Oligonucleotide Biology Platform

mRNA, RNA, messenger RNA molecule, siRNA, antisense oligonucleotide, micro RNA

Oligonucleotide, oligo, conjugation, formulation, synthesis, GalNAc, siRNA, micro RNA, ASO, lipid, PMO, miRNA

Monomer/linker/conjugate synthesis

Synthesis of bridged and bicyclic nucleic acids, novel amidites, and GalNAc ligands
Oligonucleotide custom synthesis of siRNA, miRNA, ASO/gapmers, aptamers, CpG ODN, PMO, and DNA
GalNAc-siRNA conjugation, for targeted delivery to hepatocytes
Lipid conjugation, to increase cellular uptake and enhance stability of oligonucleotides
Peptide conjugation of ASOs and siRNAs, to improve cell targeting and permeability
Conjugation of oligos to small molecules, toxins, PEG, and antibodies

Oligo Library

Library of GalNAc-conjugated siRNAs
Custom oligonucleotide library synthesis services, including siRNA duplex libraries and PMO libraries
Comprehensive library of special monomers
High-throughput oligo synthesis and purification

Oligo Optimization

Nucleobase and sugar-skeleton modifications
Sequence substitution and back bone optimization
Optimization of conjugation, for delivery and release
Stability testing of siRNAs and conjugates

Target Validation and Sequence Design

In silico sequence selection
CRISPR library in vitro and in vivo screening
sgRNA library screens (sgRNAi and sgRNAa)
Cell-line engineering: overexpression, shRNA knockdown, CRISPR KO/KI
Phenotypic profiling: cell proliferation, cell death, cell cycle profiling
Genetically engineered mouse models

In vitro and in vivo efficacy/early tox screening, off-target

Knockdown effect in cell lines and primary cells (mRNA & protein assessment)
Functional evaluation via cell cycle analysis and downstream signaling pathways
Assessment of cell-based delivery using GalNAc/ASGPR binding & uptake assays
In vitro toxicity: immune response profiling and cytotoxicity assessment in primary human hepatocytes
Off-target effects (transcriptome analysis): off-target profiling by RNAseq
Quantification of siRNA in the RNA-induced silencing complex (RISC)
In vivo PD studies in HDI, transgenic, and AAV mouse models; confirm cellular location of knockdown
In vivo PD studies in NHPs, determine duration of knockdown effect
Analysis of biomarkers to predict PD/efficacy studies of oligos

ADME and in vivo PK

PK studies in mice: siRNA levels in plasma, kidney and liver quantified by stem-loop qRT-PCR
Plasma protein binding assays and biodistribution studies
Metabolic stability assessment and metabolite profiling/identification
Mass balance and excretion analysis (including biliary excretion)
Bioanalytical platforms for measuring oligo levels, including LC-MS/MS, LC-HRMS, qPCR, and ligand binding assays

Formulation

Polymeric, lipid nanoparticles (LNPs) for siRNA and ASO delivery
Advanced technology offering high homogeneity, robust reproducibility, and scalability for all types of liposomes
LNPs exhibiting high encapsulation efficiency and narrow particle size distribution at <100 nm

Learn more on oligo.wuxiapptec.com

Scale-up and Manufacture

State-of-the-art facilities supporting oligo process development and manufacturing at any scale, from nmol to mol
Covering a variety of oligo types including, ASOs, siRNAs, aptamers, oligo conjugates, PMOs and PPMOs
Oligo production services from lab scale to commercial scale, with more than 10 R&D production lines

Learn more on oligo.wuxiapptec.com

Toxicology

Full toxicology capabilities, core battery of testing available
Discovery toxicity studies include exploratory, genetic, and in vitro assays
General toxicology studies (rodent, rabbit, dog, NHP, minipig) for 14-days, 28-days, and 13-weeks, 26/39-weeks
TK bioanalytical method development and validation
Immunology-related tox services, including tissue cross reactivity and Immunohistochemistry studies

Target Selection

Off-target, in silico, bioinformatics, oligo sequence, homology, SNP, transcripts, RNA, ASO

Selection and validation of target and modality

Bioinformatics for in silico sequence pre-selection

• Overview of target: expression pattern, isoform, SNPs
• Homology comparison cross species
• Off-target consideration
• Efficiency prediction

Oligo design

Semi-automated in silico oligo sequence and modification design

siRNA, RISC complex, knockdown, FRET, qPCR, GPCR, Tm, splicing

Sequence Design for Candidate siRNAs

Transcripts of target gene Expression & Isoform	*Genomic information* SNP & genomic sequence
Fragmentation	• SNP avoidance • GC ratio/distribution • Position at transcripts
Off-target filter in human	• Genome-wide • Transcriptome-wide • Target microRNA
Homology comparison	• NHP • Mouse/Rat
Efficiency evaluation	• Sequence context • Thermodynamic features

