In vivo drug delivery of oligonucleotides presents significant challenges due to their inherent physicochemical properties and biological barriers. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are large, negatively charged molecules that struggle to cross cell membranes on their own. Once in the bloodstream, they are susceptible to rapid degradation by nucleases and clearance by the kidneys, limiting their bioavailability. Additionally, their negative charge can hinder uptake by target cells and efficacy. Off-target effects can trigger toxicity and immune responses, adding another layer of complexity. In this next article in our WHAT’S NEXT IN OLIGO MANUFACTURING series, we look at delivery tools that seek to overcome these barriers.

Conjugations 

In addition to phosphorodiamidate backbone modifications, oligonucleotide conjugations, such as GalNAc, peptide nucleic acids (PNAs) and antibody-oligonucleotide conjugates (AOCs) are an emerging third generation form to further improve on these in vivo challenges. GalNAc (N-acetyl galactosamine) siRNA conjugates are already well-known and established in use for LEQVIO® (inclisiran), GIVLAARI® (givosiran), AMVUTTRA® (vutrisiran), and other approved oligo therapeutics. Around thirty (30) GalNAc siRNA and ASO conjugates are in the pipeline due to their proven superiority to target liver hepatocytes, opening up many disease treatment areas. PNAs, which replace the sugar-phosphate backbone of DNA with a cell penetrating peptide-like structure, exhibit higher stability and binding affinity to target RNA or DNA sequences and are being investigated in indications ranging from osteoarthritis to amyloidosis. The synthesis of PNAs typically involves solid-phase peptide synthesis techniques, where the PNA monomers are sequentially added to a growing chain anchored to a resin.

AOCs are designed to recognize a cancer cell surface protein that can be linked to an siRNA, enabling precise delivery to tumor cells while sparing healthy tissues. For AOC, the synthesis often involves chemical linking strategies, such as using click chemistry or maleimide-thiol reactions, to attach the oligonucleotide to the antibody.

Lipid Nanoparticles

Lipid Nanoparticles (LNPs) are another promising third-gen delivery solution. LNPs are tiny (typically 100 nm), spherical vesicles composed of lipids that encapsulate oligonucleotides. The lipid composition, ionizability, hydrophobicity and morphology can be tailored to enhance oligo payload, stability, cellular uptake, and enable endosomal escape – a critical step for releasing oligonucleotides into the cytoplasm where they can exert their therapeutic effects at an order of magnitude lower dose. The implementation of ionizable lipids marks a significant advancement over early cationic lipids. LNPs have been successfully used in clinical applications, such as the delivery of siRNA in the FDA-approved drug patisiran and prexigebersen ASO in clinical trials. Lipid nanoparticles (LNPs) are synthesized using methods like high pressure homogenization, microfluidics, sonication or ethanol injection followed by extrusion, TFF and filtration to form stable, uniform nanoparticles.

LNP’s lipid composition, ionizability, hydrophobicity and morphology can be tailored to enhance oligo payload, stability, cellular uptake, and enable endosomal escape – a critical step for releasing oligonucleotides into the cytoplasm where they can exert their therapeutic effects at an order of magnitude lower dose.

Despite these advancements, manufacturing these delivery systems at scale presents several challenges. Synthesis of GalNAc conjugates at large scale is fairly well established using either presynthesis by coupling to solid support early on or post synthesis by amide and phosphoroamidite chemistry. Many companies have established their own large scale platforms. However, as more and more GalNAc conjugate oligos become approved over time, improvements such as greater manufacturing efficiency, greener chemistry, and solution phase may be pursued. For PNAs, the cost and complexity of synthesizing and purifying long sequences can be prohibitive. Antibody-oligonucleotide conjugates face hurdles related to batch-to-batch consistency, ensuring the stability of the conjugate while maintaining the biological activity of both components.

LNPs can be produced and encapsulated at room temperatures. However, encapsulation efficiency may vary with modified phosphoroamidite structures, helper lipids and ionizable amino acids. Often LNP formation around the RNA may be needed early in the manufacturing than later due to inherent hydrolytic instability of the RNA drugs. LNPs are also shear sensitive which makes mixing and TFF challenging at large scale. Overall, they require stringent control over particle size, encapsulation efficiency, aggregation and stability during production, lyophilization, storage and transportation. 

Therefore, suppliers can look to offer differentiated solutions in one or several of these areas to become a leading player in this growing subsegment of oligonucleotides. Technology vendors may offer solid phase resins geared towards peptide and antibody conjugations. For LNP, lipid libraries, point of use LNP, and controlled mixing and filtration technology solutions can be offered. CROs could provide linking chemistry, linker reagents, PEGylation, and lyoprotectants along with detailed characterization, mixing, filtration and filling validation services following proper understanding of regulatory requirements. CDMOs may look to specialize in each of the niche area of GalNAc, PNA, AOC and LNP in both upstream and downstream as key competencies. These innovative modalities in the oligo field also represent an opportunity for current biologics CDMOs.

They may branch out in the oligo segment by focusing entirely on liquid phase-based platforms, e.g. biocatalytic, enzymatic, conjugation mixing solutions paired with liquid API skipping solid phase synthesis altogether. It should also be recognized that lyophilization of LNP is still a topic needing much optimization and expertise after taking lessons from Pfizer’s liposomal Covid-19 vaccines, an area where final fill CDMOs could also consider for expansion. With over 200 ongoing clinical trials using LNP delivery, it is also high time that Big Pharma could lead consortiums to guide and set standards for LNPs. All in all, these third generation conjugate and LNP oligonucleotides offer many avenues where suppliers could gain deeper insights and build their unique selling points. 

To Summarize:

In vivo drug delivery of oligonucleotides presents significant challenges due to their inherent physicochemical properties and biological barriers. Multiple conjugation and lipid nanoparticle technologies seek to overcome these barriers to better target the delivery to oligo and reduce dosage for the patient. Suppliers and service provides have ample opportunity to develop products, manufacturing services, and testing services to address the industry’s undermet needs in the drug delivery supply chain.

Are you interested in custom market research services to gain deeper insights into future oligonucleotide manufacturing trends? Reach out for a consultation.

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