Custom Peptide-conjugated PMO (PPMO) Synthesis Service
Introduction
Oligonucleotide therapeutics face poor cellular uptake and endosomal trap issues. Creative Biolabs' Custom Peptide-conjugated PMO Synthesis Service provides custom CPP-PMO delivery vectors via advanced proprietary solid-phase synthesis, creating stable, high-purity conjugates for targets like the CNS and muscle.
This validated approach aids diseases such as DMD and drug-resistant bacterial infections, delivering optimized vectors for stability and endosomal escape. CPPs covalently conjugated to charge-neutral PMOs effectively bypass limitations, with proper CPP design key to delivering active PMO to cytoplasm or nucleus.
Custom Peptide-conjugated PMO Synthesis Service
Synthesis Methods
Creative Biolabs utilizes state-of-the-art solid-phase synthesis techniques for both the PMO and the CPP components. This provides the highest control over sequence and purity for each molecule. Conjugation is typically achieved through highly efficient and orthogonal chemistry, such as:
- Thioether Linkages: Highly stable, non-cleavable bonds preferred for long-term in vivo stability.
- Disulfide Linkages: Chemically cleavable bonds that can be reduced within the cell, potentially releasing the cargo in a targeted manner.
- Strain-Promoted Click Chemistry: Bio-orthogonal reactions that achieve high yields with minimal side-product formation.
Fig.1 Various types of linkers and spacers used for coupling peptides with oligonucleotides.1
Advantages
The Creative Biolabs process is built for therapeutic development, offering key advantages over standard lab-scale synthesis:
- Purity & Characterization: We guarantee purity typically greater than 95%, crucial for minimizing toxicity and ensuring reproducible biological activity.
- Scalability: Our established methods allow for seamless scale-up from milligram research quantities to gram-scale production for preclinical studies.
- Complex Peptide Handling: We specialize in the synthesis of highly challenging, arginine-rich CPPs, which are often difficult to synthesize and purify, such as the (RXR)4 sequence validated in published data for its endosomal escape properties.
Applications
This service is critical for advancing therapeutics targeting tissues that are traditionally hard to reach:
- Splicing Correction: Developing PMOs for genetic diseases like DMD that require robust delivery to the muscle cell nucleus to facilitate exon skipping.
- Antimicrobial Therapy: Designing vectors capable of crossing the protective outer membranes of multidrug-resistant Gram-negative bacteria.
- Central Nervous System (CNS) Disorders: Synthesizing conjugates optimized to cross the blood-brain barrier for the treatment of neurological conditions.
Workflow
Our comprehensive workflow is designed for transparency and high-quality outcomes, providing a clear path from molecular design to the final therapeutic candidate.
Required Starting Materials
To initiate a project, clients typically provide:
- PMO Sequence and Length: The specific base sequence of the PMO (e.g., 2'-O-methyl, PMO backbone) and its desired length.
- CPP Amino Acid Sequence: The chosen amino acid sequence of the Cell-Penetrating Peptide (e.g., arginine-rich sequences like (RXR)4 or R9).
- Desired Conjugation Linker/Chemistry: Specifications for the chemical linker (e.g., stable thioether, disulfide, or biocompatible "click" chemistry) required to join the two molecules.
Initial Consultation & Design Review
We begin by reviewing the complete molecular specifications, including the ON/CPP sequences, linker chemistry, and required in vivo stability profile. This ensures the construct is optimized for its intended therapeutic application and target cell type.
Solid-Phase PMO Synthesis
The PMO backbone is synthesized using automated solid-phase techniques, ensuring precise control over sequence fidelity and the final backbone chemistry.
CPP Synthesis & Functionalization
The custom peptide is synthesized in parallel and chemically functionalized with the necessary linker group (e.g., maleimide or azide) to enable site-specific conjugation.
Conjugation Reaction & Purification
The PMO and CPP are chemically linked via optimized, highly efficient conjugation chemistry. Rigorous multi-step Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) purification is then performed to isolate the final, pure CPP-PMO conjugate.
Quality Control (QC) & Characterization
The final conjugate undergoes extensive QC to verify its identity and purity. This includes confirmation of molecular weight and assessment of solubility for downstream use.
