At the beginning of 2016, Peter Kim, PhD, was at the Ventura Beach Marriott Hotel in Southern California for a Gordon Research Conference, not long after he had resigned from his position as president of Merck Research Laboratories (MRL). Given his seminal research elucidating the way in which proteins enable viral membranes to fuse with cells as a professor of biology at Massachusetts Institute of Technology and a member of the Whitehead Institute before his time at Merck, Kim was drawn to a presentation given by professor Dehua Pei, PhD, showcasing data on a novel class of peptides, developed with his postdoc Leo Qian, PhD, at the Ohio State University. In his new role as an advisor at 5AM Ventures, Kim was looking into the use of peptides in medicine.
According to Pei’s studies, a class of small cyclic cell-penetrating peptides (CPPs), consisting of amino acids strung together to form a ring, can transport all major drug modalities into a cell, from small molecules to large proteins. The early endosome is a common cellular bottleneck where many therapeutics become trapped before being trafficked to the lysosome, the cell’s natural garbage disposal.
These CPPs, however, appeared to sidestep this check. In a nutshell, cyclic CPPs bind to the endosomal membrane and form CPP-rich lipid domains. These domains then cause the growth of small vesicles that leave the endosome. Once the vesicles detach from the endosomal membrane, they disassemble into amorphous peptide-lipid aggregates and release their contents into the cytoplasm.
Pei then co-founded Entrada Therapeutics, named after the Spanish word for entrance, on the simple premise that a therapeutic compound must first reach and enter a specific cell in order to reach its intended intracellular molecular target. In 2018, Entrada raised $59 million in a Series A financing co-led by 5AM Ventures and MPM Capital, along with Roche Venture Fund, MRL Ventures Fund (MRLV), and Agent Capital. Kim was later brought on as a member of the Board of Directors at Entrada.
“If you can’t get into the cell and evade the natural mechanisms to identify and get rid of a foreign substance, you’ve lost, yet many therapeutics are not designed with a mechanism to get through the cell membrane and into the cell,” said Dipal Doshi, who was recruited in 2017 to be Entrada’s president and CEO. “Many companies have been just inundating the body with more and more drugs, hoping that some of it comes through the cell membrane and then maybe a little bit gets to the target.”
Kim enlisted the help of Natarajan Sethuraman, PhD, whom he worked with at Merck following the acquisition of GlycoFi back in 2006, to conduct research at Entrada on wild-type mice to track these CPPs, now known as Endosomal Escape Vehicles (EEVs). These preliminary experiments demonstrated the remarkable muscle penetration of EEVs.
“We were blown away by the amount of cargo—in this case, an oligonucleotide, that we could actually get to the muscle,” said Doshi. “What we’ve been able to show is the ability to get a lot there at much lower doses, which we think is much better for the patient in the long run.”
Biological bullseyes
As EEVs are modular, they can incorporate targeting sequences, such as nuclear targeting sequences, to achieve subcellular targeting resolution, and they can be used to treat a wide variety of diseases.
“The modularity of the EEVs allows us to add on mechanisms to get to where we want to go,” said Doshi. “We’ve got a mitochondrial disease that we are working on, and we were able to engineer a mitochondrial targeting sequence.”
The company has made significant improvements to the platform and amassed what they consider to be best-in-class preclinical data in their lead therapeutic areas of Duchenne muscular dystrophy (DMD) and myotonic dystrophy type one (DM1), the most common form of muscular dystrophy for which there are no FDA-approved treatments.
DM1 is brought on by a mutation in the 3′-untranslated region (UTR) of the DM protein kinase (DMPK) gene, which contains a large, expanded trinucleotide (CTG) repeat sequence. Instead of the usual 5-35 triplets, mutant DMPK alleles can have up to several thousand. Consequently, in DM1, two therapeutic camps have emerged: one focuses on knocking down DMPK, while the other takes a more specific, individualized approach to preventing the triplet repeats. Entrada is going after the triplet repeats with their EEV strategy.
In December 2022, Vertex Pharmaceuticals agreed to a collaboration deal with Entrada to develop EEV therapeutics. As part of the in-licensing agreement, Entrada received an upfront payment of $224 million and an equity investment of $26 million. “We’ve been neighbors of Vertex since they’ve been in the Boston Seaport,” said Doshi. “They approached us about the EEVs with a very strong interest in these rare monogenic diseases. So, we started talking to them about DM1, which was their specific interest.”
When it comes to DMD, Entrada uses an oligo in combination with the EEV, while Sarepta Therapeutics’ gene therapy approach, which received accelerated approval from the FDA a few weeks ago, relies primarily on an oligo alone. According to Doshi, the data Entrada has collected on DMD is “astronomical.”
Oligonucleotides and onward
According to Doshi, it is crucial to advance this oligonucleotide core strategy, introduce it to the clinic, and provide patients with a therapeutic that will actually benefit them. “You just have to be good at something; you can’t just be good at a bunch of different things, and then all of a sudden, eight years from now, you’re still a preclinical company,” said Doshi. “The key is to address the disease and find a solution or treatment for the patients and their families, who so desperately deserve it. At the end of the day, DMD and DM1 are the tip of the spear for this company.”
Due to the versatility of EEVs, Doshi has stated that Entrada 2.0 will feature not only lead programs in DMD and partner programs in DM1, but also a variety of approaches. Doshi predicted the platform’s continued success, saying, “The different tools that we can bring to the table because of the modularity and flexibility of the platform are going to lend themselves well to future growth.” He added that Entrada has “quietly announced in an SEC filing that now we’re starting to begin really interesting work around gene editing” with some exciting preliminary results.
Entrada went public in late 2021 and currently employs approximately 130 people. The preclinical company recently moved into new offices in the Innovation and Design (ID) Building. Of the roughly 80,000 square feet of space, Entrada is using 75% of it and subleasing the remaining space. Doshi said that what’s important about that 60,000 square feet of space is the amount of space that’s dedicated to the labs. Besides large-scale manufacturing, most everything is done internally. Confirmatory studies are conducted at Entrada’s facilities. “We have the R&D capabilities of a very big research team,” said Doshi. “Most of the company is really focused on R&D, and we are building a clinical organization as we get closer and closer to the clinic.”
But Doshi said that if someone can do something better, more efficiently, then that deserves a conversation, especially if it’s a non-core asset. For example, Doshi said that Entrada has no business dabbling in oncology. “We are not geared to be an oncology company, nor are we trying to build one,” he said.
“There are a lot of companies that are interested in intracellular oncology targets that are really attractive but very, very difficult to address. What I don’t want to do is license a really important program to a company for a lot of money just to have it set up. That’s not the point of the company. The point of the company is to follow the science and get drugs made.”
