Addressing challenges in nucleotide therapeutic delivery

Types of Nucleotide Therapeutics

There are multiple nucleotide therapeutics, each with its benefits and challenges.

“All nucleotide therapeutics are drugs made up of either DNA or RNA,” began Strapps.

According to a 2021 article in Nature, four dominant types of nucleotide therapeutics comprise the approved or late-stage nucleotide therapeutics: antisense oligonucleotides, ligand-modified short interfering RNA (siRNA), lipid nanoparticles, and adeno-associated virus (AAV) vectors.

Strapps explained that oligonucleotide therapeutics are a broad class of therapeutic interventions initially developed to target short pieces of DNA or RNA that interfere with transcription at the transcriptional level.

“Antisense oligonucleotides and siRNAs, for example, target the messenger RNA (mRNA). They bind to a particular mRNA by different mechanisms, but the result would be the same: destroying that mRNA,” he offered as an example.

Strapps explained that oligonucleotide therapeutics have broadened recently, with one of the most well-known examples being mRNA vaccines, which were critical throughout the COVID-19 pandemic.

“[The vaccines] can deliver a full mRNA, and the mRNA, once it enters the cell, is translated into a protein,” explained Strapps. “In the case of the vaccines, [the protein] immunizes people against the virus itself.”

Another layer of oligonucleotide therapeutics is gene editing, commonly using the CRISPR/Cas9 gene editing system. Typically, these systems deliver short pieces of messenger RNA “that directly target DNA within the cell, usually causing a cut in the DNA, which is repaired imperfectly, which causes gene disruption.”

On the other hand, more specific gene editing technologies make single nucleotide changes that can revert a mutant allele of the gene into the wild-type gene.

Challenges in Nucleotide Therapeutics

One of the primary challenges of nucleotide therapeutics is delivering these products to specific organs or locations.

“Most oligonucleotide therapeutics at this point have focused on targets in the liver because that is an organ that is relatively easy to deliver to,” explained Strapps.

While this is hugely beneficial for diseases that are in the liver, it presents a barrier to delivering therapeutics to other organs or cell types.

“Broadly speaking, the approaches that have been used to deliver all of the nucleotide therapeutics are often referred to as conjugates or single chemical entities or viral delivery methods.”

Conjugates or single chemical entities have two primary limitations. First, the delivery location is challenging because the ligands most commonly used are taken up the best in the liver. The other concern is the length of the nucleotides. This method requires that the nucleotide can be chemically synthesized, which limits the length.

 "The next delivery technology is likely what I'll broadly refer to as viral. AAV stands as perhaps the most commonly utilized example of this," he remarked. "It enables the encapsulation of specific DNA cargo within a viral shell, with the flexibility for modifications. While various organs can uptake it, efficiency remains a challenge."

According to Strapps, these delivery techniques require relatively high viral doses to deliver the intended segment of DNA. They also need local delivery, meaning providers must inject the therapy exactly where they want it, which poses challenges for more delicate organs.

“The cargo [delivery method] can only use DNA, so it must be coding for something [providers] want to express. And there's a limit on how long the message can be within that virus,” he said.

While other viruses, such as lentivirus and herpes simplex virus, can be used to deliver therapeutics, they also have challenges. While herpes simplex virus delivery methods have a larger pairing capacity, research on this tool is still in the very early stages. Additionally, lentivirus has limited systemic delivery applications. Strapps noted that it is only really effective in ex vivo applications. The industry standard for viral delivery is AAV.

Another delivery tool is lipid or polymer nanoparticles. According to Strapps, lipid nanoparticles have been the primary focus in this arena; they are used to deliver the COVID-19 vaccine.

“It essentially self-assembles a complex that can be administered systemically and delivered to the cells.”

However, lipid nanoparticles also pose a concern regarding delivery. Early iterations of lipid nanoparticles go to the liver.

“They deliver very effectively to the liver, but they don't deliver very effectively to other organs. However, there is essentially no limit on the capacity within the lipid nanoparticles. Still, it is limited to the liver.”

A New Type of Testing

Strapps explained that Liberate Bio is looking for ways to develop lipid nanoparticles that can be administered systemically and reach beyond the liver.

He noted that the traditional methods of testing these products are much like conventional drug development. First, the delivery is assessed in cell cultures, followed by mice and nonhuman primates. However, this process is not very effective. Strapps explained that the translation from cells to mice and then nonhuman primates for lipid nanoparticles is poor.

“Essentially, while you might get a positive result in a cell and a kind of a midline result in a mouse, its ability to translate into a non-human primate for various biological and biophysical regions is challenging.”

Additionally, going through the standard process is costly and time-consuming.

Instead of continuing with the standard burdensome approach, Liberate Bio is taking a new approach to lipid nanoparticle delivery.

“The approach that we've taken is to essentially front load a lot of the risk associated with identifying novel lipid nanoparticles,” explained Strapps. “What that means is we're using non-human primates from the very beginning because non-human primates translate, as far as we know, relatively well to humans.”

“We make hundreds of novel lipids. We put a cargo inside the lipid nanoparticle with a unique barcode within the oligonucleotide cargo. Then, we make a hundred of these lipid nanoparticles and put them together,” he continued.

After injecting the animal, a postmortem analysis is conducted to extract the organs of interest, and next-generation sequencing is used to identify the presence of the barcodes. This allows researchers to examine a wide range of nanoparticles and quickly identify those that would be most advantageous for further research.

"This approach significantly reduces both cost and time in identifying delivery agents tailored for specific organs. In non-human primates, according to our calculations, the screening of a hundred lipid nanoparticles using conventional methods would typically take 2–2.5 years and cost ten times as much as our current approach." Strapps revealed. “We did it in three months, once we built the platform, and we could screen 150 novel lipid nanoparticles.”

As Liberate Bio and other companies continue to explore new methods for optimizing nucleotide therapeutic delivery, researchers may consider leveraging these tools in early-stage development and pre-clinical studies.