrophy) or by downregulation of transcript levels (e.g., Inotersen acts on the transthryretin mRNA to reduce its expression; it is prescribed for the treatment of hereditary transthyretin amyloidosis) in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed. Full article

(This article belongs to the Special Issue Transcriptional Therapy and Nucleic Acid-Based Therapeutics: From Molecular Targets to Preclinical Studies)

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