536. The Viral Transduction Enhancer Vectofusin-1 Is a Nanofibrillar Peptide Capable of Increasing the Contact between Viral Vectors and Target Cells

Abstract

Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using HIV-1-based lentiviral vectors (LVs) has several therapeutic applications ranging from monogenic diseases, infectious diseases and cancer. In such therapeutic context, the gene therapy could be improved by enhancing transduction levels of target cells and by reducing the amount of LVs used on the cells for greater safety and reduced costs. We recently identified a new cationic amphipathic peptide, Vectofusin-1, with viral transduction enhancing capacity, enabling higher transduction levels with low amounts of LV. Vectofusin-1 promotes the entry of several retroviral pseudotypes into target cells when added to the culture medium and is not toxic to HSPCs. Here, we present the first insights into the mechanism of action of this new transduction enhancer. First, a viral pull down assay showed that viral particles were easily pelleted by low speed centrifugation in presence of Vectofusin-1, suggesting that this latter may form unsoluble nanofibrils, trapping lentiviral particles. Atomic force (AFM) and electron microscopy (EM) of Vectofusin-1 confirmed that this peptide is rapidly forming annular aggregates and nanofibres in culture medium. Furthermore, these fibres were shown to be auto-fluorescent in medium with high-protein content (X-Vivo20), allowing the observation of a nanofibrillar network of Vectofusin-1 using confocal microscopy. Next, Vectofusin-1 was shown to strongly interact with Congo Red, especially in presence of lentiviral particles, but labeling with Thioflavin T was inefficient, suggesting that Vectofusin-1 fibres are not amyloid-type fibrils. Structural studies by circular dichroism confirmed this result. The capacity to form nanofibrils appears to be essential for the mechanism of action of Vectofusin-1 since a defective mutant called LAH2-A4, unable to promote lentiviral transduction (Majdoul S. et al (2016) J. Biol. Chem.), was also unable to form nanofibrils. In conclusion, biophysical, nanoscopic and microscopic observations have helped us to define Vectofusin-1 as a new nanofibrillar peptide capable of enhancing lentiviral transduction of target cells in conditions well-adapted to cGMP and scalable gene therapy protocols.