What is gene silencing?

What is Gene Silencing?

Gene silencing is a biological process where gene expression is suppressed or eliminated through the targeted degradation or translational repression of messenger RNA (mRNA), primarily mediated by double-stranded RNA molecules that trigger sequence-specific interference with gene function. 1

Fundamental Mechanism

Gene silencing operates through RNA interference (RNAi), a double-stranded RNA-dependent, post-transcriptional process that regulates gene expression by inhibiting mRNA with small non-coding RNA molecules. 1 The mechanism involves:

• Small interfering RNAs (siRNAs) that recognize and cleave target mRNA through complementary sequence matching 1
• MicroRNAs (miRNAs) that mediate translational repression or direct mRNA cleavage 1, 2
• RNA-induced silencing complex (RISC) formation, where the antisense strand is activated via selective removal of the sense strand by Argonaute 2 (Ago2) endonuclease 1

The Molecular Process

The silencing cascade proceeds through distinct steps:

• Double-stranded RNA (dsRNA) serves as the initiator molecule, processed by Dicer (an RNase III-type endonuclease) into short segments of 19-25 nucleotides in length 1
• Dicer cleavage produces two complementary single-strand RNA segments, but only the guide strand integrates with Argonaute protein 1
• RISC assembly occurs when the single-stranded siRNA binds to complementary target mRNA and catalyzes its destruction or selectively inhibits translation 1
• Target mRNA cleavage happens at a specific site corresponding to the center of the siRNA antisense strand 1

Two Major Silencing Strategies

Gene silencing employs fundamentally different approaches depending on the stage of intervention:

• Transcriptional gene silencing (TGS) prevents RNA synthesis and is often associated with DNA methylation in most eukaryotes 3
• Post-transcriptional gene silencing (PTGS) degrades existing RNA after transcription has occurred, exploiting cellular mechanisms where transcripts with sequence similarity to dsRNA molecules undergo degradation 3, 4

Therapeutic Applications

RNAi has evolved from a research tool to a clinically validated therapeutic class, with four FDA-approved siRNA therapeutics as of 2021 (patisiran, givosiran, lumasiran, and inclisiran), all targeting the liver and administered parenterally. 1

The technology offers distinct advantages:

• Broader applicability than gene addition or gene editing because translation repression is theoretically suitable for all gene-related diseases 5
• Straightforward targeting governed by Watson-Crick base pairing rather than complex DNA integration 5
• Non-viral delivery options (lipid nanoparticles, GalNAc conjugates) that lack mutagenic risk and offer enhanced targeting capability 1, 5

Critical Design Requirements for Therapeutic siRNAs

For effective gene silencing in mammalian cells, siRNAs must simultaneously satisfy specific sequence conditions:

• A/U at the 5' end of the antisense strand 1
• G/C at the 5' end of the sense strand 1
• At least five A/U residues in the 5' terminal one-third of the antisense strand 1
• Absence of any GC stretch exceeding 9 nucleotides in length 1

siRNAs with opposite features produce little or no gene silencing in mammalian cells. 1

Major Clinical Limitations

Off-target gene silencing represents the primary obstacle to clinical translation, occurring through partial binding to non-target mRNA with incomplete homology or improper loading of the sense strand into RISC. 5

Additional safety concerns include:

• Innate immune activation through binding to cytosolic receptors and toll-like receptors, triggering type I interferon and inflammatory cytokine release 5
• Interferon response when long dsRNA is introduced into mammalian cells, making siRNA the preferred reagent over long dsRNA 1

Pharmacokinetic Characteristics

Approved siRNA therapeutics demonstrate unique PK/PD properties:

• Rapid tissue distribution with plasma elimination half-life of minutes to several hours, but tissue half-life of days to months 1
• Major clearance route through tissue uptake rather than renal elimination 1
• Delayed onset of action with long-lasting pharmacological effect reflecting target tissue concentration rather than transient plasma levels 1