Nanoscale Virus Altered to Deliver Peptide Drugs to Cells

Researchers at Rice University exploring a viral protein have discovered a path toward virus-like, nanoscale devices that may be capable of supplying drugs to cells.

The protein is one of three that make up the protective shell, known as the capsid, of natural adeno-associated viruses (AAV). By gradually making smaller versions of the protein, the researchers created capsids with exceptional abilities and learned a significant deal about AAV's mechanisms.

The research has been published in the American Chemical Society journal ACS Nano.

Rice bioengineer Junghae Suh investigates the manipulation of non-disease-causing AAVs to carry helpful cargoes like chemotherapy drugs. Her research has paved the way to the development of viruses that can be stimulated by light or by extracellular proteases related with some diseases.

AAVs are small -- approximately 25 nm -- and contain a single strand of DNA within tough capsids that comprise a variety of proteins known as VP1, VP2 and VP3. AAVs have been used to deliver gene-therapy payloads, but nobody has guessed how AAV capsids physically reconfigure themselves when activated by external stimuli, Suh said. That was the opening point for her lab.

"This virus has intrinsic peptide (small protein) domains hidden inside the capsid," she said. "When the virus infects a cell, it senses the low pH and other endosomal factors, and these peptide domains pop out onto the surface of the virus capsid.

"This conformational change, which we termed an 'activatable peptide display,' is important for the virus because the externalized domains break down the endosomal membrane and allow the virus to escape into the cytoplasm," Suh said. "In addition, nuclear localization sequences in those domains allow the virus to transit into the nucleus. We believed we could replace that functionality with something else."

Suh and lead author and Rice graduate student Nicole Thadani think their mutant AAVs can become "biocomputing nanoparticles" that detect and process environmental inputs and create controllable outputs. Adapting the capsid is the principal step.

Of the three natural capsid proteins, only VP1 and VP2 can be activated to reveal their functional peptides, but neither can form a capsid on its own. Shorter VP3s can create capsids by themselves, but do not exhibit peptides. In natural AAVs, VP3 proteins are more than each of their counterparts 10-to-1.

That restricts the number of peptides that can be exposed, so Suh, Thadani and their co-authors set out to alter the ratio. That led them to truncate VP2 and synthesize mosaic capsids with VP3, resulting in positive alteration of the number of exposed peptides. Based on earlier research, they inserted a common hexahistidine tag that made it easy to observe the surface display of the peptide region.

"We wanted to boost the protein's activable property beyond what occurs in the native virus capsid," Thadani said. "Rather than displaying just five copies of the peptide per capsid, now we may be able to display 20 or 30 and get more of the bioactivity that we want."

They then made a truncated VP2 able to form a capsid on its own. "The results were quite surprising, and not obvious to us," Suh said. "We chopped down that VP2 component enough to form what we call a homomeric capsid, where the entire capsid is made up of just that mutant subunit. That gave us viruses that appear to have peptide 'brushes' that are always on the surface.

"A viral structure like that has never been seen in nature," she said. "We got a particle with this peptide brush, with loose ends everywhere. Now we want to know if we can use these loose ends to attach other things or carry out other functions."

Homomeric AAVs display nearly 60 peptides, while mosaic AAVs could be programmed to react to stimuli specific to particular tissues or cells and display a smaller preferred number of peptides, the researchers said.

Viruses have evolved to invade cells very effectively. We want to use our virus as a nanoparticle platform to deliver protein- or peptide-based therapeutics more efficiently into cells. We want to harness what nature has already created, tweak it a little bit and use it for our purposes.

Junghae Suh, Rice Bioengineer

Co-authors are Rice Ph.D. graduate Christopher Dempsey and alumna Julia Zhao and former lab manager Sonya Vasquez are co-authors of this research. Suh is an associate professor of bioengineering.

Support to the research was provided by a National Science Foundation Graduate Research Fellowship to Thadani and a National Institutes of Health Nanobiology Interdisciplinary Graduate Training Program grant to Dempsey.