Peptide Oligonucleotide Conjugates – Why the Excitement?
By Dr Jordan Fletcher (Technical Director of Peptides) and Dr Cameron Thorpe (Oligos Team Lead)
The Rationale for Peptide Oligonucleotide Conjugates
Peptide Oligonucleotide conjugates (POCs) have been garnering a lot of attention over recent years, and for very good reason. The rationale behind their use is incredibly simple: take the exquisite precision of oligonucleotides – able to silence, repair, or modulate gene expressions – and couple it with the ability of peptides to deliver a cargo to its site of action. It is a marriage made from necessity, because for all the sophistication of antisense oligonucleotides and siRNAs, their biggest problem has always remained delivery. On the other hand, peptides are one of nature’s messengers, able to bind cell-surface receptors, navigate the body’s transport systems, and often able to pass through membranes. By linking the two modalities together we create a hybrid molecule, bringing together targeted peptide cell delivery, with effective therapeutic oligonucleotides.
Why Now?
Although the concept of POCs has been around for decades, recent years have seen a surge of interest. Several factors have converged to create this momentum. There are many reasons at play, but one of these is very simple: timing. After several generations of oligonucleotides hitting the market, the challenge facing oligonucleotide therapy has changed. There is no doubt that gene-targeted medicines are of utility in humans, but now the primary hurdle has shifted from “can we make them potent” to “can we get them to their site of action in a targeted fashion”.
Peptides are seen as part of the answer to this conundrum and in recent years the chemistry has matured. Advances in synthetic strategies – for both peptide and oligonucleotide assembly – as well as a multitude of orthogonal protection and conjugation strategies, has made the manufacture of POCs more accessible than it has been in the past. Finally, Pharma is now far more open to considering that oligonucleotide conjugates and POCs could be the answer to their questions in healthcare. In this respect, nothing brings more confidence to this than the overwhelming success of GalNAc-oligonucleotide conjugates which offer targeted delivery to hepatocytes and has shown that with targeted delivery oligonucleotide therapies unlocks a multibillion-dollar industry. The hope now is that peptides will enable similar delivery to tissues beyond hepatic targets.
Conjugation Strategies and Synthetic Challenges
Despite recent advances, synthesis of peptide oligonucleotide conjugates is not without its challenges, often requiring Pharma, Biotechs or CROs to bring together multidisciplinary teams of peptide and oligonucleotide scientists to tackle these hybrid modalities together. There are considerable differences in the chemistries used in their assembly: Although both biopolymers are often synthesised using solid phase synthesis, peptides are typically prepared using Fmoc/tBu methodologies on a polystyrene support, while oligonucleotides are assembled via phosphoramidite chemistry on controlled-pore glass. Each of these methods have been honed over decades and – apart from caveats around their green credentials – are highly refined and fit for purpose. As such, the synthesis of each component is typically optimised on its own, and the peptide and oligonucleotide components then conjugated together via a chemical linker. The formation of the hybrid modality is often done in solution, but with thoughtful use of orthogonal protection strategies, may also be completed on the solid support before final deprotection and cleavage. Either way, there are now several refined chemistries available for such conjugations, and an assortment of ways in which chemically addressable handles may be incorporated. Popular options include conjugations between a cysteine thiol with a maleimide-containing oligonucleotide, the ever-popular azide/alkyne “click” reaction” – in either its copper-catalysed or strain-promoted guise – as well as other methods including oxime ligation, disulphides, and hydrazone formation. Aside from offering a convenient means to assemble POCs, the choice of conjugation strategy can also offer extra levels of control. Indeed, stability, or the deliberately introduced instability of the linker is a key design consideration. Disulphides are a classic example here allowing for the reducing environment of the cytosol to be exploited and leading to the intracellular cleavage of the peptide and oligonucleotide components. Other linker strategies, such are triazoles, are robust by comparison and will remain intact throughout.
Clinical Proof-of-Concept
What has been so pleasing to see in this field over recent years, is that this hypothesis – that peptides can guide the targeted delivery of oligonucleotides – has grown from “good in theory”, to a point now that their clinical potential is clear. In the quest to develop therapeutics to treat Duchenne muscular dystrophy (DMD), the conjugation of phosphorodiamidate morpholino oligomers (PMOs) to polycationic cell-penetrating peptides gives rise to enhanced dystrophin production in muscle fibres following systemic administration.1,2 Albeit, this is also seen with unconjugated morpholino analogues, but only at much higher and less practical doses.
In oncology, the classic integrin-binding peptide, cyclic RGD,3 has been conjugated to siRNAs to target the integrin-rich vasculature of tumours to deliver gene-silencing therapeutics with reduced off-target effects. In a similar vein, other researchers have made use of peptides known to bind other receptors, such as SSTR2 for treatment of neuroendocrine tumours,4 HER2 receptor for treatment of breast cancer,5 as well as bone marrow targeting motifs for haematological disorders.
A Modular Platform
What makes these clinical advances so exciting is generalised proof-of-concept that is implied. POCs are endlessly adaptable, in both the peptide and oligonucleotide component. The peptide component can be swapped to target the oligonucleotide to a different tissue, or for one that evades the immune system for example. The oligonucleotide can be fine-tuned through chemical modification as well, leading to enhanced stability, binding affinity, and potency. This is a modular system of “plug-and-play” meaning POCs are a true platform therapeutic.
Challenges and Outlook
The progression of peptide oligonucleotide conjugates from bench to the clinic is not without its challenges. Although it may soon change, we are yet to see POC make it all the way through clinical trials. So, all this excitement does need to be tempered slightly. Cationic CPPs can be toxic at high doses and can be problematic when working alongside polyanionic oligos. Endosomal escape will continue to be a hurdle with the frustration that, although the POC has been delivered intracellularly to the target tissue, the oligonucleotide payload remains trapped in intracellular vesicles after entry. Industrial-scale manufacture may also prove a challenge, will require manufacturers to overcome significant engineering challenges, alongside potential re-evaluation of chemistry, which although robust for early phase devolvement, may not be suitable for larger scale production. Anyway, when a therapeutic platform such as this shows such clear potential the manufacturing sector will find a way.
Conclusion
Despite the above obstacles, the future for POCs remains incredibly bright. The push for extrahepatic oligonucleotide delivery is strong, and peptides are an ideal candidate to replicate the success of GalNAc – indeed, the wealth of small, receptor-specific peptides allows scope to target almost any tissue type. Coupled with an array of tools now at our disposal, across both fields, to tackle hurdles that come our way, there is little wonder there is such excitement – something that has not been lost on us here at CatSci either.
At CatSci, with our combined expertise in peptides, oligonucleotides, and biosciences, we are uniquely position to drive innovation in this field – and we look forward to sharing moer on our work in this space over the coming months. It is an exciting time to be involved.
- Hong M. Moulton, et al. Bioconjugate Chemistry, 2004, 15 (2), 290-299
- Alter, J., Lou, F., Rabinowitz, A. et al. Nat Med, 2006, 12, 175–177
- Xiaoxia Liu. et al. Nucleic Acids Research, 2014, 42, 18, 11805–11817
- Hoppenz P, Els-Heindl et al. Front Chem. 2020, 8, 571
- Mittendorf, E.A. et al. Annals of Oncology, 2016, 27, 7, 1241–1248