Link to the paper: https://onlinelibrary.wiley.com/doi/full/10.1002/adhm.202301364

Peptide Amphiphiles as Biodegradable Adjuvants for Efficient Retroviral Gene Delivery

Kübra Kaygisiz, Lena Rauch-Wirth, Aysenur Iscen, Jan Hartenfels, Kurt Kremer, Jan Münch, Christopher V. Synatschke, Tanja Weil

Abstract

Retroviral gene delivery is the key technique for in vitro and ex vivo gene therapy. However, inefficient virion-cell attachment resulting in low gene transduction efficacy remains a major challenge in clinical applications. Adjuvants for ex vivo therapy settings need to increase transduction efficiency while being easily removed or degraded post-transduction to prevent the risk of venous embolism after infusing the transduced cells back to the bloodstream of patients, yet no such peptide system have been reported thus far. In this study, peptide amphiphiles (PAs) with a hydrophobic fatty acid and a hydrophilic peptide moiety that reveal enhanced viral transduction efficiency are introduced. The PAs form β-sheet-rich fibrils that assemble into positively charged aggregates, promoting virus adhesion to the cell membrane. The block-type amphiphilic sequence arrangement in the PAs ensures efficient cell-virus interaction and biodegradability. Good biodegradability is observed for fibrils forming small aggregates and it is shown that via molecular dynamics simulations, the fibril-fibril interactions of PAs are governed by fibril surface hydrophobicity. These findings establish PAs as additives in retroviral gene transfer, rivalling commercially available transduction enhancers in efficiency and degradability with promising translational options in clinical gene therapy applications.

Schematic representation of three selected PAs A C16-AGGRVK-NH2, B PA1, and C PA2, that show distinct aggregation properties. Laser scanning microscopy images of Proteostat stained PAs (red) and HeLa cells (nucleus in blue) reveal formation of µm-sized PA clusters for PA1 and PA2. The PAs (20 µg mL-1) were added to cells for 30 min, washed and samples were analyzed. PA1 and PA2 revealed association with cellular membranes while C16-AGGRVK-NH2 did not associate with cells, scale bar 200 µm. D – F Coarse grained molecular dynamics simulations. Snapshots show cross-sectional (top) and side views of selected PAs self-assembled into single fibers after 15 µs from randomly dispersed PAs in solution in a simulation box with a size 13 nm × 13 nm along the fibril cross-section (top view) and 6.5 nm along the lateral direction (side view). The snapshots for the interaction of multiple fibers are shown after self-assembly following an additional 10 µs simulation time in a box with a size 18 nm × 18 nm along the fibril cross-section (top view) and 6.5 nm along the lateral direction (side view). Color codes represent alkyl chains in yellow, nonpolar amino acids in red, polar amino acids in purple and polar, cationic amino acids in blue. Water and ions in the simulation box are omitted in the snapshots for clarity. For the single fibril system, the molecules in the simulation box are shown in color, while the periodic image is shown in white. The simulation box is shown in black for the multi fibril system. D Simulation of C16-AGGRVK-NH2 and E PA1 reveal stable fibril formation with high-density of polar groups (blue) on the fibril surface that prevents fibril-fibril interaction. F In contrast, PA2 displays hydrophobic amino acids (red) on the fibrils surface that is likely the main driving force for multi-fibril networks formation.