Viral protein assemblies provide a unique scaffold for cell growth. They are biocompatible and provide a structured surface ideal for tissue cell growth. This study presents the construction, characterization, and brain implantation of a nano-biorganometallic material called ?ChipVP6-Au, comprised of viral protein nanotubes (rotavirus nVP6) functionalized with gold nanoparticles (nVP6-Au) printed on a poly(vinyl alcohol co-vinyl acetate) chip as electric in series or in parallel nVP6-Au circuits. Coating of PAcVA with nVP6-Au increased its conductivity by 169-fold. Atomic force microscopy (AFM) analysis revealed accumulation zones of nVP6-Au on PAcVA with an electrical conductivity of -60 pA. Additionally, nVP6-Au increased the roughness of PAcVA, creating depressions and crests measuring -6 ?m and 3.9 ?m, respectively. A549 and mHypoE-N1 cell lines were cultured on nVP6, nVP6-Au, and ?ChipVP6-Au, exhibiting a similar cell growth than on commercial culture plates. No cytotoxicity or oxidative stress was observed, demonstrating the innocuousness of nVP6-Au for mammalian cell cultures. Furthermore, as a proof of concept, ?ChipVP6-Au loaded with L-DOPA was intracranially implanted in the brain of a mouse model of Parkinson’s disease and in control mice. Motor coordination was restored at 8 days post-implantation, and it was comparable with the control group. ?ChipVP6-Au is a potential versatile scaffold for cell culture, enabling cell replacement therapies, in situ drug release, and electrical stimulation. This study presents the first functional nano-bioorganometallic material based on viral protein assemblies for brain implantation.

Ferritin (Ft) nanoparticles provide a stable, self-assembling scaffold suitable for antigen display. Two complementary strategies have been established to enable precise and functional antigen display for vaccine development, with broader applicability to other biotechnology fields. Tyrosinase-mediated catalysis was applied to Pyrococcus furiosus ferritin (PfFt), a cysteine-free archaeal nanoparticle. Through targeted mutagenesis, single cysteine residues were introduced to enable site-specific, irreversible antigen conjugation under physiological conditions. This method preserved nanoparticle structure and allowed repetitive antigen distribution, as confirmed by native PAGE, DLS, HPLC, and mass photometry. Biolayer interferometry demonstrated nanomolar affinities to neutralizing antibodies, indicating maintenance of conformational epitopes. This platform is compatible with microbial expression systems and broadly applicable to areas requiring precise, site-specific protein–protein conjugation. In parallel, a genetic fusion approach was used to express antigen-Ft constructs in animal cells. Flexible glycine-serine linkers promoted structural separation, contributing to uniform antigen display, proper protein folding, and nanoparticle assembly, validated by cryo-EM analysis. An in vitro immunological evaluation pipeline was established using THP-1-derived dendritic cells. Nanoparticle uptake was visualized by confocal microscopy, and immune activation was evaluated through flow cytometry of surface markers, cytokine profiling using Olink® multiplex assays, and transcriptional analysis by real-time qPCR. Together, these strategies offer modular and versatile Ft-based platforms for precise antigen display and immune characterization. Their compatibility with various expression systems and analytical pipelines underscores their relevance to vaccine development, immunotherapy, and targeted bioconjugation applications.

AAV based gene therapy has revolutionized medicine and provided curative treatments for several indications. The lack of adequate platforms and the complex nature of the drug has affected COG significantly owing to low yield, poor quality of viral vectors and high required dosages to reach therapeutic effects. This results in low accessibility and hinders targeting of larger indications through novel AAV gene therapies. In the multidisciplinary GeneNova innovation milieu, we innovate the manufacturing of AAV by simultaneous targeting of several limitations in the AAV development pipeline. Here we report on improvements of AAV2 manufacturing using rational domain swopping of AAV2 and AA8 as well library approaches allowing for selection of improved variants with >10x improved secreted titers at the expense of lower infectivity. We further exemplify the ability to restore infectivity and steer tropism using modular grafting of 58 aa Affibody scaffolds onto the surface (VR4 or VR10) of the AAV. This platform allowed for selective targeting of cell types of interest including EGFR, HER2, HER3, IGF1R, PDGFRb or VEGFR2 positive cells lines, while reducing off targeting to unwanted tissues such fibroblasts. We show that this is of particular utility for applications delivering toxic cargo such as in the case of AAVs packed with herpes simplex virus thymidine kinase used in conjunction with ganciclovir as a cytotoxic drug for oncolytic therapies. To increase upstream yield, we performed a systems biology approach comparing AAV production in more efficient adherent 293T cells with less productive suspension HEK293F cells. Transient validation studies aimed at up/down regulating genes of key pathways correlating with improved AAV productivity, e.g. relating to viral response and cell homeostasis, was performed followed by development of stable cell lines and balancing of such genes in HEK293. Productivity fold-improvements and packaged yield for engineered HEK293 will be presented. To assure correct packaging of desired target ssDNA, adequate sequencing methods fit for AAV are needed. To avoid introduction of bias present in typical NGS methods we designed a fragmentation and amplification free sample preparation and data analysis pipeline for quality control of encapsidated viral genomes using Oxford Nanopore sequencing. We show the ability capture full lengthsequences of encpasidated DNA of both transgene and undesired DNA sequence originating from the bioprocess.

Interest in therapeutic viruses, such as oncolytic viruses, is growing with the first oncolytic therapeutics on the market and several candidate viruses in clinical trials. Currently doses of up to 1012 infectious viruses/dose are envisioned for use of these viruses and remain a challenge for the upstream processing. Oncolytic viruses are naturally occurring, or genetically engineered viruses that selectively infect and kill cancer cells. To tackle the coming demand, intensified production processes need to be developed. Here, we present development of a high-yield perfusion process for the production of oncolytic Newcastle disease virus (NDV) in suspension EB66 cells. EB66 duck cells (Valneva SE) were cultivated in shake flasks and in a DasGip 3 L reactor (Eppendorf) using CDM4 Avian medium at 150 rpm, 37°C and a pH of 7.2. Infections were performed at a MOI of 10-4 with the NDV-GFP LaSota strain. Small-scale batch and semi-perfusion experiments were used for process characterization and optimization. In perfusion mode, cells were fed at a perfusion rate of 36 pL/(cell x day) and a TrypLE concentration of 2.5 U/mL, to cleave and activate the NDV fusion protein. Tangential flow depth filtration (TFDF, Repligen) was used for cell retention. Infectious virus titers were determined by TCID50 assay with adherent Vero cells, total virus titers by HA assay. With optimized batch conditions, maximum titers of up to 4.2 x 108 TCID50/mL were achieved. Infection of EB66 cells at 20.1 x 106 cells/mL resulted in a 3-fold higher maximum titer of 1.3 x 109 TCID50/mL with a 1.5-fold higher volumetric virus productivity and increased cell-specific virus yield, compared to batch processes. Moreover, TFDF allowed direct clarification and harvesting of virus through the depth filter, enabling upstream and downstream process integration with a reduction of one unit operation. This resulted in continuous virus harvest with 99.4% cell retention and 90% turbidity reduction. In a next step, we aim to produce NDV at very high cell densities >100 x 106 cells/mL to challenge the EB66 cell specific productivity and the TFDF system.