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Page 26 of 35 Scherman. Rare Dis Orphan Drugs J 2023;2:12 https://dx.doi.org/10.20517/rdodj.2023.01

[135]
This technology is now in a clinical trial for a Type 1 myotonic dystrophy RNA drug .

Another PK/PD challenge paradoxically comes from the tremendous efficacy of the last generation of RNA
drugs. The remarkable duration of the siRNA silencing effect after a single dose results from the siRNA
metabolic stability and increased affinity to RISC, which have been brought by the optimized chemistries
described in Figures 4 and 5. In addition, grafting functional groups such as 5’vynil phosphonate or methyl
phosphonate facilitates guide strand interaction with the RISC complex [Figure 6]. In this way, the guide
strand can remain stably bound to RISC and undergo sustained long-term recycling [Figure 3, left side].
The fact that a single administration can lead to 6 to 12 months of activity raises the necessity to find ways
to terminate the treatment on demand, and to do this fast enough in case of severe adverse events. The
stopping drug effect can be obtained by promoting guide strand dissociation from RISC. Such a reversal
system has been described in the liver. It uses short synthetic high-affinity oligonucleotides complementary
to the siRNA guide strand that can compete with RISC binding to the guide strand . The authors reported
[163]
that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets to displace
the guide strand from RISC and to stop the interference reaction. This 9-mer is targeted to the liver through
a tri-GalNac moiety.

Concern must also be considered about the risk of potential toxicity effects of ASOs / siRNA. The first type
of toxicity effects might result from direct interactions between the ASOs / siRNA and cellular proteins via a
hybridization-independent mechanism. This potential toxicity has to be studied in a similar way than for
any classical drug.

The hybridization of ASOs, siRNA/RISC complex, or of free single-strand siRNA to non-intended RNAs
sharing some sequence homology with the targeted mRNA is called “off-target effect”. For instance, ASO
hepatotoxicity in mice has been described to be partly mediated via the RNase H-dependent degradation of
off-target RNAs [164,165] . Some strategies are being developed in order to alleviate the ASO off-target effects.
Optimization of the ASO nucleotide length by extending its size from a 14-mer to an 18-mer has been
shown to reduce the number of off-target candidates, presumably by decreasing the number of matching
with non-intended mRNAs .
[166]
siRNA might suppress the expression of unintended mRNAs with partially complementary sequences by a
similar mechanism to that of miRNA-mediated RNA silencing. This siRNA-mediated off-target effect
occurs mainly from similarities in the siRNA seed region (nucleotides 2-8). An in-depth analysis of which
base pairing was responsible for the off-target effect involving a machine learning technique and using a
random sampling procedure led to the conclusion that nucleotides 2-5 were mostly responsible for siRNA
off-target effects on RNAi . Enhanced stabilization chemistry has been recently proposed to substantially
[167]
[68]
reduce siRNA seed-mediated binding to off-target transcripts while maintaining on-target activity .
Another cause of off-target silencing is improper strand selection by RISC. Passenger-strand silencing can
be avoided by selecting siRNA sequences with high thermodynamic asymmetry or by chemically modifying
the sense strand, as described above [168,169] .

Abundant siRNAs or shRNA can overload the endogenous RNAi pathway leading to toxicity, which has
been mostly reported in hepatocytes. Precise dosing must be carefully optimized to avoid this saturation of
the RISC system and limit hepatotoxicity of short- and long-term clinical gene silencing by both
approaches [169-171] .