One-Stop Antisense Oligonucleotide (ASO) Development Services

Overview

Small interfering RNA (siRNA), antisense oligonucleotides (ASOs), and aptamers are called Oligonucleotide Drugs. Compared with normal chemotherapy drugs, biological therapeutics, and small molecule drugs, oligonucleotide drugs have a number of advantages, such as targeting at the level of genes, wider target range, shorter development time, more controllable synthesis method compared to other biological drugs (antibodies, viral vectors, etc.), high intracellular accessibility (can be delivered to the interior of cells), multiple mechanisms of action (gene silencing, exon skipping, and other RNA regulatory mechanisms), lower immunogenicity, and the ability to combine with other drug types such as antibodies and peptides to achieve synergistic effects. Due to its ability to regulate gene expression and protein function with low toxicity, it has achieved certain results in the treatment of neurodegenerative diseases, rheumatoid arthritis, psoriasis, kidney transplant rejection, inflammatory bowel disease (Crohn's disease), tumors, rare genetic diseases, etc.

ASOs are less than 30 nucleotides, synthetic single-stranded RNA (ssRNA) or single-stranded DNA (ssDNA) molecules, designed to have a sequence that complements their target. ASO depends heavily on RNase H to play a role. RNase H-dependent ASOs produce a DNA/RNA duplex upon binding to their complementary site on a target mRNA. Double-stranded recruits the ubiquitous RNase H, resulting in RNA strand degradation and reduced protein levels, with a degradation rate of 80-95%¹.

Fig.1 Schematic representation of the action of therapeutic oligonucleotides^1,3

Advantage of Our Service

• Experienced technicians can use knowledge bases and algorithms to jointly implement ASO sequence design modifications and gram-level production of ASO;
• Customized screening and validation protocols;
• Extensive project experience with fast speed of delivery;
• We provide a one-stop development service that supports streamlined processes with multiple advantages, such as time-saving, reduced overhead, centralized technical support, fewer Intermediaries, etc.

When you need to order ASO, you only need to provide a background introduction of the target gene or related diseases and applications, and we can design the ASO sequence accordingly, use common modifications to make it have better properties and effects, and provide a series of related experiments such as subsequent formulation and functional analysis, etc.

Antisense Oligonucleotide (ASO) Design and Synthesis

• The First Generation ASO —— PS ODNs

The molecular mechanisms of toxicity of chemically modified phosphorothioate ASO, facilitate cellular uptake and protect against nuclease degradation, extending their half-life from minutes to days.

• The Second Generation ASO —— 2'-MOE

The sugar backbone was modified by the addition of alkyl groups at the 2' position of the ribose. 2'-O-methyl (2'-OMe) and 2'-O-methoxyethyl (2'-O-MOE) modifications are considered the standard. The 5-10-5 gapmer substitution is usually chosen.

Fig.2 Several forms of second-generation ASO^2,3

Gapmer refers to a form of oligonucleotide that contains an internal region "gap" of approximately 10 nt and is not modified with a 2 '- O -alkyl (or other modifying group) group, allowing this region to retain high affinity for RNAse-H enzyme, while their "wings" have approximately 5nt and be modified with a 2' - O-alkyl group to alter their properties. Gapmer modification increases the resistance of ASO drugs to nuclease degradation while ensuring RNase H activity. Chemical analogues of natural RNA are derived from bases modified with LNA, 2 ' - OMe, or 2' - F. These modifications can enhance enzyme resistance of nucleic acids, reduce immunogenicity, and decrease toxicity. Gapmer can also generate higher affinity for target mRNA, which reduces off-target effects, non-specific binding, and unwanted gene silencing.

Fig.3 Gapmer molecular structure Distributed under CC BY-SA 4.0, from Wiki, without modification.

2'-F

2 '- F modification is a chemical modification suitable for siRNA. High electronegative fluoride increases the binding affinity of modified siRNA to target mRNA sequences, and also exhibits good nuclease tolerance. 2 '- F can be used for full sequence modification of RNA, which does not affect binding to RISC, thus providing a more stable and efficient double-stranded structure.

2'-OMe

2 '- OMe modification is one of the most widely used modifications in oligonucleotide drugs, which can increase the binding affinity to target RNA and improve nuclease stability. 2 '- OMe modification is also very suitable for siRNA because it has good nuclease tolerance and can reduce siRNA immune stimulation.

Combining these modifications with gapmer design can effectively improve the properties of ASO drugs.

• The Third Generation ASO

LNA:

LNAs utilize a methylene bridge linking the 2'-oxygen and 4'-carbon of ribose to enhance stability, and binding affinity, and inhibit backbone hydrolysis through conformational constraint.

PNA

PNAs are ONs characterized by the substitution of the phosphodiester backbone with a polyamide backbone, composed of repetitive units of N-(2-aminoethyl) glycine, wherein the nucleobases are linked by a methyl carbonyl linker.

Substituting ribose rings with morpholino rings and phosphodiester bonds with phosphorodiamidate bonds, makes high solubility in an aqueous solution.

Fig.4 Schematic diagram of common ASO modification methods^2,3

Antisense Oligonucleotide (ASO) Conjugate

The significance of ASO antibody conjugation lies in utilizing the specific action of antibody drugs to solve the targeting problem of ASO, enabling it to enter cells and target mRNA; By utilizing the stable structure of antibody drugs, the stability of ASO can be improved; Combined with the specificity of small nucleic acid targets, it ultimately promotes the safe, effective, and selective treatment of diseases with drugs.

