Advancing oligonucleotide drug delivery through endosomal escape
Controlling endosomal escape could be the key to enhancing therapeutic oligonucleotide delivery, researchers say.
The therapeutic scope of oligonucleotide drugs has expanded in recent years. This includes treatments Tides Global previously reported on, including for neurodegenerative conditions like Parkinson’s and nano-rare diseases like KIF1A-associated neurological disorder.
Despite ongoing research in this field, effective delivery to target organs remains an obstacle to the widespread use of oligonucleotide-based therapeutics. One of the main culprits: inefficient endocytosis.
Researchers have proposed that facilitating endosomal escape could address this crucial rate-limiting step. Tom Baldini, principal scientist at AstraZeneca, and Leo Qian, co-founder and vice president at Entrada Therapeutics, presented their respective teams’ strategies at TIDES Europe 2024.
Escaping the endosomal chasm
Unlike small molecules, oligonucleotides cannot passively diffuse across the membrane of their target cell. Instead, the macromolecules are taken up by a process called endocytosis. Originally coined by Christian deDuve in 1963, a 2018 Journal of Immunological Sciences article by Xiaofeng Ding describes endocytosis as “a process in which a cell internalizes non-particulate materials […] by engulfing them in an energy dependent manner.”
This is where the problem lies. Very few endocytosed oligonucleotides are released from the organelle’s lipid bilayer into the cytoplasm. For the rest, their fate is endosomal entrapment.
“Less than 1% of an oligo-drug dose leads to productive uptake” and “therefore higher doses are often required,” Baldini and co-authors reported on their poster. In turn, “high doses may lead to undesired side effects such as proinflammatory responses.”
Some scientists have proposed using pH-responsive polymers – such as the cationic poly(ethyleneimine) – to exploit the acidic conditions of endosomes. Known as the proton sponge effect, polycationic compounds act as a buffer against endosomal acidity through the uptake of protons. A continuous buffering action increases osmotic pressure within the endosome. This ultimately leads the organelle to rupture and release the captive oligonucleotide drug into the cytosol.
The scientific community has yet to reach a consensus on the validity of this hypothesis. Some studies even challenge it.
This ongoing issue led researchers like Baldini and his colleagues to investigate a different approach. Specifically, his team sought to improve the endosomal escape of oligonucleotide therapeutics and optimize their use in vivo. Their method uses what they call endosomal escape enhancers (EEEs).
Baldini’s study is yet to be published and peer reviewed. Therefore, the team has declined to reveal the specific methods used to design and develop their EEEs. However, clues may be found in a related study – also published by AstraZeneca scientists – which previously demonstrated the efficacy off EEEs in conjunction with extracellular vesicles containing therapeutic cargo.
The team did, however, detail the purported capabilities of their technology.
“Our EEEs can enhance endosomal escape to the same extent as RNAiMAX without toxic side effects,” Baldini and his colleagues claimed. Further, these EEEs reportedly “work for any oligo-type” such as “sequence-specific oligonucleotides, antisense oligonucleotides” and “primarily act in the release, not the uptake.”
RNAiMAX is a cationic lipid formulation that facilitates the delivery of small interfering RNA (siRNA), single guide RNA (sgRNA), and microRNA (miRNA) into cells in culture, although it is known to be toxic. The compound is commonly used as a gold standard in a laboratory setting for in vitro experiments.
If Baldini and his team’s method is indeed comparable in efficiency to RNAiMAX while remaining safe for cells, this could represent a significant step forward in drug development.
AstraZeneca applied for a patent earlier this year (WO2024084394) protecting various compounds they have developed to facilitate the delivery of oligonucleotides into the cytosol. This may hint at the work that is to be released by Baldini and his colleagues once the study passes review in Molecular Therapy Nucleic Acids.
Without more details to understand exactly how these EEEs work, it is impossible to say whether this approach will bear out in animal models. Still, the possibility of overcoming these hurdles is tantalizing.
Entrada, not entrapped
Elsewhere at TIDES Europe, Entrada Therapeutics’ Leo Qian outlined his company’s endosomal-based strategy to overcome the limitations of oligo therapeutic delivery.
He presented on the capabilities of Entrada’s “Endosomal Escape Vehicle” (EEV). The aim of the technology is to deliver oligonucleotide therapeutics to skeletal and cardiac muscle tissue, both notoriously hard to reach.
“Fit-for-purpose EEVs can be designed for target indications and modalities through iterative optimizations of EEV peptides,” he told the audience. By way of an example, Qian described how Entrada’s “first-generation EEV1 peptide-phosphorodiamidate morpholino oligomer (PMO)” facilitated antisense oligonucleotide (ASOs) delivery in HeLA EGFP-654 cells. This approach prevented aberrant splicing and restored eGFP expression in vitro, indicating their ASOs reached their target transcript.
“Our EEV-therapeutic candidates have demonstrated favorable pharmacological properties,” he continued. These characteristics include “efficient intracellular delivery, significant uptake in target tissues, and potent pharmacodynamic outcomes.”
Early success encouraged Entrada to specialize in specific areas of disease. The company chose Duchenne muscular dystrophy (DMD) because the condition “has a substantial patient population with a significant unmet clinical need,” reflecting Tides Global’s past reporting.
DMD is often caused by frameshift mutations which lead to incorrect gene splicing and dystrophin depletion. The team at Entrada focused their efforts on developing an exon-skipping therapy. By skipping mutated exons, the gene can shift back in-frame, producing a truncated but functional dystrophin protein.
The company claims that its EEV technology increases cellular uptake of oligonucleotides. These principles are at work in their new drug, ENTR-601-44, which is currently undergoing clinical trials.
The FDA maintains a clinical hold on Entrada’s drug – despite an appeal from the company – preventing further clinical trials in the United States. Therefore, the company has relocated its development efforts to the UK.
Entrada’s recent press release outlined preliminary Phase I results. The report emphasized pharmacokinetics and target engagement; in comparison to placebo drugs, ENTR-601-44 successfully reached muscle tissue and significantly induced exon 44 skipping. This follows a preclinical trial, which investigated its efficacy in human dystrophin-expressing (hDMD) mice and nonhuman primates.
“Based on the cumulative data to date, we expect to see a significant accumulation of exon skipping and dystrophin production in patients,” said Dipal Doshi, CEO at Entrada, “which we believe will lead to an improvement in functional outcomes after multiple doses.”
He continued: “We believe that the flexibility of our EEV-based approach will allow the therapeutic to be tailored to meet the changing needs of growing pediatric and young adult patients.”
If forthcoming clinical trials generate positive results, this treatment could eventually make its way to DMD patients. Approximately six per 100,000 individuals live with this deadly condition in the United States and Europe. Entrada “would like to decrease this figure by leveraging the EEV platform,” Qian shared.
The field of oligonucleotide therapeutics is making strides toward overcoming drug delivery by improving targeting and cellular uptake. Although significant hurdles remain, early successes with EEEs and EEVs highlight the potential of these technologies to develop more effective therapies.
