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Page 4 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

truncated form of the SMN2 mRNA is produced. An ASO which sterically blocks the intronic splice silencer
present on the SMN2 gene favors pre-mRNA maturation towards the complete form of the SMN2 mRNA,
thus leading to the expression of a functional SMN protein. This approach has represented a historically
major success as the first therapy for SMA [5,7,8, and more references in Spinal muscular atrophy].

ASO-induced mRNA degradation
RNA primers are required to initiate the synthesis of both the leading strand and Okazaki fragments on the
lagging DNA strand during genome replication. Moreover, DNA replicases occasionally incorporate
ribonucleotides into DNA. Finally, R-loops generated as a by-product of transcription when nascent mRNA
molecules hybridize with the template DNA represent another example of naturally occurring RNA/DNA
duplexes. Cells are ubiquitously equipped with ribonucleases H type 1 (called here RNase H), whose
function is to remove RNA moieties from DNA, because such RNA/DNA duplexes might cause
chromosomal instability and cell lethality during replication [12,13] . The RNases H nuclease belongs to the
nucleotidyl transferase superfamily which relies on divalent cations to catalyze nucleophilic substitution
reactions. These enzymatic reactions specifically hydrolyze either a single ribonucleotide or stretches of
RNA in a diverse range of nucleic acids, such as RNA/DNA hybrids, R-loops, and double-stranded DNA
with an embedded single ribonucleotide, etc. . Other enzymes such as transposase, retroviral integrase,
[14]
Holliday junction resolvase, and RISC nuclease Argonaute involved in the mechanism of RNA silencing
belong to the same nucleotidyl transferase superfamily as RNase H.

As already mentioned, antisense oligodeoxynucleotides annealing to complementary mRNA sequence
create an intracellular DNA/RNA heteroduplex to which RNase H binds, leading to cleavage and
subsequent degradation of the targeted mRNA [Figure 2A]. After mRNA cleavage at the complementary
site, the mRNA strand is released from the ASO, which thus becomes available for further association with
another target mRNA.

RNase H-dependent ASOs are of interest for treating diseases caused by dominant-negative genetic
variants. An important feature is that RNase H requires a non-modified deoxyribose moiety on the center of
the complementary ASO sequence (the “seed” sequence) to maintain its catalytic efficiency. Thus, an
efficient ASO must be either a pure deoxy oligomer bearing only unmodified ribose sugars, or a gapmer
containing a stretch of about 10 natural ribose sugars with a variable number of modified sugars on the 3’
and 5’ ends.

mRNA degradation by RNA interference RNAi
The second strategy to suppress a targeted mRNA in a sequence-specific manner makes use of the RNA
interference process (RNAi) [15-18] . As indicated above, interference is the natural microRNA-mediated
process (mi-RNA) that is central to the post-transcriptional silencing regulation of many basic cellular and
developmental programs. This process is schematized in Figure 3.

The basic steps of miRNA maturation involve the transcription by RNA polymerase II of primary miRNAs
(pri-miRNAs). The miRNAs derive from regions of RNA transcripts that fold back on themselves to form
short hairpins whose main characteristic is the presence of one or several mismatches in the self-
complementary sequence. The primary transcript known as pri-miRNA is processed in the cell nucleus into
~70 nucleotides pre-miRNA by the microprocessor complex Drosha/DGCR8. The microprocessor complex
subunit DGCR8 (DiGeorge syndrome critical region 8) contains an RNA-binding domain that stabilizes the
primary miRNA for processing by the second microprocessor subunit Drosha, an RNase III enzyme. The
pri-miRNA is cleaved by the Drosha/DGCR8 complex to a characteristic stem-loop structure known as a
pre-miRNA. Pre-miRNAs have a hairpin structure with stems containing interspersed mismatches and are