In vitro activity

In vitro cell-based knock down assessments (transfection and free uptake)
Splicing analysis
Functional assays
Tm (melting temperature) determination
Quantification of siRNA in RISC complex

Cell Lines for Knockdown Assessment

> 400 cell lines available lines in WuXi Biology cell bank

Patient derived cell lines
Customized stable cell lines with lentivirus system

Primary Cells for Knockdown Assessment

Primary human and animal hepatocytes
Primary monkey, rat and mouse fibroblast cells; neuronal cells and muscle cells
Customized additional primary cells available upon request

Transfection Method

Commercial reagents
Electroporation

Reporter assays	psiCHECK reporter assay and other reporter assays
Evaluation of knockdown effect	• mRNA assessment by RT-qPCR, bDNA, ddPCR, capillary electrophoresis, Sanger sequencing and NGS • Protein assessment of by ELISA, Luminex, MSD, CBA, Western/Capillary Western, FACS, HCS, IF, HiBiT luciferase reporter assay, AlphaLISA, and ICW
Tm (melting temperature) determination	FRET method with qPCR
Cell-based functional assays	• GPCR & other receptor assays • Ion channels & transporters • Apoptosis/cell cycle • Angiogenesis • Downstream signaling pathway analysis • Cell structure and morphology analysis • Virus infection assays • Customized assays

RT-PCR, mRNA, electroporation, primary cells, LDH

siRNA, gapmer, aptamers, lentivirus, RNAseq, psiCHECK reporter assay

Early toxicology

Cytotoxicity assesment

Cancer cell lines (HUH-1, HEPG-2)
Non-cancer cell lines (HEK-293, HK-2, HFL-1, HUVEC)
Cryopreserved primary human hepatocytes

Immunogenicity

HEK-Blue-hTLR3, 5, 7, 8, 9, RIG-I, MAD5 cell-based reporter assays
Cytokine induction and immune cell activation in PBMCs

Off target profiling

In silico analysis
NanoString, single-cell RNAseq analysis
psiCHECK reporter assay

Cell viability and apoptosis	• CellTiter-Glo • LDH assay • Caspase-Glo 3/7
In vitro immunogenicity assays	• Cell-based reporter assays: TLRs, RIG-I and MAD5 reporter • Cytokine stimulation in hPBMCs or animal immune cells/whole blood
Off-target effects	• PPHs transfected with siRNA; RNA extraction and analysis by RNAseq • Generation of volcano plots, heatmaps, CNET plots • Analysis of pathways not involving the target gene

siRNA, psiCHECK, RT-PCR, NHP models, in silico, GalNAc-ASGPR delivery, SPR screening

siRNA activity in RT-PCR reporter assay, KD

In vivo Efficacy and PK/PD studies

Humanized mouse models
- Transgenic
- HDI
- AAV mouse models
NHP models
Pharmacology models
Various dosing routes
Short and long-term KD efficacy/pharmacology readouts

Humanized mouse models, transgenic, HDI, AAV mouse models, NHPs

Delivery and Analysis

GalNAc-ASGPR delivery system
SPR screening
Customized assays for other delivery systems
In vitro stability and protein-protein binding
Delivery/RISC PK in mouse, rat, NHP

GalNAc-ASGPR delivery system, SPR screening, RISC PK, protein-protein binding

Oligonucleotide Synthesis & Conjugation

A One-stop Shop to Create Your ‘Monomer-Oligo-Conjugate’

Focus on Discovery Scales to Support Early-stage Development | Accelerate Oligo-based Drug Discovery
Delivering a broad spectrum of special monomers, diversified oligonucleotides, and custom-tailored solution for oligo-conjugation
- Special monomer synthesis bicyclic nucleic acid, stereo-defined amidite, GalNAc, PMO monomers
- Comprehensive panel of oligonucleotides: siRNAs, antisense oligonucleotide (ASO), CpG, aptamer , PMO
- Modified oligonucleotides: various bicyclic nucleic acids, 2’-modification , inverted abasic, 5’-VP
- Linker-attached oligonucleotides: single-strand and duplex with 3’- or 5’-linker modification, including amino, azido, alkyne, tetrazine, and maleimide, toward antibody conjugation.
- Long RNA sequences: up to 100 bp, >90% purity
- Oligo conjugation: siRNA and ASO with 3’- or 5’-conjugation, including GalNAc, [eptide, small molecule, cholesterol, lipid, PEG, and fluorescent tags