Cell-penetrating peptides as a delivery system for oligonucleotides

CDMO in peptides and oligonucleotides manufacturing, Bachem, has started building a new future for oligonucleotides: solving the industry’s main challenges around capacity, sustainability and cost-effectiveness.

Emerging as a new treatment option in rare and orphan disease areas, oligonucleotide therapeutics have matured into a drug class with a broad indication spectrum. The promise of oligonucleotides making a difference to an increasing number of patients is driving Bachem’s rapid implementation of manufacturing capabilities and capacities for oligonucleotide-based APIs.

Oligonucleotides act on the RNA level through different molecular pathways, but there is a major drawback when using them as drugs: their poor bioavailability and cellular uptake. These considerations limit their application as therapeutics.

To address these problems, it’s vital to develop delivery systems that enable oligonucleotides to reach their targets: cell-penetrating peptides (CPPs) present a potential solution to this issue. Here, Bachem explores the science behind this approach.

What are cell-penetrating peptides?

CPPs are short peptides that can translocate cargo molecules across cell membranes2. These peptides typically contain up to 30 amino acids and can be cationic, amphipathic or both.

Over the years, a range of different CPPs have been developed, including natural translocating proteins through to newly-designed computer-prediction sequences3.

How are cell-penetrating peptides used?

When using a CPP as a delivery system, it must first be conjugated to its cargo molecule and internalised through endocytosis. The CPP can then be transported to the endosomal compartment, where it will be entrapped. Finally, the CPP will leave the endosomal compartment to deliver the cargo molecule to the chosen destination.

Ensuring that the CPP can escape this endosomal compartment is crucial for its bioavailability and bioactivity properties.

How are cell-penetrating peptides conjugated to oligonucleotides?

There are two vectorization strategies which can be used for CPPs: covalent conjugation and nanoparticle formation.

To form a covalent linkage between the CPP and its cargo molecule, thiol-maleimide coupling is used. This is a common reaction within peptide chemistry and it has been approved by regulatory authorities for various antibody-drug conjugates4. Bachem has spent time studying and researching this reaction, along with the side-reactions that happen during the coupling stage.

However, this method does come with one major drawback. As CPPs are positively charged and oligonucleotides are negatively charged, they can be prone to aggregation, which can sometimes lead to precipitation of the conjugate.

On the other hand, the nanoparticle formation approach is based on electrostatic and hydrophobic interactions between the CPP and its cargo. This method is generally used to deliver small interfering RNA (siRNA), but it can also be used for larger oligonucleotides too.

Unfortunately, the particles formed though this method are unstable in physiological fluids. To overcome this challenge, the CPP will need to be chemically modified.

Examples of using cell-penetrating peptides to deliver oligonucleotides

Currently, only a small number of CPP-based oligonucleotide delivery examples have been reported for in vivo application.

The table below includes some of these examples.

CPPOligonucleotideConjugation strategy
WRAP5siRNANanoparticles 5
PepFect6Anti-miRNANanoparticles 6
cRGDsiRNACovalent (thiol-maleimide) 7
GLP1RASOCovalent (disulfide bridge) 8

In a recent publication from scientists at the University of Bordeaux, a CPP-based nanoparticles approach was reportedly used to deliver siRNA into cancer cells of solid tumours.

In a second publication by the scientists at Bordeaux, CPP-based nanoparticles were applied as a vector for an anti-miRNA (AMO). AMO are used to neutralise microRNA (miRNA), which are short complementary sequences involved in the suppression of the translation process.

CPP-AMO nanoparticles can also be used for tumour imaging8, where the major component is a PepFect6 peptide that encapsulates the AMO labelled by a radiotracer.

cRGD-oligonucleotide conjugates have also been used in past examples, opening up doors to further opportunities. The cRGD (cyclic(arginine-glycine-aspartic)) peptide is commonly used to target αvβ3 integrin receptors, which are involved in angiogenesis and tumour metastasis.

In one example of a CPP-oligonucleotide conjugate, a derivative of cRGD peptide, cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (MAL: maleimide), was covalently conjugated to a siRNA9. Plus, in numerous in vitro experiments, the conjugate has been able to specifically enter αvβ3 positive human cells and silence the targeted genes.

The promising cRGD-siRNA conjugate has also been injected into tumour-bearing mice, proving to be well tolerated and resulting in a significant reduction of the tumour volume. This example makes clear the potential that cRGD-oligonucleotides have as anti-tumour therapeutics.

In a final example, glucagon-like peptide-1 receptor (GLP1R) was used as a vector for delivering antisense oligonucleotides (ASOs). Originally, GLP1R was viewed as unsuitable for selective drug delivery, due to its low abundance and restricted ability to internalise a large amount of drug conjugate.

Despite this drawback, in 2018, researchers reported a new approach which utilised GLP1R as an internalisation inducer for delivering an ASO to the pancreatic β-cells8. In this approach, the covalent conjugation through a disulfide bridge between GLP1R and the ASO enhanced the selective cellular uptake of the ASO.

This work shows great potential for new treatment options for diseases caused by an aberrant gene expression in pancreatic β-cells, such as diabetes.

Making the transition to peptides

CPPs hold so much potential to overcome the current problems surrounding oligonucleotide delivery. These peptides ensure that the cargo molecules can reach their target cells and provide easier access to challenging tissues, such as muscle and bone marrow.


(1) H. J.-P. Ryser and W. C. Shen, Proc. Natl. Acad. Sci. U. S. A. 75(8), 3867–3870 (1978)https://www.pnas.org/content/75/8/3867

(2) T. Lehto et al. Adv. Drug Deliv. Rev. 106, 172–182 (2016) https://www.sciencedirect.com/science/article/pii/S0169409X16301922
(3) M. Hansen et al. Adv. Drug. Deliv. Rev. 60, 572-579 (2008)https://www.sciencedirect.com/science/article/abs/pii/S0169409X07002918?via%3Dihub
(4) L. Perez et al. Drug Discov. Today 19(7), 869-881 (2014)https://www.sciencedirect.com/science/article/abs/pii/S135964461300398X
(5)I. Ferreiro et al. Pharmaceutics 13(5), 749 (2021)https://www.mdpi.com/1999-4923/13/5/749
(6) Yang et al. Mol. Pharmaceutics 18(3) 787–795 (2021)https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.0c00160
(7) X. Liu et al. Nucleic Acids Res. 42(18) 11805–11817 (2014)https://academic.oup.com/nar/article/42/18/11805/2435428?login=false
(9) X. Liu et al. Nucleic Acids Res. 42(18) 11805–11817 (2014)https://academic.oup.com/nar/article/42/18/11805/2435428?login=true

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