Human Protein Used To Deliver Molecular Therapies – Technology Networks

A collaborative team of researchers have developed a novel system known as SEND that harnesses human proteins to deliver molecular therapies.

Proteins are often referred to as the "workhorses" of the cell. There are many different types of proteins expressed in the human body, such as enzymes, receptors and signaling molecules. Proteins are encoded by DNA. The central dogma of molecular biology specifies that DNA is transcribed to RNA, which is then translated to proteins. This is a highly simplified summary but enables you to understand where proteins come from. If there is a mutation or an error that occurs during this process, it can result in a faulty or absent protein, which can lead to human disease. By developing therapeutics that target the molecular processes that result in protein production, we can work to treat the cause of a disease, rather than just the symptoms. To learn more about transcription and translation, visit our summary piece.

Examples of such therapeutics include gene therapies and RNA-based therapies. The COVID-19 global pandemic has cast a spotlight on RNA, as the first vaccines to receive authorization for human use were mRNA-based. However, using RNA in a therapeutic context is not a novel idea. The authorization of mRNA-based COVID-19 vaccines is a culmination of many decades of research effort from groups across the world. Ultimately, there have been many barriers to overcome in the process of developing RNA therapeutics, and many challenges remain.

Studies that knock out the PEG10 gene have demonstrated that the subsequent protein plays a role in embryonic development, binding to cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), a type of RNA that is important in placentation (the forming of the placenta inside the uterus).Previous research had shown that another retrotransposon-derived protein known as ARC could form structures that resemble viruses and were able to transfer RNA between cells. Would it therefore be possible to engineer retrotransposon proteins to become a "courier" for genetic material? It was considered but had not yet been proven.

"Working with Eugene Koonin and his team at NCBI, we identified a number of retroelement- derived proteins in the human genome that were predicted to form capsids, including PEG10. We screened these proteins to find one that not only formed capsids, but also exhibited specificity for what mRNA was packaged inside the capsids. PEG10 fit the bill," Blake Lash, graduate student in the Zhang lab, and co-first author of the study, told Technology Networks. "It mostly had its own mRNA inside the capsids, which told us that there was a specific mechanism guiding the packaging process, and we hoped we would be able to take advantage of that to reprogram PEG10 packaging."

The engineering involved a number of steps. First, the researchers had to search for molecular sequences within the PEG10 mRNA that it is able to identify and package. These signals were utilized to modify PEG10 so that it would selectively package specific types of RNA. Fusogens were then attached to the surface of the PEG10 capsules. These are proteins that are found naturally on the surface of cells, and act like a "binding glue". The fusogens help SEND to target a particular cell, tissue or organ. Zhang said that mixing and matching different components within the system will open the door for developing therapeutics for different diseases.

"To test if our cargo was being delivered, we used assays to see if the cargo was functional in the recipient cell. For example, we delivered the mRNA encoding a fluorescent protein, and we could read out the delivery of that cargo by looking to see if the receiving cells started to fluoresce (this can be done visually with a microscope)," Segel said. "We also delivered the mRNA encoding the CRISPR gene editing protein Cas9 and the guide RNA that directs Cas9 to its targets. In that case, we tested to see if SEND worked by looking for gene editing at the target site in the genome of the receiving cells." These testing processes occurred in both mouse and human cells, where SEND was successful across both types of cells.

Both a limitation and a feature of the delivery system is that it does not deliver DNA, it delivers RNA. RNA is rapidly degraded, while DNA persists for longer. This is a typical feature of RNA delivery vectors and it is a property that has been harnessed to create therapeutics that can make reversible changes to human physiology. Ultimately, the therapy can be readministered as needed to ensure the intended therapeutic effect is maintained.

Zhang concluded, "The realization that we can use PEG10, and most likely other proteins, to engineer a delivery pathway in the human body to package and deliver new RNA and other potential therapies is a really powerful concept."

Feng Zhang, Michael Segel and Blake Lash were speaking to Molly Campbell, Science Writer for Technology Networks.References:1.Segel M, Lash B, et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for intercellular mRNA delivery. Science. 2021. doi: 10.1126/science.abg6155.

2.Kaczmarek JC, Kowalski PS, Anderson DG. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Medicine. 2017;9(1):60. doi: 10.1186/s13073-017-0450-0.

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Human Protein Used To Deliver Molecular Therapies - Technology Networks