Bachem Cell-Penetrating Peptides (CPPs) as delivery systems for Oligonucleotides

Bachem has been developing Cell Penetrating Peptides (CPP) as short peptides that are able to translocate ‘cargo molecules’ across cell membranes into the cell’s endosomal compartment and from there to find their therapeutic target.

CPP technology is particularly exciting for the targeted delivery of drugs based on oligonucleotides that have hitherto been challenging for both bioavailability and cellular uptake.

Releasing Oligonucleotide potential

Oligonucleotides, also called oligomers or oligos, are short single strands of synthetic DNA or RNA. In nature, these ‘small RNA’ molecules function in the regulation of gene expression, for example as microRNAs or miRNAs, or as degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Oligonucleotides can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning and as molecular probes. Further, because small RNA oligos act on the RNA level through different molecular pathways as described in the January 2020  Oligonucleotide Trends article, they form a new class of therapeutics with huge possibilities to treat diseases that were incurable before. The use of oligonucleotide therapeutics is well tolerated by the body, selective to their target, and with reduced secondary effects.

However, there are two major drawbacks when developing oligonucleotide-based drugs: their poor bioavailability and cellular uptake. Overcoming these challenges will require development of  delivery systems that can ‘carry’ oligonucleotides to their intended targets. In a previous article GalNAc: Delivering Promise of Oligonucleotide on oligonucleotide conjugates, Bachem has described the use of the trimeric GalNAc (N-acetylgalactosamine) molecule as a carrier of oligonucleotides to specific cells, the hepatocytes.

Bachem research is also focusing on a delivery system that was first described almost 40 years ago, the cell-penetrating peptide (CPP). The first use of CPP, a poly L-lysine, was for the delivery of an anticancer drug in vitro and in vivo of mouse model¹. It has opened the way for generation of CPP-drug conjugates and has potential as well for oligonucleotides².

What are Cell-Penetrating Peptides?

A CPP is a short peptide that is able to translocate a small ‘cargo molecule’ across cell membranes³. CPPs usually consist of no more than 30 amino acids and are cationic and/or amphipathic, rich in arginine and lysine amino acids.  A variety of different CPPs exists, ranging from natural translocating proteins as the earliest developed CPP to newly designed sequences based on computer prediction⁴.

In all cases, the mode of action is similar: First, the CPP, conjugated to its cargo molecule, is internalized through endocytosis. It is then trafficked to the cell’s endosomal compartment where it is entrapped. Finally, the CPP should be able to escape the endosomal compartment to deliver the cargo molecule to the target. The ability of the CPP to escape this compartment is key for its bioavailability and bioactivity properties. Important criteria for a CPP system delivery are to enhance the cellular uptake, endosomal escape, cell membrane receptor binding, and nuclear localization.

Conjugating CPPs to Oligonucleotides

There are two main vectorization strategies for CPP:

1. Covalent conjugation: A covalent linkage is formed between the CPP and its cargo molecule, with thiol-maleimide coupling adopted as method of choice. It is a widely used reaction in peptide chemistry and approved by the regularity authorities for several antibody-drugs conjugates⁵. Bachem has extensively studied this reaction and the side-reactions that occur during the coupling onto a peptide, with observations posted in a recent blog article and also the subject of a recent webinar. This method faces a major challenge when it comes to purifying the CPP-oligonucleotide conjugate. Since the CPP is positively charged and oligonucleotide is negatively charged, they are prone to aggregation leading to precipitation of the conjugate. Recently, a methodology to couple a CPP to an antisense oligonucleotide efficiently via the thiol-maleimide reaction has been published⁶. The researchers demonstrated that highly cationic CPPs can be conjugated to 2‘-O-(2-Methoxyethyl) phosphothioate oligonucleotides, and that they can be isolated in good yields.
2. Nanoparticle formation: The nanoparticle formation-based approach is based on electrostatic and hydrophobic interactions between the CPP and the cargo. Typically, this strategy is used for the delivery of small interfering RNA (siRNA). One big advantage is that it can be applied for larger oligonucleotides. However, the particles formed are unstable in physiological fluids, requiring the CPP to be chemically modified.

Examples of CPP-based oligonucleotide delivery

Although the delivery of oligonucleotides by CPP represents a very attractive approach, only a handful of examples have been reported in the literature for in vivo application. Some recent examples are described in the table below.

In a recent publication, scientists from university of Bordeaux reported on a CPP-based nanoparticles approach, developed to deliver siRNA into cancer cells of solid tumors. They showed in vivo delivery efficiency of a new peptide WRAP5 that is able to wrap the siRNA and then form nanoparticles⁷.

In a second work, the Bordeaux team applied CPP-based nanoparticles as a vector for anti-miRNA (AMO) synthetically designed oligonucleotides that are used to neutralize the microRNA (miRNA) short complementary sequences to messenger RNA (mRNA) and involved in the suppression of the translation process. CPP-AMO nanoparticles were used for tumor imaging¹¹. The major component is a PepFect6 peptide encapsulating the AMO labeled by a radiotracer used for imaging the miRNA-21 expression in lung adenocarcinoma xenografts. PepFect6 improves the cellular delivery of the conjugated oligonucleotides and has shown promising results for in vivo imaging of miRNA.

