Gene Therapy and Small Molecules Used in the Treatment of Cystic Fibrosis
Yashwant Pathak in Gene Delivery, 2022
Since the CFTR gene mutation causes hyperactivation of the epithelial sodium channel (ENaC), the absorption of Na+ ions is enhanced and the lung airway mucus becomes dehydrated. Hence, the inhibition of ENaC expression could serve as a promising therapeutic approach for the treatment of cystic fibrosis. One method for the inhibiting the expression of ENaC-encoding genes (SCNN1A, SCNN1B, SCNN1G, and SCNN1D encoding α, β, γ, and δ ENaC subunits, respectively) involves the use of a single strand nucleic acid known as antisense oligonucleotide (ASO) (Almughem et al., 2020). When this oligonucleotide is hybridized to mRNA, RNase H is triggered to slice the hybridized mRNA. Targeting the α-subunit of ENaC in the respiratory organ, using ASO might inhibit the cationic channel activity (Almughem et al., 2020). Another study showed the possibility of using aerosolized ENaC antisense oligonucleotide containing wing modifications to inhibit ENaC mRNA in CF-like mice models (Hajj and Whitehead, 2017). This aerosolized ENaC antisense oligonucleotide helped to cure various cystic fibrosis symptoms, like airway hyper-responsiveness and inflammation (Crosby et al., 2017).
Recent Advances In HIV/AIDS
Anne George, K. S. Joshy, Mathew Sebastian, Oluwatobi Samuel Oluwafemi, Sabu Thomas in Holistic Approaches to Infectious Diseases, 2017
The HIV pol gene encodes a number of enzymatic functions related to reverse transcription. First, a DNA polymerase (the RT) assembles a DNA strand complementary to the viral RNA. Then the RNA strand must be removed to allow a complementary DNA strand to form double-stranded DNA. The C-terminal region of the HIV RT enzyme contains a ribonuclease, RNase H, which serves this function. Mutations in the RNase H domain of the RT have been shown to abolish infectivity. Researchers are looking forward into the development of inhibitors of RNase H (Yu et al., 2008). Certain natural compounds such as β-thujaplicinol and manicol, derived from heartwood of cupressaceous trees have been identified with anti-HIV activity affecting Ribonuclease H at sub-micromolar concentrations (Budihas et al., 2005).
Synthetic DNA-Based Compounds for the Prevention of Coronary Restenosis: Current Status and Future Challenges
Eric Wickstrom in Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
Non-sequence-dependent effects are due to the avid binding of negatively charged oligonucleotides (e.g., phosphorothioate) to proteins. The increase in length and concentration of oligomers appears to enhance nonspecific inhibition (Cazenave et al., 1989; Gao et al., 1992; Guvakova et al., 1995). At higher doses oligonucleotides inhibit RNase H activity, thereby eliminating an important mechanism aiding the inhibition of gene expression by antisense. Longer sequences (28-mer) of phosphorothioate homopolymers of cytidine or thymidine have also demonstrated the ability to interact with extracellular growth factors (e.g., bFGF) and their binding with cell surface receptors (Guvakova et al., 1995). This is consistent with their polyanion structure, thereby reducing cell proliferation in non-sequence-dependent manner in vitro and neointimal formation in the rat model in vivo (Guvakova et al., 1995; Wang et al., 1996).
Hyaluronan-based delivery of therapeutic oligonucleotides for treatment of human diseases
Published in Expert Opinion on Drug Delivery, 2019
Steric blockage mechanism of ASOs can be also executed in cytoplasm when acting on mRNA (Figure 1(b)) or microRNA (Figure 1(c)). Steric blocker ASOs were proposed to inhibit translation by disrupting ribosomal assembly at the 5ʹ-cap region of mRNA through hybridizing to it [9]. However, the most successful way of inhibition of mRNA translation by ASOs was found to occur via RNase H dependent mechanism. RNase H is an enzyme that cleaves the RNA strand in DNA:RNA duplex and hence RNase H dependent ASOs should contain at least eight 2ʹ-deoxynucleotides long stretch, which is normally flanked with chemically modified RNA bases to increase affinity and reduce susceptibility to degradation by nucleases (gapmer strategy) [10]. After hybridization to target mRNA by Watson-Crick base pairing, RNase H recognizes the mRNA:DNA duplex and cleaves the mRNA strand (Figure 1(d)) via hydrolysis of internucleotide phosphodiester bond, which is followed by release of the ASO and its binding to a new mRNA molecule. This catalytic mechanism recruiting RNase H greatly amplifies the therapeutic potency of ASOs.
Flavonoids and Acid-Hydrolysis derivatives of Neo-Clerodane diterpenes from Teucrium flavum subsp. glaucum as inhibitors of the HIV-1 reverse transcriptase–associated RNase H function
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Benedetta Fois, Angela Corona, Enzo Tramontano, Simona Distinto, Elias Maccioni, Rita Meleddu, Pierluigi Caboni, Costantino Floris, Filippo Cottiglia
Since the neo-clerodane flavuglaucin B was not able to inhibit the RDDP function and apparently it does not contain any functionality able to bind to the RNase H active site coordinating the Mg2+ cofactors, we supposed that this compound might bind an allosteric RT site. In order to verify this hypothesis, it was chosen to perform site-directed mutagenesis, determining the independent impact of several amino acid substitutions on the potency of the compound to inhibit the RNase H function. All the selected aminoacids are localised in the RNase H domain and are potentially crucial for the binding of RNase H function inhibitors. To verify a possible interaction for flavuglaucin B in the allosteric site described by Himmel et al.16, residue V108 was replaced by a phenylalanine in order to reduce the binding available space for the compound. Results showed a slight increase in IC50 when flavuglaucin B was assessed against V108F, compared with the wild type enzyme (Figure 4).
Antisense techniques provide robust decrease in GnRH receptor expression with minimal cytotoxicity in GT1-7 cells
Published in Systems Biology in Reproductive Medicine, 2018
Maurice Andre Recanati, Hongling Du, Katherine J. Kramer, Maik Hüttemann, Robert A. Welch
Unlike the nonspecific inhibitors, antisense oligonucleotides reduce the expression of a single protein product without disrupting other proteins responsible for normal cell function. Phosphorothioates are oligonucleotides with a sulfur substituted for one of the oxygens in the phosphodiester bonds between the nucleotides. This modification increases the stability of the oligonucleotides, especially in aqueous solutions, and prevents cellular nuclease enzymes from rapidly degrading them. Phosphorothioates can function in vivo by a number of mechanisms. At the RNA level they generate a substrate for RNase H activity by forming a thermally stable DNA-RNA duplex with a specific sequence and thus render the mRNA accessible for RNase-H cleavage (Stein 1998). In addition, antisense oligonucleotides can form triple helices that interfere directly with transcription and may also inhibit translation (Cazenave et al. 1989). In general, HPLC purified oligonucleotides ranging in size from 14 to 28 bases offer excellent specificity without being subject to self-annealing (Sczakiel 1997).
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