BOSTON Following the recent release of a preprint on the cause of death of Duchenne muscular dystrophy (DMD) patient Terry Horgans in a closely monitored gene therapy trial, plausibly in part the result of an immune reaction to the high dose of gene delivery of the vector adeno-associated virus (AAV), there is renewed scrutiny over the long-term safety of virally delivered therapies. So, it’s fitting that my first two interviews at BIO 2023 are about the safety of genetic medicines.

Dietrich Stephan, PhD, CEO of NeuBase Therapeutics in Pittsburgh, echoes my concerns. How many more viral gene therapies do we have to give people before we know they are one time only and that some people will be prepared to have a sharp response against them? he asked.

And Tuyen Ong, MD, CEO of Ring Therapeutics (and CEO partner of Flagship Pioneering), reiterated the importance of mocking his company’s viral delivery platform.

I essentially asked both of them: What would a genius (or an AI platform) give back if you wanted the perfect genetic medicine?

Nuclease-free gene editing

Stephan believes the answer to safe genetic medicines lies in overcoming the barriers of delivery, tolerability, selectivity, manufacturability, shelf life and scalability. Just a couple of weeks ago at the American Society of Gene and Cell Therapy (ASGCT) conference, NeuBase revealed its stealth editors and demonstrated preclinical data that this non-viral, nuclease-free platform does not elicit cell-based immunity and may be a non-immunogenic in vivo solution.

The stealth editor system is based on the NeuBases Peptide-Nucleic Acid Antisense Oligonucleobase (PATROL) platform, which uses small peptide nucleic acid (PNA) molecules consisting of approximately 20 nucleotide modified DNA oligos with peptide-based backbones. By inserting modifications to PNAs that allow for Hoogstein base pairing (instead of the traditional Watson-Crick method), this system can distort the DNA double helix and disrupt polymerase binding. Each of these events sends out a molecular glow that triggers a molecular shear-and-patch mechanism called global genome nucleotide excision repair (GG-NER).

Stephan elaborated: All of a sudden this enzyme complex comes down on the locus and cuts that PNA-DNA event so much and throws it away. Now, you have your single strand break, and if we add a donor oligonucleotide with a corrected base in the middle that will slide to where that single strand break is, it will install the fix. If you have a gain-of-function mutation, you target the mutant sequence, stop the RNA polymerase from transcribing it, and you’re done. You can do the reverse and open the promoters so they are more transcriptionally active and increase genome output, which becomes interesting for haploinsufficiencies.

As of now, NeuBase can inject the bare oligo system, but Stephan said they are working to encapsulate PNA and DNA in clinically validated lipid nanoparticles (LNPs). With these LNPs, NeuBase will target in vivo gene editing, starting in the liver and then in the bone marrow, joining the race to safely treat hemoglobinopathies, such as sickle cell disease, that have plunged some companies.

The field is getting crowded and it’s a really bad macroeconomic climate for early-stage biotech companies, said Stephan. But I think it’s worth keeping pushing. (Neubases stock price peaked at over $11 a share in February 2021, but has since plummeted, currently trading at around $0.20 a share.)

In another bold move, Stephan said that NeuBase has a gene silencing program based on transforming PNA into a molecule that can cross the cell membrane without the need for a cell penetrating peptide (CPP), as Sarepta or others use. and also engineers out the toxicity associated with CPPs by one or two orders of magnitude.

Peter Nielsen, who developed this technology in Copenhagen, has published a first report in Science in 1991 demonstrating the exquisite sequence selectivity and stability of molecules in biological fluids. The next major innovation was conducted at Carnegie Mellon University, which made the molecules soluble and bioavailable. This is where NeuBase, founded in mid-2019, stepped in and licensed the technology.

There’s a lot you can do with these backbones, Stephan said. Indeed, there is almost infinite complexity.

Viruses commensal inside all of us

Instead of evading viral delivery altogether, Ong is looking into the human commensal virome at Ring Therapeutics. When Ong learned of the commensal viruses inside us, a light bulb went on in his mind about how to deal with the safety and delivery of programmable genetic drugs.

We got closer [programmable genetic medicines] variously with viral, typically AAV, and non-viral delivery systems, such as LNPs, which are still lacking in terms of tropism, Ong said.

By looking at the viral equivalent of the gut microbiome, Flagship Ong and colleagues found ringworms named after the Latin word for circular or ring (hence the company’s name). The virus has single-stranded circular DNA, Ong said. We didn’t just find a random virus and Frankenstein in a vector! It was actually thinner than that. And there is no known disease associated with it.

Scientists at the Ring, founded in 2017, have discovered a tremendous amount of genetic diversity in ringworms that is driving phenotypic diversity. These viruses, for one reason or another, have figured out a way not to reside in different tissues in our bodies—they’re tropics for different tissues, Ong said. They somehow evaded our immune response as well. So far we have collected a library of approximately 5,000 ringworm sequences from different body tissues. Our hypothesis is that the sequence that Mother Nature has given us allows us to have those properties.

From there, Ring incorporated machine learning to create a platform for creating tissue-specific vectors. Nobody else is working on ringworms, so we’re taking a very coordinated approach and trying to de-risk scientific understanding in our development plan, Ong said.

A limitation of these ring viruses is that they can only pack a few kilobases of nucleic acids. But Ong believes that, given the challenges already associated with gene editing and delivery, it is best to address the challenges of delivery and tropism. People have not been able to get away from the gravitational pull of the liver and I think we have the ability to do so.

Ong said about 100 Ring employees are working hard to get the first patient dose. In terms of preclinical data, we generated ringworms from different tissues, vectored them, and showed that we can repeat doses and target tissues of interest, Ong said. We’re starting to build that profile of going to the clinic very deliberately. We have identified indications where we can differentiate from a delivery perspective.

One small step for man, one giant leap for humanity

NeuBase and Ring are two examples of bringing genetic medicines to fruition by facilitating specific human attributes to create safer therapies. On the one hand, Ring Therapeutics is exploiting the harmless viruses inside us. On the other hand, NeuBase is facilitating a DNA repair mechanism used by human cells in contrast to the CRISPR-Cas system, which comes from a bacterial immune system. Prokaryotic and eukaryotic cells separated over two billion years ago, with eukaryotic species becoming increasingly complex in terms of genomes and the machinery they’ve had to evolve to maintain those genomes in a high-fidelity state.

The DNA repair protein family, dozens and dozens of different proteins acting in different ways with different mutations in aggregate clean up about a million mutations per cell per day and do it on trillions of cells pretty well, Stephan said. I think this gives you an intuitive sense of the exquisite fidelity of those machines running constantly against some CRISPR-Cas based and prime editor reports in terms of error rates of 0.1% vs. 10-6 at 10-9 error rates.

It’s a critical time for gene therapy not to fall into another dark era, like the one that followed Jessie Gelinger’s death in 1999, especially with so much investment. In the field of background editing, for example, David Liu noted at the ASGCT that it takes only six years from first publication to a successful clinical intervention, which is a tribute to the 3,000 labs and more than 4,000 field publications that are based on this core technology.

NeuBase and Ring are just two of many efforts in genetic medicines approaching the clinic. The more these approaches can be mocked, the greater the chances of having a positive impact on patients’ lives.


#BIO #Derisking #Genetic #Medicines #NeuBase #Ring #Therapeutics

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