How to Enhance the Promising Potential of siRNA Drugs

What is a Nucleic Acid Therapeutic?

Nucleic Acid Therapy is a highly promising treatment approach that holds great potential. It involves introducing a segment of nucleic acid sequence to upregulate, downregulate, or correct target genes, enabling more precise disease treatment. Nucleic acid drugs can be categorized based on their composition into two main classes: DNA drugs and RNA drugs. Among RNA drugs, there are antisense oligonucleotides (ASO), small activating RNAs (saRNA), small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), and nucleic acid aptamers (Aptamer).

What is a siRNA Drug?

siRNA, or small interfering RNA, is a type of artificially synthesized double-stranded RNA consisting of a sense strand and an antisense strand. Typically, siRNA is around 21 nucleotides in length. The antisense strand forms a complete complementary base pairing with mRNA, resulting in mRNA degradation and exerting its post-transcriptional regulatory function. The mechanism of siRNA-mediated mRNA degradation in cells is similar to that of miRNA. Synthesized siRNA enters the cell through endocytosis, and a small portion of siRNA can escape from endosomes into the cytoplasm. Once in the cytoplasm, siRNA forms a complex with Dicer and TRBP, creating the RLC complex. The RLC complex recruits AGO2, and subsequently, the sense strand is degraded. The antisense strand binds to the mRNA sequence with complete complementarity, and AGO2 then cleaves the mRNA, ultimately leading to mRNA degradation.b

How to Optimize siRNA Drugs?

From the design and synthesis of siRNA to the final drug product, several challenges need to be overcome.

(1) Specificity of the target sequence

(2) Off-target effects of siRNA

(3) Immunogenicity of siRNA

(4) Toxic side effects due to the degradation of non-target organ-specific genes after siRNA systemic administration

(5) Efficiency of siRNA entry into cells

(6) Lysosomal escape of siRNA

These issues above can primarily be addressed through the following approaches to enhance the drugability of siRNA drugs.

(1) Sequence Optimization and Selection

The primary purpose of siRNA sequence selection is to achieve two main objectives: first, to reduce the likelihood of off-target effects, and second, to have a highly effective knockdown effect on the target protein. There are three ways in which siRNA can have off-target effects. The first is the miRNA-like off-target effect of the antisense strand of siRNA, where siRNA acts similarly to miRNA by specifically binding to mRNA sequences through seed sequences, leading to mRNA degradation or translation inhibition. The second is due to mismatches in the antisense strand of siRNA, as a certain degree of mismatch is allowed when siRNA binds to mRNA, causing the degradation of mismatched mRNA sequences. The third way off-target effects occur is when the sense strand, which originally needs to be degraded, enters the RISC complex, leading to the knockdown of non-target genes. Therefore, when designing siRNA, it is crucial to assess the siRNA sequence first and then predict off-target effects for all siRNAs that meet the criteria. Off-target prediction primarily involves the Mismatch score and miSeedScore. By calculating weighted scores for these two aspects, the designed siRNA sequences can be ranked, and those with higher scores can be eliminated.

(2) Chemical Modifications

Enhancing the drugability of siRNA can be achieved through chemical modifications. Chemical modifications, such as GNA, LNA, 2′-MOE, can effectively improve the immunogenicity of siRNA, reduce off-target toxicity, and increase siRNA’s effectiveness. Chemical modifications can be categorized based on their different positions, including phosphate modifications, ribose modifications, and base modifications. Typically, all three types of modifications may appear in siRNA simultaneously.

STC Modification Scheme: The STC modification scheme includes ribose modifications, specifically 2′-deoxy-2′-fluoro (2′-F) and 2′-OMe, as well as phosphate modifications using PS (thiophosphate). The STC modification is a universal modification approach that enhances the stability of siRNA and its affinity for mRNA.

ESC Modification Scheme: The ESC modification scheme, compared to the STC modification, incorporates four additional PS modifications. Another significant change is the reduction in the number of nucleotides with 2′-F modifications. This reduction in 2′-F modification is primarily due to concerns about the potential increased toxicity of siRNA with excessive use of 2′-F modification. Compared to the STC modification, the ESC modification scheme significantly improves the effectiveness and half-life of siRNA. The ESC modification scheme results in a superior pharmacokinetic (PK) and pharmacodynamic (PD) profile for siRNA drugs while reducing the required dosage.

Advanced ESC Modification Scheme: In the Advanced ESC modification scheme, the 2′-F modification is further reduced, and the ratio and modification sites of 2′-F and 2′-OMe are optimized. The Advanced ESC modification enhances the stability of siRNA without affecting its effectiveness.

(3) Delivery Systems

The delivery systems for siRNA drugs include lipid nanoparticles, GalNAc, exosomes, peptides, conjugated polymers, and more. Among them, lipid nanoparticles (LNP) and N-Acetylgalactosamine (GalNAc) have received clinical approval and are widely used in clinical applications.

LNP primarily consists of four components: cationic lipids, cholesterol, helper lipids, and PEG lipids. The main differences among LNPs used for siRNA delivery lie in the cationic lipids and PEG lipids, with cationic lipids being the core component of LNPs. LNPs are effective in protecting siRNA, preventing it from degradation by nucleases and clearance by the kidneys, and efficiently facilitating the transfer of siRNA into target organs and cells.

GalNAc is a ligand for the asialoglycoprotein receptor (ASGPR), which is a specific and highly expressed endocytic receptor in liver cells. Therefore, ASGPR is an ideal liver-targeting receptor. By covalently attaching trivalent or tetravalent GalNAc to siRNA, efficient delivery of siRNA to the liver can be achieved. The connection between GalNAc and siRNA can vary, such as attaching GalNAc to the 3′ end of the sense strand of siRNA, or to the 5′ end of the sense strand, or directly forming a GalNAc tetramer based on siRNA.

Exosomes, as an important tool for intercellular communication, have gained increasing attention. Most cells can release various types of small vesicles (EVs), which are loaded with a wide range of materials such as proteins, lipids, and various types of nucleic acids. Based on the process of their formation, extracellular vesicles can be broadly classified into apoptotic bodies, microvesicles (MVs), and exosomes. Exosomes, as endogenous extracellular vesicles, have a diameter similar to that of nanoscale carriers. They can carry different signaling molecules (RNA and proteins), making them potential candidates for drug delivery carriers. Moreover, compared to exogenous nanocarriers, exosomes have advantages such as minimal immunogenicity and lack of biological toxicity.

Antibody-drug conjugates (ADCs) are created by chemically linking a biologically active small molecule drug to a monoclonal antibody (mAb). The mAb serves as a carrier to transport the small molecule drug to the target cells. Recently, the concept of ADCs has been extended to create Antibody-Oligonucleotide Conjugates (AOCs) where siRNA is connected to an antibody. AOCs leverage the characteristics of antibody molecules, such as high specificity, good drugability, and ease of production, to achieve liver-specific and diverse tissue delivery of siRNA.

PNP refers to a polymeric nanoparticle composed of artificially designed and synthesized arginine peptides. Its advantage lies in the ability of each PNP nanoparticle to carry multiple siRNA molecules, enabling combined delivery to multiple target points.

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