The commonly used antibody conjugation mechanisms currently include the use of fish sperm protein, RNA binding protein, and the strong interaction between biotin and avidin. There are two main ways of fish sperm protein conjugation: one is to directly express a fusion protein (scFv/tP) containing antibody scFv and fish sperm protein fragments, and the other is to use a bifunctional crosslinking agent - sulfo SMCC, in which the NHS ester reacts with primary amines to form amide bonds and then reacts with hydrophobic groups to form stable thioether bonds, thereby achieving antibody oligonucleotide conjugation. Similarly, the mechanism based on RNA binding proteins also has two ways of action. Firstly, it directly expresses a fusion protein containing antibodies and RNA-binding proteins. Secondly, crosslinking agents such as sulfo SMCC are used to link the separately expressed antibodies and RNA-binding proteins.

Antisense Oligonucleotide (ASO) Delivery

In Vitro ASO Delivery

*GalNAc (N-acetylgalactosamine) is a high-affinity targeting ligand for the sialoglycoprotein receptor (ASGPR), an endocytogenic receptor that is highly specifically expressed on the membrane surface of liver cells. ASGPR and clathrin-mediated endocytosis can effectively transport GalNAc from the cell surface to the cytoplasm. The disadvantage of the GalNAc delivery pathway is that it is only effective on liver cells expressed by ASGPR, and there is no effective delivery pathway in other cells/tissues.

**Polymer-based nanoparticles are a relatively innovative delivery method with nanoparticles coated with RNA with dendritic macromolecules, polylactic acid-glycolic acid copolymers (PLGA), and other Polymer carriers. It has the physical and chemical properties of biodegradability, biocompatibility, high water solubility, and stable storage. Similar to LNP, the delivery principle of polymer is that therapeutic RNA is released under acidic conditions after the cell membrane is internalized to form endosomes. At present, more research materials include PLGA, polyethylenimide (PEI), poly (β-amino ester) (PBAE), poly (amide amine) (PAMAM), and so on.

After ASO is delivered into cell lines, we can provide a variety of experimental methods (flow cytometry, immunofluorescence, WB, qPCR, Elisa, etc.) and experimental results to detect the silencing efficiency of ASO on target genes, cell activity after administration, and off-target effects. In addition, we also provide other personalized validation experimental designs.

Off-target Analysis of ASO

The off-target effect is mainly caused by the following factors:

• Non-specific binding of guide RNA (gRNA) or DNA binding domains;
• The DNA binding domain recognizes and binds to non-target sites that are similar to the target sequence, causing accidental DNA cutting;
• Endonuclease may still cut DNA after binding to non-target sites, resulting in off-target effects.

The detection of off-target effects can be divided into extracellular detection and intracellular detection. Extracellular detection methods are the most direct, purifying and cutting the genome in vitro, and then capturing the sites where off-target cutting occurs. The advantage of the intracellular assay method is that the purified genome has been digested and removed with histone proteins, so its structure is looser than chromatin in the cell, and theoretically more off-target sites can be easily found. Off-target effects after gene editing can be detected by:

• Whole Genome Sequencing (WGS): Through whole genome sequencing, all variations in the genome are comprehensively detected and off-target effects are identified.
• Targeted Capture Sequencing: High-throughput sequencing of predicted off-target sites to verify variation at specific locations.
• Genome-wide Unbiased Identification of DSBs Enabled by Sequencing: Identification of some sequence/protein-induced double-strand break sites by integrating small double-stranded oligonucleotide labeling and high-throughput sequencing.
• PCR-based Detection Methods: Identify off-target effects by detecting heterozygous mismatch sites in PCR products.

Custom In Vitro ASO Screening

We can provide customized and personalized delivery and screening processes for customers to choose from. As mentioned earlier, there are currently multiple ways to deliver ASO in vitro, such as viral vectors, non-viral vectors, and electroporation technology. In addition, we can provide various cell model platforms, such as primary cells, immortalized cell lines, iPSC-derived disease models, organoids, etc. You can choose suitable cells for transfection based on the function of the target RNA. Finally, for the selection of ASO transfection concentration and cell number, we will provide as detailed and appropriate a ratio as possible for pre experiments, in order to obtain an experimental plan with high efficiency and high cell activity.

Custom In Vivo Study of Antisense Therapeutics

Regarding commonly used animal models in vivo, we can provide mdx mice, R6/2, YAC128, SMN Δ 7, SOD1-G93A, TTR V30M, and so on.

The most widely used animal model for DMD is the muscular dystrophy protein deficient mdx mouse, which has a mutation in the anti-muscle atrophy protein gene, resulting in reduced expression of fully functional muscular dystrophy protein. The Dmd mdx mutation in mice has a stop codon on exon 23, which can produce truncated proteins and exhibit the muscular pathological features of DMD.

R6/2 and YAC128 mice, as common animal models for studying Huntington's disease, have shown significant neuropathological changes in the caudate putamen and other brain regions.

The SMN Δ 7 mouse model was used to study spinal muscular atrophy, and the pathology of SMA in the mouse cerebellum was evaluated using structural and diffusion magnetic resonance imaging, immunohistochemistry, and electrophysiology.

SOD1-G93A mice can be used to study neuromuscular diseases such as amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease).

References

1. Dume, Bogdan, Emilia Licarete, and Manuela Banciu. "Advancing Cancer Treatments: The role of oligonucleotides-based therapies in driving progress." Molecular Therapy-Nucleic Acids (2024).
2. Scherman, Daniel. "RNA antisense and silencing strategies using synthetic drugs for rare muscular and neuromuscular diseases." Rare Disease and Orphan Drugs Journal (2023).
3. Distributed under Open Access license CC BY 4.0, without modification.