Page 61 - ITPS-7-3

INNOSC Theranostics and
Pharmacological Sciences Designing miRNA ONTs for cardiometabolic pandemics

functions, distribution, and half-lives. Coupling an ONT
108
to a cellular targeting agent dramatically alters the systemic
PK/PD profile of the conjugate and reduces concerns about
serum protein binding, as shown for the generation 2.5
N-acetyl-galactosamine conjugates. 95,109
4.6. Criterion 6: Optimize and simplify the chemical
synthesis of ONTs
Various chemical modification and conjugation strategies
have been introduced to improve ONT drug characteristics,
such as nuclease resistance, RNA-binding affinity, and PK
profiles. 110,111 PNA compounds are synthesized using a
straightforward method similar to peptide synthesis. This
cost-effective solid-phase peptide synthesis method is
detailed in the literature. 106,112 Furthermore, using a Fmoc/
Boc protection scheme for both PNA monomers and
Figure 3. Various chemical modifications are applied to the structure of amino acid building blocks, in association with microwave-
peptide nucleic acids (PNAs). Note: B=Nucleic acid base. assisted solid-phase synthesis, allows continuous assembly
of PNA-peptide conjugates. 113,114
modifications, such as PMOs, also exhibit chirality. This
issue of chirality is a matter of concern for regulatory To efficiently select PNA sequences of interest, a
authorities. Since the 1980s, there has been a regulatory group of scientists recently developed machine learning
100
preference to approve single enantiomer drugs. algorithms and automated synthesis technology to predict
101
Consequently, strategies were implemented to reduce the PNA and peptide-PNA conjugates’ synthesis efficiency
number of PS modifications or synthesize stereopure and to guide rational PNA sequence design. 115,116 Another
9
ONTs. 98,102 Scientists from Alnylam Pharmaceuticals group of scientists incorporated an ultrasonication step in
®
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demonstrated that stereo-defined PS linkages at the the solid-phase synthesis of PNAs. This “United States
termini of siRNAs improved their in vivo pharmacology. (US)-PNAs” approach improves the purities and yields of
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Scientists from Wave Life Sciences Company reported synthesized PNAs, such as short or medium compounds
that stereopure PS and phosphoryl guanidine backbone (5-mer and 9-mer), complex purine-rich sequences, and
chemistries increase the potency and durability of mRNA longer oligomers.
silencing. They showed that a GalNAc-siRNA targeting 4.7. Criterion 7: Conjugate the ONTs to a fatty acid
human HSD17B13 leads to 80% silencing for at least or a short peptide for greater and active targeted
14 weeks after administration of a single subcutaneous delivery to the tissues and organs of interest at a
dose in transgenic mice. 104
much-reduced therapeutic dose
Another option is to rely on chemical modifications that
do not result in chirality, as is the case for PNA compounds, For therapeutic efficacy, after systemic or local
administration, an ONT must be selectively delivered to
which have an achiral protein-like backbone. 105,106
the organs/tissues of interest, efficiently transported into
4.5. Criterion 5: Limit binding to serum proteins cells, and translocated to the proper cellular compartment
to interact with its molecular target. The rapid renal
The tissue bioavailability and potential toxicity of clearance and potential toxicity of unconjugated ONTs
ONTs are modulated by plasma protein binding. 91,107 PS can be mitigated by using local targeted delivery methods.
modifications increase plasma stability and plasma protein Therefore, the conjugation of ONTs is crucial to achieve
binding. In contrast, ONTs that lack charges or are less safe and effective druggability. 13,41,110
extensively bound to plasma proteins (e.g., unmodified
and unformulated siRNA, PNAs, and PMOs) are rapidly For instance, ONTs targeting the liver, the central
cleared from the blood through either metabolism or nervous system, and muscles have been successfully
excretion in urine. The systemic distribution of generations developed and approved for clinical use. 9,109,117,118 The
1 and 2 ONTs is broad, with the highest concentrations use of triantennary N-acetylgalactosamine (GalNAc3)
achieved in the liver and kidneys. Gapmer PS-ASOs with ligand and the hepatocyte asialoglycoprotein receptor
constrained ethyl, LNA, and/or 2’-MOE modifications illustrates the advantages of receptor-mediated uptake
bind with high affinity to cellular proteins, modifying their of ligand-conjugated antisense drugs targeting RNAs in

Volume 7 Issue 3 (2024) 8 doi: 10.36922/itps.3025