The cRGD (cyclic(arginine-glycine-aspartic)) peptide is a common CPP used to target αvβ3 integrin receptors. Integrin αvβ3 is involved in angiogenesis and tumor metastasis and is up-regulated in tumor cells of many cancer types¹². Some examples of cRGD-oligonucleotide conjugates have been reported in the literature to enhance cellular uptake, subcellular distribution, and pharmacological effects of the cargo oligonucleotide¹³. In the following example of a CPP-oligonucleotide conjugate, a derivative of cRGD peptide, cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (MAL: maleimide), has been covalently conjugated to a siRNA¹⁴. In in vitro experiments, the conjugate has demonstrated to specifically enter αvβ3 positive human cells and to silence the targeted genes. Following these promising results, the cRGD-siRNA conjugate has been injected in tumor-bearing mice and has shown very positive results. In addition to being well tolerated and well distributed in the tumor tissues, injections induced a down-regulation of corresponding messenger RNA and protein resulting in a significant reduction of the tumor volume, down to 90%. It thus appears that cRGD-oligonucleotide conjugates have potential as anti-tumor therapeutics.

A last example highlights glucagon-like peptide-1 receptor (GLP1R) as a vector for delivering antisense oligonucleotides (ASOs). In the literature, GLP1R is described to be unsuitable for selective drug delivery mainly due to its low abundance and limited ability to internalize a large amount of drug conjugate. In 2018, researchers described a new approach using GLP1R as an internalization inducer to deliver an ASO to the pancreatic β-cells¹⁵. The covalent conjugation via a disulfide bridge between GLP1R and the ASO has enhanced the selective cellular uptake of the ASO in the targeted cells. The peptide conjugation also improves the potency of the ASO in a GLP1R-dependent manner and induces the gene expression silencing that leads to a reduction in protein levels. Furthermore, it has been demonstrated that GLP1R is not only able to deliver the ASO to the pancreatic β-cells but also into the pancreatic islets in the liver with an enhanced uptake. This work opens the door to a new treatment options for diseases caused by an aberrant gene expression in pancreatic β-cells, like diabetes.

Peptide-Oligonucleotide Transition

CPPs represent a great opportunity to overcome the delivery obstacles and poor bio-distribution of oligonucleotides. They enable the specific delivery of their cargo molecule to their target cells and tissues. Either linked covalently or complexed into nanoparticles with their cargo, CPPs provide a huge mobility advantage and a critical access to the most challenging tissues, such as muscle, bone marrow and barriers, or through the blood-brain barrier. CPP-oligo conjugates combine two core competencies of Bachem, peptide and oligonucleotide synthesis.

For more than half a century, Bachem has provided high quality peptides for various applications, amassing a track record of about 80 Drug Master Files (DMFs) filed. Recently that Bachem heritage and expertise has been expanded to oligonucleotides that also demand expert knowledge in solid-phase synthesis and protecting group chemistry. Downstream processing typically includes the same steps as peptides, such as chromatography, ultra- and diafiltration techniques, precipitation and lyophilization.

Bachem has seen demand for oligonucleotides steadily increasing as the bases for new drugs to cure rare and genetic diseases and has built up its capabilities for large scale cGMP production of oligonucleotides. With its strong innovation program, Bachem can offer its customers the solutions they need to supply large volume of oligonucleotide drugs in sustainable ways, helping pharma and biotech companies to develop new therapies that can transform patient’s lives.

References

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2. Degols et al. Bioconjugate Chem. 5(1), 8–13 (1994)
3. Lehto et al. Adv. Drug Deliv. Rev. 106, 172–182 (2016)
4. Hansen et al. Adv. Drug. Deliv. Rev. 60, 572-579 (2008)
5. L. Perez et al. Drug Discov. Today 19(7), 869-881 (2014)
6. Halloy et al. ChemMedChem 16 (2021)
7. Ferreiro et al. Pharmaceutics 13(5), 749 (2021)
8. Yang et al. Mol. Pharmaceutics 18(3) 787–795 (2021)
9. Liu et al. Nucleic Acids Res. 42(18) 11805–11817 (2014)
10. Ammala et al. Sci. Adv. 4, eaat3386 (2018)
11. Yang et al. Mol. Pharmaceutics 18(3) 787–795 (2021)
12. Pasqualini et al. Nature Biotech. 15, 542–546 (1997)
13. Md R. Alam et al. Bioconjugate Chem. 22(8) 1673–1681 (2011),  Zhou et al. Mol. Ther. Nucleic Acids, 25, 603-612 (2021), K. Nakamoto et al. ACS Omega 3(7) 8226–8232 (2018)
14. Liu et al. Nucleic Acids Res. 42(18) 11805–11817 (2014)
15. Ammala et al. Sci. Adv. 4, eaat3386 (2018)

Resources

Click on Expanding into Oligonucleotides to watch interview with Bachem Peptides & Oligonucleotides Business Development Manager Seamus White.
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Click on Thiazine Formation: The Elucidation of a Thiol-Maleimide Side-Reaction to see video.
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