Page 59 - ITPS-7-3

INNOSC Theranostics and
Pharmacological Sciences Designing miRNA ONTs for cardiometabolic pandemics

modifications that are not hampered by these potential PNAs to evade immune surveillance is a substantial benefit
toxicities are needed. over ASOs. 68
PNAs were discovered by Nielsen and Nielsen PNA antagomirs have been successfully tested to target
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et al. in 1991. PNAs are synthetic DNA mimics with miR-155, 74-76 miR-122, 77,78 miR-122-3p, miR-21, 80,81 miR-
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the deoxyribose phosphate backbone replaced by N-(2- 101-3p, miR-145-5p and miR-335-5p, miR-210, miR-
aminoethyl)-glycine units (Figure 2). PNA molecules 584-5p and miR-425-3p, miR-141-3p, miR-155-5p
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combine the properties of both peptides and nucleic acids. and miR-221-3p, miR-10b and 21, and miR-33. 88,89 As
The neutral pseudopeptide backbone of PNAs enhances reported recently by Pradeep et al., PNAs targeting mRNA,
binding affinity, improves biological stability, and prolongs pre-mRNA, and miRNA can be used to treat various
half-life. Nucleobases (adenine, guanine, cytosine, and human pathologies. 68
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thymine/uridine) are attached to the backbone through a The therapeutic relevance of PNAs was initially
carboxy methylene linkage. The chemical properties of PNA hampered by their poor cellular permeability and the
ensure a similar distance between nucleobases, allowing absence of efficient delivery methods. Recent advances
tight hybridization with their complementary nucleic acid in delivery methods of PNAs, using lipid or peptide
targets through Watson–Crick base-pairing. A given PNA conjugates or nanoparticles, have improved their in vivo
recognizes its complementary nucleic acid strand with efficacy and safety. 74,90 This topic is specifically addressed
high affinity and excellent specificity, surpassing those of in Criterion 7 below.
natural nucleic acids and their analogs. Unlike gapmer
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ASOs, PNA–RNA hybrids are not targets for RNase H1 4.3. Criterion 3: Maintain resistance to nucleases
enzyme degradation. and proteases/peptidases
The initial PNA structure included a neutral and achiral In contrast to small molecules and protein drugs, which
backbone, which is hampered by poor cellular penetration. are respectively metabolized or catabolized, ONTs can
Consequently, several chemical modifications (⍺-PNAs, be degraded by endogenous RNases. ONTs share similar
β-PNAs, and γ-PNAs) were implemented to improve absorption, distribution, metabolism, and excretion
their hybridization properties and cellular penetration (ADME) profiles within each chemistry category. 91,92
(Figure 3). ONTs undergo prompt cellular uptake enabled by
chemical conjugates and delivery methods, leading to a
Gamma backbone modifications convert a randomly
folded PNA into a helical structure. 70,71 The introduction short circulating blood half-life. Such conjugated ONTs
have a unique pharmacokinetic/pharmacodynamic (PK/
of an L-serine side chain at the γ-position produces a PD) profile whereby their local PD effects in the targeted
right-handed helix PNA and enhances hybridization organs and tissues are distinct from their systemic PK.
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with its target. Regarding the risks of on/off-target After cellular/tissue uptake, the intracellular concentration
effects, a PNA–RNA heteroduplex is more stable and of the ONT, not its systemic concentration, interacts with
more susceptible to mismatches than a PNA–DNA the targeted mRNAs, leading to an extended PD half-life
heteroduplex. A single base-pair mismatch in a PNA–RNA and dosing frequency schedule. The PK/PD and ADME
heteroduplex significantly diminishes the thermal melting features of GalNAc-conjugated siRNAs are similar across
temperature by approximately 12°C. PNAs require only 9 species, predictable, and precisely scaled up to human
– 12 monomers to effectively discriminate and efficiently subjects, allowing for safe and effective dosing regimens
bind to their specific targets. For reference, a 17-mer before assessing human liver PK profiles. 94
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oligonucleotide sequence is expected to occur only once in
the human genome. Therefore, a 17 – 22-mer PNA ONT The peculiar backbone of PNAs provides resistance
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would not trigger on/off-target effects. against both peptidase and nuclease degradation. 68,95 No
known endogenous nucleases and/or proteases recognize
Mechanistically, PNA antagomirs bind to their target PNAs as substrates. 96
miRNAs by Watson–Crick base pairing, consequently
sterically blocking downstream miRNA-mRNA 4.4. Criterion 4: Avoid chirality
interactions. Thus, our single-stranded miR-22-3p ONTs ONTs often include a PS backbone to decrease nuclease
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act by Watson–Crick base-pairing complementary to the degradation. Given that the substitution of oxygen in the
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human miR-22-3p sequence in a specific manner.
ONT backbone with sulfur introduces chirality, a full PS
The preclinical efficacy and safety of chemically 16-mer ONT includes 2 (32,768) distinct stereoisomers,
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modified PNAs have been established for multiple targets with inherent risks of inconsistent therapeutic effects
in several animal models. Furthermore, the capacity of and unintended off-target effects. 97-99 Other chemical

Volume 7 Issue 3 (2024) 6 doi: 10.36922/itps.3025