A GEENIE in a Bottle: CyGenica’s Unique Non-Viral Cargo Delivery Platform

And how did this discovery come about?

Professor Ueno had invited me to join his lab in iCeMS, the establishment of which was part of a national Japanese interdisciplinary initiative to ignite new discoveries by having biologists do chemistry, chemists do biology, physicists do chemistry, and so on. The idea was that a scientist coming from another discipline would approach a problem differently, and that really triggered my curiosity.

By this stage, I knew how to synthesise compounds and I had a good understanding of proteins and how they fold. But in Kyoto I got the unique chance to investigate exactly what happens when drugs/molecules are added to cells, and I found a protein with a very intriguing structure that entered into living cells.

A molecular drill that bores through the cell membrane

So CyGenica started because of a single protein? What was so special about this protein?

This protein could go through living cells by a unique non-endocytic helical mechanism, which completely defied the known mechanisms of cell penetration. Cell penetration usually occurs through the endocytic pathway whereby positively-charged molecules are designed to be cell-penetrating. But this protein was a negatively-charged molecule that was still going through the cells, and that caught my attention.

I hypothesised based on that discovery that it should be possible to make almost any molecule cell-penetrating. This idea brought me back to India in 2016, where I was awarded an Indian grant to try and prove my hypothesis. Within a year, I could prove it - if I designed a molecule based on the hypothesised criteria, it became cell-penetrating!

Around this time, I was accepted into the RebelBio accelerator programme in Ireland, and the company received accelerator funding followed by pre-seed funding from SOS Ventures in Ireland as well as several grants from Indian agencies.

You mentioned that the protein you found in Kyoto would inspire CyGenica’s GEENIE platform. Can you tell us more about what GEENIE looks like today?

First of all, GEENIE stands for ‘enablinG safE and targEted iNtracellular delIvEry’. We envision that GEENIE will be used to deliver therapeutics for rare genetic diseases, cancer, and infectious diseases.

GEENIE can be thought of as a protein drill that bores through the cell membrane and brings its cargo along with it. It is a 48-KDa protein that is taken into cells by a non-endocytic mechanism that doesn’t damage the cells. It delivers its cargoes to the nucleus, which is highly relevant for gene-editing therapeutics.

Another attractive feature of the platform is that we can easily control how fast the cargo is released from GEENIE once inside the cell by influencing the chemistry, i.e., by carefully choosing an appropriate linker to fuse GEENIE to the cargo. Some linkers will be broken down quite fast inside the cell, while others will persist for longer, resulting in a slower release of the cargo.

The protein that I found in Kyoto is not the protein behind GEENIE. Based on the structure and features of the protein I found, we found another similar protein elsewhere, and engineered it until it became what we now call GEENIE. To use the platform, we make a fusion between the GEENIE protein and its intended cargo, which could be CRISPR reagents, an anti-cancer drug or even an antibiotic.

Strong safety profile and no immunogenicity

Within gene editing, just a few in vivo programmes have advanced, mainly for the liver and the eye. For other organs, it seems to be a challenge to deliver the gene-editing cargo in high enough quantities to achieve efficient editing at a safe dosing level. How safe is GEENIE?

We have shown that GEENIE is non-toxic even at an extremely high dose of 1,800 mg per kg in mice. To put that into perspective, the FDA recommends 2,000 mg per kg as a maximum dose in single-dose safety studies. We tested at 1,800 mg because of a material shortage and found no signs of toxicity or immunogenicity whatsoever in vivo in mice.

In ex vivo studies, we observed a very statistically insignificant cytokine response. The lack of immunogenicity sets GEENIE apart from viral delivery methods such as the adeno-associated viral vectors and lentiviral vectors that are often used in gene therapies. Beyond the safety advantage, as a simple protein, GEENIE is also much easier to work with and more cost-effective than therapeutic-grade viral culture.

You mentioned that GEENIE has a good distribution profile after 24 to 48 hours? In terms of safety, for how long does it ‘hang around’ in cells? For gene editing, it won’t be needed once the editing is done.

Since GEENIE is a protein, it will automatically be degraded by proteases inside the cell. We don’t know exactly how many days this takes but we can see from our biodistribution studies in mice that there is very little GEENIE present after 48 hours.

Giving new life to old antibiotics

We focused mainly on gene-editing therapeutics today, but I also want to touch upon a feature of GEENIE that I find very intriguing. I read that it it may help to overcome acquired antibiotic resistance. How would this work?

We’ve seen that GEENIE can cross the cell membrane barrier of multi-drug resistant bacteria and carry fluorescent dyes into the cells. When it comes to antibiotic resistance, there are two main mechanisms. The first is that the drugs do not penetrate the bacterial cell membrane at all, and the second is that the drugs penetrate but are pumped back out again by protein-based efflux pumps located at the bacterial cell membrane.

We haven’t gone into this area heavily yet, but we see potential for using GEENIE here in two ways. By conjugating GEENIE to a non-penetrable drug, we may be able to deliver the drug inside resistant bacterial cells. In addition, conjugating an antibiotic with GEENIE will render the resulting complex far too large to be effluxed by the pumps. If we can conjugate antibiotics to GEENIE without altering their modes of action, we may be able to revive the antibiotics that have become redundant because of widespread drug resistance.

Partnerships with drug developers

As a start-up company with a unique platform that has potential applications across many areas, what is the long-term vision for CyGenica? Will the company eventually develop its own therapeutic pipeline?

We envision proving GEENIE as a platform by building a pipeline of candidates that we will advance to a certain extent. For instance, we are now working on early pre-clinical studies for glioblastoma and HER2+ breast cancer, using GEENIE to deliver chemotherapeutic drugs, and we hope to take one of these to a first-in-human trial in 2025. At the same time, we are actively looking for partners to engage in R & D partnerships, co-development partnership, as well as partners for out-licensing agreements.

What does CyGenica need to do to get to a first-in-human trial by 2025?

We need to compile a package of data from IND-enabling studies before we can apply to the FDA to start a clinical trial. We have already performed many of these studies sporadically with encouraging results, for instance the MTD (maximum tolerated dose) level and the single-dose toxicity study, so we are confident that we can provide the full package within 2 years, once we have proof-of-concept that GEENIE works in animals. But, of course this all depends on funding.

This has been really interesting, Nusrat. Thanks for the chat and I look forward to seeing what happens next for CyGenica!