Antisense-Based Gene Silencing

Innovative Chemistry & Molecular Design

The AUMsilence platform represents a leap forward in RNA silencing technology. ASOs are designed as short (18-22 nucleotide) sequences that are complementary to the target RNA. They feature a gapmer architecture: a central DNA-like "gap" region flanked by modified RNA analogs that increase binding affinity and protect the oligo from degradation.

The central region, once bound to the target RNA, creates an RNA:DNA duplex that is recognized by RNase H1, an endogenous enzyme present in the nucleus and cytoplasm. RNase H1 cleaves the RNA strand of the duplex, fragmenting the target RNA and preventing its translation into protein. The ASO itself is not degraded in this process and can dissociate and bind additional RNA molecules, acting in a catalytic fashion.

From mRNA to Protein Silencing

By cleaving messenger RNA transcripts, AUM's ASO technology effectively reduces or abolishes the production of the target protein. Within hours of ASO introduction, target mRNA levels drop, and corresponding protein levels follow suit (often within 1-2 days, depending on protein half-life). This is a precise mode of action — only transcripts containing the exact complementary sequence are targeted, which means minimal impact on other genes.

Nuclear and Cytoplasmic Action

A key advantage of antisense mechanism is that it works in both the nucleus and cytoplasm. siRNAs and shRNAs primarily act in the cytoplasm on processed mRNA, whereas AUM ASOs can also bind precursor mRNAs or lncRNAs in the nucleus. This broad cellular reach allows silencing of RNAs at multiple stages (from pre-spliced nuclear RNA to mature cytoplasmic mRNA) and is particularly useful for targeting nuclear-retained RNAs.

AUM sdASO™ vs. siRNA (RNAi)

Small interfering RNAs (siRNAs) rely on the RISC complex to bind and cleave target mRNA. Unlike siRNAs, which typically require cationic lipid transfection, AUM sdASO™ products are self-delivering and have shown efficient uptake even in primary immune cells and neurons without any carrier.

siRNAs need to unwind and integrate into RISC, which can sometimes lead to unintended gene silencing via seed-based off-target effects. AUM ASOs avoid RISC entirely; their specificity is encoded in the direct Watson-Crick base pairing to the target RNA.

For in vivo use, delivering siRNA often requires lipid nanoparticles or viral vectors; AUM sdASO™ products can be used in vivo without formulation or conjugates, simplifying animal studies.

AUM sdASO™ vs. CRISPR/Cas9 Gene Editing

CRISPR systems (Cas9 protein or mRNA plus guide RNA) are large and often require advanced delivery methods (viral vectors, electroporation), which can be inefficient and toxic to certain cell types. AUM sdASO™ products are much smaller molecules that readily enter cells on their own.

CRISPR achieves permanent gene knockout by causing DNA mutations. This permanence can be problematic if you only want a transient knockdown or if a knockout is lethal. AUM's ASO-induced knockdown is transient and tunable: stop treatment and gene expression will return.

CRISPR off-targets involve unintended DNA cuts at similar genomic sequences, potentially causing mutations in untargeted genes. AUM ASOs do not alter DNA, and their off-target at the RNA level is minimal due to sequence specificity.

AI-Driven Target Site Selection

Not all regions of an RNA are equally targetable. AUM BioTech's AI algorithms identify ideal target sites based on:

• RNA Structure: Targeting accessible, single-stranded regions rather than highly structured areas
• Sequence Uniqueness: Ensuring the chosen sequence doesn't have significant homology to other transcripts
• Optimal GC Content: Balancing binding strength with specificity (typically 40-60% GC content)
• RNase H Activation: Designing the "gap" region to efficiently recruit RNase H for RNA cleavage

AUM BioTech's platform combines these factors to select target sites that will result in maximum knockdown efficiency with minimal off-target effects. This advanced targeting is one reason why AUMsilence ASOs consistently outperform traditional RNA silencing tools.

Chemical Modifications for Enhanced Performance

The AUMsilence platform incorporates strategic chemical modifications that provide several key advantages:

• Phosphorothioate Backbone: Replacing oxygen with sulfur in the oligonucleotide backbone dramatically increases resistance to nuclease degradation
• Advanced Sugar Modifications:Chemical modifications enhance binding affinity while maintaining RNase H compatibility
• Gapmer Architecture: The central "gap" region promotes RNase H recruitment, while modified flanking regions enhance stability and cellular uptake
• Self-Delivery Enhancements: For sdASO™ products, additional modifications enable direct cellular uptake without transfection reagents

These carefully balanced modifications are applied according to the specific needs of each product in our lineup, optimizing for different RNA targets and experimental contexts.

In Vivo Delivery (No Vehicle Required)

Traditional RNA therapies often need lipid nanoparticles or viral delivery for in vivo use. AUM's sdASO™ products, however, can be administered naked (without a delivery vehicle). Studies have shown effective uptake in tissues when oligos are injected via routes such as:

• Subcutaneous injection
• Intraperitoneal injection
• Intravenous injection
• Intrathecal injection
• Oral gavage for gut targets

For example, to knock down a gene in a mouse model, a researcher can simply dissolve the AUM sdASO™ in saline and inject it subcutaneously; the oligo will distribute and enter target cells to silence the gene. We typically recommend testing a dose range (e.g., 3-30 mg/kg in mice) to find the optimal silencing effect.

Pharmacokinetics and Tissue Penetration

Chemical modifications provide high stable in biological fluids, giving AUMsilence sdASO™ a relatively long half-life in vivo. AUMsilence sdASO™ can circulate and penetrate tissues such as liver, muscle, tumor xenografts, and even cross leaky areas of the blood-brain barrier at effective concentrations.

For widespread systemic targets, intravenous (IV) injection can be used. For localized action (e.g., knocking down a gene in the CNS), intrathecal injection can introduce the ASO directly into the cerebrospinal fluid. No special formulations or conjugates are needed to achieve tissue uptake, though formulations can be used to further enhance delivery if desired.

In Vivo Efficacy Examples

AUMsilence sdASO™ technology has been used in animal studies for a variety of applications. In one example, mice injected with an AUMsilence ASO targeting an inflammatory cytokine showed a drastic reduction in that cytokine's mRNA in spleen and liver, correlating with lower protein levels and an amelioration of disease symptoms in a model of autoimmune disease.