Cystosolic Delivery Macromolecules: Vehicle Design and Lessons Learned in Delivery Limitations
Suzie Pun, PhD, Washing Research Foundation Professor of Bioengineering, University of Washington
Polymers have huge potential for the oligonucleotides sector, according to the research team behind a new pH sensitive “virus-inspired” delivery technology.
Suzie Pun, a Washington Research Foundation Professor of Bioengineering at University of Washington, spoke about the technology known as VIPER at the TIDES oligonucleotide and peptides conference in May.
“The delivery of macromolecules starts with internalization, usually by some kind of endocytic mechanism into vesicles, which then require an escape step in order to reach the cytosol,” Pun said.
The escape step can be a major stumbling block, according to Pun, who cited a 2013 study that showed, in some cases, only a fraction of the material that enters a vesicle escapes into the cytosol.
“Even with this highly effective LNP formulation, the endosomal release efficiency is less than 2%. So we wanted to develop a material that could improve on endosomal release,” she said.
Adenovirus
The team working on VIPER took inspiration from viruses, specifically the adenovirus.
“Looking in the literature, what we found out is that adenovirus is really effective at escaping and that attribute is conferred by a protein called protein 6 … It’s a teardrop-shaped, tiny yellow dot that’s inside embedded in the protein nano capsule,” Pun said.
“And what you’ll see is that protein six is actually membrane lytic, but it is hidden inside the capsid. And then after the adenovirus is taken up inside the cell, the protein capsid undergoes a conformational change that then exposes this lytic protein.”
This mechanism shows how the adenovirus is internalized by cells without damaging the extracellular cell membrane before then disrupting the endosomal membrane.
The VIPER technology is designed to mimic this process, according to Pun, who said, “This polymer is pH sensitive so that itself assembles into nanoparticles. And we have conjugated to the interior, a membrane lytic entity that is just like adenovirus hidden inside the particle.
“After it’s taken into the endosome, when the endosome acidifies, it triggers a transition of the polymers so that the assembly falls apart, exposes the lytic peptide and allows for endosomal release.”
The polymer works because it is highly sensitive to pH changes specifically when the pH changes to 6.4, which is the critical level for endosomal release.
Oligonucleotide delivery
VIPER has obvious application in oligonucleotide delivery, according to Pun, who shared details of how the team is using the technology in combination with peptides.
“The application that we’ve started off with is peptide antigens and, again, in this case we are making our hydrophilic portion either with PEG or with mannose for targeting dendritic cells.”
“By combining with mannose with the lytic peptide we are able to get a much higher antigen-specific T-cell response,” Pun said, adding “if we have our endosomal release agent can get around 3% of the T-cells in the spleen to be antigen specific versus our controls. If we add in an adjuvant, we get over 10%.”
The VIPER technology has also been used to deliver nucleic acids, according to Pun, who highlighted various in vitro trials the team has completed.
“So what we’ve seen is that VIPER works really well for almost all cultured cells, including stem cells. For example, we work a lot with urine derived stem cells, and we can see that viper transects those really well,” she said.
“We can do direct intra-tumoral injection, and we can see gene delivery there. We do delivery into the cerebral spinal fluid, and we can see gene expression in the brain. Olivia Merkel at LMU in Germany has formulated VIPER for pulmonary delivery, and she has been able to deliver siRNA through that route of administration.”
Shortcomings
The team has not achieved systemic delivery of nucleic acids using the polymer, according to Pun. “We can get a hundred percent mRNA delivery without any drop of serum, but once [serum is introduced and] nucleases are present, we get zero.
“And so VIPER does not protect mRNA well enough in its formulation probably because actually VIPER forms micelles first and then the micelles interact with the nucleic acids” Pun said.
T-cell delivery is another area in which the team encountered some disappointment.
“The other surprise to us was that VIPER works so well on almost all cell types … but it didn’t work on T-cells. So we went through a whole series of studies to try to figure out what was going on.
“And what it turned out to be is that T-cells, to our great surprise, do not acidify nearly as much as most other types of cells. And so if we measured the acidification of cultured and primary T-cells we saw that they actually never hit that 5.7 pH, which is our trigger for VIPER,” Pun said.
“So now we are designing polymers that trigger at a higher pH to see if we can correct that issue with T-cells.”