Final Deliverables
Upon project completion, the client receives:
- Purified CPP-PMO Conjugate: The ready-to-use product, typically at greater than 95% purity.
- High-resolution Mass Spectrometry Report: Detailed data confirming the exact molecular mass and successful conjugation.
- Certificate of Analysis (CoA): A comprehensive report detailing the chemical purity, yield, and specific batch characteristics.
Estimated Timeframe
The typical timeframe for custom synthesis ranges from 4 to 8 weeks, heavily dependent on the complexity of the peptide sequence, the length of the PMO, and the specific purification challenges associated with the chosen conjugation chemistry.
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What We Can Offer
Full-Stage One-Stop Synthesis Support
Cover research-grade (milligram-scale), preclinical-grade (gram-scale) to IND-enabling (multi-gram-scale) production, meeting diverse project needs from early R&D to late-stage development;
Highly Customizable Technical Solutions
Support tailored peptide modifications, conjugation of CPPs/targeting ligands (GalNAc, TfR ligands), and synthesis of multi-component chimeras (peptide-PMO-PNA), adapting to specific delivery and functional requirements;
GMP-Aligned Quality Assurance System
Integrate QbD (Quality-by-Design) and PAT (Process Analytical Technology) to establish a standardized quality system, ensuring stable batch-to-batch performance of conjugates;
Strict Aseptic & Comprehensive QC
Implement strict aseptic operations throughout the synthesis process, and conduct end-to-end quality testing (HPLC, ESI-MS, LAL) to eliminate endotoxin and free peptide/PMO residues;
Professional Sequence Optimization Service
Expert computational team customizes CPP/PMO structures to enhance endosomal escape efficiency and tissue-specific delivery (e.g., CNS, muscle), improving therapeutic potential;
Efficient Scale-Up Capability
Realize rapid transition from lab-scale high-throughput screening (supporting 50+ CPP sequences) to gram-scale GLP study production in 6-8 weeks, accelerating drug discovery timelines.
Customer Reviews
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FAQs
Q: How do conjugation methods (amide/disulfide bonds) affect peptide-PMO conjugate stability/activity? What's the optimal selection?
A: Amide bonds offer high hydrolysis resistance (ideal for muscle/CNS long-term use) but are irreversible. Disulfide bonds enable intracellular PMO release (for precise delivery) but risk premature blood cleavage. Click chemistry suits multi-modification but needs copper ion removal. Select per use case; verify via HPLC (>95% conjugation efficiency) and CD (PMO base-pairing).
Q: How to ensure CPP-PMO batch consistency from mg to gram-scale IND production? Key QC indicators?
A: Use automated solid-phase synthesis, uniform monomers, SOPs, and verify >99% stepwise conversion. Key QC: >95% purity (HPLC), <0.01% MW deviation (ESI-MS), 1:1±0.05 peptide-PMO ratio (UV), ≥50% splicing efficiency (RT-PCR), <0.1 EU/mg endotoxin; 3-month storage degradation <5%.
Q: Major challenges in complex peptide-PMO synthesis (dual-peptide, chimeras) and solutions?
A: Challenges: multi-component regioselectivity, purification efficiency. Resolve regioselectivity via stepwise Fmoc/Boc protection (>98% selectivity). Boost purification via affinity tags (e.g., 6×His) and enzymatic removal (>96% purity). Validate via in vitro activity, animal models, and PK/PD to avoid non-functional products.
Creative Biolabs' Custom Peptide-conjugated PMO Synthesis Service delivers high-quality, customized delivery vectors to turn specific oligonucleotide science into therapeutic reality. Mastering CPP-PMO conjugate synthesis, we solve core challenges of cellular uptake and endosomal escape, accelerating your drug discovery.
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Reference
- Malinowska, Anna L., Harley L. Huynh, and Sritama Bose. "Peptide–Oligonucleotide conjugation: chemistry and therapeutic applications." Current Issues in Molecular Biology 46.10 (2024): 11031-11047. https://doi.org/10.3390/cimb46100655. Distributed under Open Access license CC BY 4.0, without modification.
