Human Protein Used To Deliver Molecular Therapies
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A collaborative team of researchers have developed a novel system known as SEND that harnesses human proteins to deliver molecular therapies.
A path towards personalized medicine
Over recent years, the biomedical research landscape has evolved, moving towards therapeutics that manipulate or change the molecular makeup of our cells to treat or prevent a disease. These advancements have been made possible due to scientific endeavors such as the Human Genome Project, completed in 2003, that progressed our understanding of genetics and how our DNA code can contribute to specific diseases.
Molecular biology basics
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.
Choosing the right courier
Developing cargo systems to safely deliver RNA has arguably been the greatest challenge faced by this research field. RNA, specifically, naked RNA, is subject to degradation by nucleases, enzymes that thrive on chomping away nucleic acids they discover in the physiological environment. Packaging RNA in a way that does not activate the human immune system is also important to prevent adverse reactions to drugs. Finally, while incredibly small in the context of what the human eye can see, RNA isn't that small in the context of molecular biology. It can be tricky to deliver it through the cell membrane.2
A simple analogy is to imagine that you are expecting an important, large and fragile parcel. Which courier will the sender of your parcel choose? They must be able to ensure your product will not be damaged en route, that it won't be picked up and taken away by a neighbor in your building or get stuck in your mailbox because of its size.
SEND opens a new delivery route
Researchers from the Massachusetts Institute for Technology (MIT), the McGovern Institute for Brain Research at MIT, the Howard Hughes Medical Institute and the Broad Institute of MIT and Harvard have presented new research that could solve this conundrum. It's called SEND, which stands for Selective Endogenous eNcapsidation for cellular Delivery, and their work is published today in the journal Science.
"Existing delivery vehicles for molecular therapies can be inefficient and lead to random integration of the gene therapy into the genome of cells, both of which can increase the risk of side effects. Moreover, some can stimulate unwanted immune reactions. We also don’t have many delivery systems that can target specific tissues or organs, making it challenging to get molecular therapeutics to the affected part of the body," Professor Feng Zhang, a pioneer of CRISPR genome-editing technology, and senior author on the study, told Technology Networks. The team believe SEND has the potential to overcome these challenges.
SEND is a novel delivery platform that is based on naturally occurring molecules in the human body, reducing the risk of an immune response occurring. To understand how SEND works, we need to take a closer look at a protein known as PEG10. This is an example of a retrotransposon-derived protein.
What are retrotransposons?
Retrotransposons are a class of transposons, fragments of DNA that possess the ability to essentially "move" around the genome. Retrotransposons achieve this mobility through a "copy and paste" mechanism, whereby a gene segment is copied into RNA. This RNA is then transferred to a target site on the genome and the RNA is transcribed back into DNA via an enzyme known as transcriptase. The result? The gene has been inserted into a new location within the genome.
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."
A mix and match approach to delivering genetic material
In this study, the scientists successfully engineered PEG10 so that it can be manipulated to selectively package and carry different types of RNA. “That's what’s so exciting,” said first author Michael Segel, a postdoctoral researcher in Zhang's lab "[…] This study shows that there are probably other RNA transfer systems in the human body that can also be harnessed for therapeutic purposes. It also raises some really fascinating questions about what the natural roles of these proteins might be.”
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.
SENDing genetic therapies into the clinic
The research is still at an early phase, but the team are encouraged by their initial results. "We need to understand how well this system can work in vivo and further engineer the system to deliver cargo to a variety of tissues and cells. We will also continue to probe the natural diversity of these systems in the human body to identify other components that can be added to the SEND platform," Blake Lash said.
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.
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.