Bilgiustam

Platforms for RNA-Based Cancer Therapeutics


RNA-based therapeutic strategies have emerged as an alternative to traditional protein-based therapies that are difficult to maintain as adapter proteins or transcription factors. Such protein molecules can be regulated by modulating mRNA levels or translation of proteins. The primary focus of oligonucleotide-based therapeutics includes gene silencing or activation and insertion modulation, which provides a wide range of potential targets beyond conventionally accessible pharmacological strategies. These methods follow the universal Watson-Crick complementarity basic matching rule, thus allowing direct querying of different default target sequences. Therefore, if the primary sequence of the target gene is available, it is easy to rationalize, design and screen specific ends. To achieve the desired functions, different oligonucleotide-based platforms have been developed, including antisense oligonucleotides, RNA interference molecules and mRNA transcripts.

Antisens Oligonükleotidler

Platforms for RNA-Based Cancer TherapeuticsAntisense oligonucleotides (ASOs) are ~ 18-30 nucleotide long, single-stranded, synthetic polymers of nucleic acids with a variety of chemicals. ASOs are small molecule drugs that target mRNAs based on complementary base pairing, interfering with different aspects of gene expression and regulation. These nucleotide sequences can interfere with DNA decoding, transcription, mRNA insertion, gene expression, and translational profile of target genes through different mechanisms. ASOs can be divided into two sub-categories depending on their mechanism of action and they are as follows:
• It acts by promoting RNA cleavage and degradation,
• Occupancy-only regulation, sometimes referred to as steric block.
Based on post-hybridization events, antisense oligonucleotides can modulate the expression of the target gene through two different mechanisms. There are different mechanisms of action for antisense oligonucleotide mediated gene silencing and they are as follows:
Occupancy mechanisms only,
• RNA degradation mechanisms,
In occupancy mechanisms alone, binding of ASOs with target RNAs does not result in RNA degradation. It modulates gene expression in several ways and these are as follows:
Addition modulation using insertion switch ASOs to perform exon skipping or exon insertion
• Inhibition of MRNA polyadenylation,
Translation modulation by means of DNA-like ASOs forming base pairs with mRNA is to inhibit translation, for example steric blocks, or to activate translation by binding to inhibitory elements such as upstream open reading frames (uORF).
For inhibition of miRNA-related function, these ASOs also modulate miRNA by base pairing miRNA (anti-miRs) or by occupying miRNA-sensitive elements (MRE) on the target mRNA to invalidate the effect of a particular miRNA. can. On the other hand, ASOs in RNA degradation pathways trigger target mRNA cleavage by either RNase H1 or siRNA mediated AGO2 RISC complex and ribozyme-mediated cleavage. The binding of ASOs to the target RNA cleaves the target at the ASO binding site, facilitating degradation of the target RNA and thereby lowering gene expression. This is one of the most widely used approaches for down-regulation of genes where overexpression is associated with the manifestation or progression of the disease. RNase H1 is a highly selective endonuclease that specifically acts on the RNA of the RNA-DNA heteroduplex. The detailed enzymatic and cellular functions of RNase H1 have now been revealed, and the substrate specificity of RNase H1 has been continuously used for the development of RNA-based therapeutics.
In mammalian cells, the RNase H1 distribution is ubiquitous, such as in the cytoplasm, mitochondria, and nucleus. It serves a variety of genomic functions, including DNA repair, resolution of R-loops, removal of chromatin-associated pre-mRNAs, transcriptional termination, preservation of genome integrity, and extraction of RNA associated with the okazaki segment. Interestingly, ASOs designed to use endonuclease activity as the mechanism of their action must have an extension of at least five DNA-nucleotides. Thus, following the RNase H-competent mechanisms currently used, ASOs are based on DNA gapmer patterns, a hybrid-type oligonucleotide sequence in which the chemically modified RNA side-sequences are inserted between the central DNA stretch known as the gap. The main advantage of using RNase H1-based ASOs is that they facilitate targeting of nuclear transcripts. These are less accessible for other approaches such as small intrusive RNA (siRNA). Platforms for RNA-Based Cancer TherapeuticsThe steric block or occupancy-only mediated mechanism uses high affinity ASOs to bind to the target RNA without inducing direct degradation of the target RNA. This class of ASO consists of nucleotides that do not form RNA-DNA that act as a substrate for RNase H1 or Ago2. Therefore, to prevent the formation of RNase H substrates and unwanted cleavage of target RNA, ASOs must be chemically modified or composed of a mixture of different nucleotide chemistries, often referred to as mixers, to expand sequential DNA-like nucleotides. Some common chemical modifications include thiophosphoroamidate, thiophosphoroamidate morpholinos, nucleoside groups and peptide nucleic acid inserts. Steric block ASOs bind to the specific target sequence and work by modulating translation, processing of RNA, splicing, RNA-protein interactions, and interactome of the target RNA. The most common application of these ASOs is to manage selective exclusion or inclusion of exons, eg exon skipping and inclusion, through modulation of alternative splicing.
Interestingly, the steric block approach of ASO can be used to disrupt the target insertion variant where the exon skipping method inhibits or downregulates translation of the target transcript. The insertion correction or inclusion approach has been used to correct or restore the translation framework to save the synthesis of therapeutic proteins. ASOs perform this function by masking the splicing signals, making the target invisible to the spliceosome, and eventually changing the spliceosome’s splicing decisions. It has common chemical modifications to antisense and RNA oligonucleotides. Different RNA modifications or RNA analogs have been identified and evaluated in antisense mechanisms. A representative structure of the dinucleotide is indicated by the marked positions at which oligonucleotides are commonly modified. The modifications commonly used in ASOs consist of sugar, base, phosphate, inter-nucleoside linkage modifications and conjugates of small or large molecules. Besides structural modifications, the therapeutic properties of several key modifications are also highlighted.

Platforms for RNA-Based Cancer TherapeuticsPreviously, antisense approaches have mainly been used to downregulate gene expression or translation, but have not been used as a broad alternative for situations where overexpression of useful proteins is required as a therapeutic strategy. More recently, combinatorial research of various approaches such as ASOs, micro / si-RNAs to increase target RNA expression or protein translation has revolutionized RNA-based therapeutic strategies for different diseases, including cancer. Further studies have demonstrated an effective approach to use ASOs for targeting microRNAs and ultimately increasing protein production. MicroRNAs inhibit gene expression or protein translation and control associated gene networks. These observations inspired the development of ASOs targeting microRNAs that block or suppress their binding properties with target RNA transcripts and resulted in the translational increase of microRNA-regulated genes.
Since microRNAs have cell or tissue specific activities to inhibit translation of multiple RNAs or targets, blocking a single microRNA can alter the expression of different proteins. Alternatively, using certain ASOs against 5′-untranslated regions of mRNAs is a more precise and targeted approach to increase protein production of associated RNA targets. Alternative splicing is a useful strategy for generating protein diversity. ASOs can be used to induce isoform change to promote expression of therapeutic / useful proteins or inhibit the expression of disease-associated proteins. Based on these mechanisms, three ASOs – golodirsen, usinersen and eteplirsen – have received FDA approval for insertion modification.

References:
ncbi.nlm.nih.gov/pmc/articles/PMC5376066/
researchgate.net/publication/333089303_Site-specific_replacement_of_phosphorothioate_with_alkyl_phosphonate_linkages_enhances_the_therapeutic_profile_of_gapmer_ASOs
sciencedirect.com/science/article/pii/S2162253117302408
springer.com/article/10.1186/s12943-021-01338-2

Writer: Ozlem Guvenc Agaoglu


Leave a Reply

Your email address will not be published. Required fields are marked *