1. Введение
Cancer remains a significant global health crisis and a leading cause of mortality worldwide
[1][1][2][3][3][4][5]
According to the U.S. Food and Drug Administration (FDA), effective GT agents should repair or replace disease-causing genes, introduce therapeutic genes, or modulate the expression of faulty genes
[6][4][7][7][8][9][10][8]
Targeting aberrant RNA splicing, which leads to the synthesis of cancer-specific protein variants, also presents a promising therapeutic avenue
[11][12][13][14][14][6]
To address these limitations, innovative approaches utilizing marker-activated antisense agents are being explored. This review focuses on binary antisense oligonucleotides (biASOs), a novel strategy designed to enhance both target specificity and the therapeutic impact of conventional ASOs. biASOs are engineered with a unique “sensing module” that recognizes cancer-specific marker RNAs, coupled with a “therapeutic module”, similar to conventional ASOs, that aims to suppress vital housekeeping genes
[6]
Graphical Abstract
GT — Gene Therapy;
FDA — Food and Drug Administration;
mRNA — messenger Ribonucleic Acid;
miRNA — micro–Ribonucleic Acid;
nc-RNA — non-coding Ribonucleic Acid;
si-RNA — single interfering Ribonucleic Acid;
RNA — Ribonucleic Acid;
biASO — binary antisense oligonucleotides;
ASO — antisense Oligonucleotide;
4WJ — Four-way junction;
RNase H —Ribonuclease H;
SNP — single nucleotide polymorphism;
KRAS — Kirsten rat sarcoma virus;
S/B — signal to background ratio.
2. Design and Mechanism of Action
Binary antisense oligonucleotides (biASOs) represent a significant departure in both design and functional mechanism from conventional antisense oligonucleotides (ASOs). Unlike their predecessors, biASOs employ a two-component system to achieve highly targeted RNA degradation
[6][6][15][16][17][18]
Mechanism of action of binary antisense oligonucleotides (biASOs)
[19][20][6][21]
By design, the biASOs are deliberately engineered to be inert in the absence of the intended activator RNA, a feature that enables their conditional activation, restricting their activity to cells expressing the specific marker RNA and thus enhancing target specificity
[6][13][22]
3. Features and advantages
The new approach of using binary antisense oligonucleotides (marker-activation strategy) during the construction of biASOs gives them a greater advantage over the conventional ASOs thus addressing some of the limitations faced by conventional ASOs. This approach gives the biASOs a high selective advantage and unlimited efficacy because the method confines the therapeutic activity to maker-positive cells and enables the targeting of vital housekeeping gens
[6][18][23][13][21]
Lastly, the biASOs are versatile and easily tuned to specific desires due to the modular design which provides a considerable flexibility. The sensing module can be easily made to adapt to recognising a variety of RNA markers including different cancer-specific transcripts, combined markers and others
[6][14][24]
Shows the key Features and Advantages of the biASO, Compared to conventional ASOs
| Key feature | Advantage | Reference |
| Enhanced specificity | The design nature of biASO which employs the 4WJ complex formation highly enhances its target specificity. According to Drozd et al. (2022) even a subtle sequence variation in the activator RNA (like a single base mismatch) can be readily discriminated by the biASO sensing module thus less background activation. | [6] |
| Improved selectivity | The marker-dependant activation greatly reduces the potential for unwanted gene suppression in healthy tissue lacking specific targeted makers hence minimizing system toxicity and side effects. | [21] |
| Tunability and Versatility | The modular design gives biASO flexibility tunability because the sensing module can be adapted to recognise a variety if RNA markers such as different cancer transcripts, and combinations of makers for increased specificity. | [24] |
4. Potential Applications of biASO-Based RNA Modulation in Cancer Therapy: In Vitro Evidence
Despite the promising design and mechanism of binary antisense oligonucleotides (biASOs), direct evidence for their efficacy in treating cancer in vivo remains to be established. Clinical trials are yet to commence, and preclinical studies in animal cancer models are anticipated to follow. However, current evaluations of biASOs’ therapeutic potential are predominantly derived from in vitro studies that rigorously examine their mechanism of action and capacity for marker-dependent RNA degradation
[6][6][25][6][26][27][28]
This allowed for the assessment of biASO activation in response to both common and cancer-specific KRAS variants. The efficiency of RNA cleavage, serving as a direct readout of biASO activity, was quantified as the percentage of GFP-RNA cleaved by RNase H in the presence or absence of the KRAS activator RNA. Initial experiments focused on optimizing the biASO design to achieve robust activator-dependent cleavage. This was done by systematic variation of the length of GFP-RNA binding sites within the biASO constructs, hence they identified that the biASO construct designated as a8-b10 were exhibiting the greatest signal-to-background ratio (S/B)
[6][12][13]
5. Challenges and Future Directions for biASO-Based Therapeutics
Even though the binary antisense oligonucleotides (biASOs) present a compelling and innovative approach to targeted RNA modulation, it is crucial to acknowledge the significant challenges that must be addressed to translate biASOs in vitro promise into clinically effective cancer therapeutics.
[14][29]
6. Conclusion
Binary antisense oligonucleotides (biASOs) represent a novel and conceptually advanced class of oligonucleotide-based therapeutics. By employing a bifunctional, two-component design and a marker-dependent activation mechanism, biASOs offer the potential to overcome key limitations associated with conventional antisense oligonucleotides. In vitro studies have provided compelling evidence for their mechanism of action and ability to selectively degrade target RNAs in a marker-dependent approach
[6]