Adeno-associated viruses (AAVs) naturally integrate into the AAVSI locus of the host genome. With a helper virus like Adenovirus (AdV), their genome is amplified, producing AAV progeny. Recombinant AAVs (rAAVs), which carry a gene of interest (GOI) instead of AAV genes, are widely used in gene therapy due to their safety and production scalability. However, current triple transfection process is expensive and leads to batch variability. Unlike wild-type AAVs with 90–100% full capsids, rAAVs show only 10–30% full-to-empty ratios, and the cause of this low packaging efficiency remains unclear. To explore key factors in rAAV production and assess the benefit of stable GOI integration, we engineered a HEK293 cell line with a single-copy “CMV-eGFP-WPRE” construct integrated at the AAVSI locus via RMCE. rAAVs were produced using this stable line with co-transfection of two plasmids (rep, cap, and AdV helper genes) and compared to the standard triple transfection in parental HEK293 cells. To study the low full-to-empty capsid ratio, both lines were also infected with replication-competent AdV. As high eGFP expression may reduce AAV yield in the stable line, the effect of siRNA-mediated eGFP repression was tested. Our experiments showed that stable cell lines produce lower amounts of filled rAAVs as compared to the standard triple transfection. However, this effect can be alleviated by AdV infection and siRNA co-transfection. Wild type AAVs are being produced in viral replication complex (VRC) in the nucleus which are formed by the helper virus. rAAV production was more efficient with the infection of AdV as compared to the helper plasmid which contained the DNA binding protein, E4 & VA RNA from adenovirus. This indicates that vital genes are missing in the helper plasmid. We have taken electron microscopy images of the VRC to investigate the differences between adenovirus infection and helper plasmid transfection. This investigation of rAAV production from a stabe cell line with a genome integrated GOI offers a lot of possibilities to explore process optimization from a molecular biology aspect. This highlights that AAV production is still not fully understood and that the helper functions play a vital role in AAV productivity.

The use of virus-like particles (VLPs) as nanocarriers for biomolecules is a promising approach in drug delivery research. CXCR4 is a cell surface receptor associated with several types of cancer. The T22 peptide is a known antagonist of this receptor that efficiently binds to and penetrates CXCR4-positive cells. In this work, we produced Gag-GFP VLPs in HEK293 cells via transient transfection. We then optimized a four-step downstream process for these VLPs, including clarification via depth and standard filtration, intermediate purification using tangential flow filtration or multimodal chromatography, capture through ion exchange, hydrophobic interaction, and heparin affinity chromatography, and final polishing with size exclusion chromatography resulting in an overall recovery of 38% and a purity of 64%, with host cell DNA and protein levels complying with regulatory standards. These VLPs were then functionalized with the T22 peptide via click chemistry. In vitro studies revealed the ability of Gag-GFP VLPs-T22 to penetrate CXCR4-positive cells in a dose- and time-dependent manner. CXCR4-positive cells exposed to 8.0 × 10 functionalized Gag-GFP VLPs/mL exhibited 30-fold higher fluorescence than those exposed to non-functionalized Gag-GFP VLPs after 24 hours of incubation, showing the internalization of functionalized VLPs. Also, the absence of Gag-GFP VLPs-T22 internalization in CXCR4-negative cells confirmed specificity via the CXCR4 receptor. The functionalization of VLPs with the T22 ligand enables selective targeting of CXCR4-positive cells, which is significant due to the established role of CXCR4 in promoting cancer progression and metastasis. By specifically targeting CXCR4, these VLPs can selectively deliver therapeutic biomolecules to cancer cells, potentially enhancing treatment efficacy while minimizing off-target effects and reducing systemic toxicity. The presented results show that this targeted nanoplatform holds great potential for the development of safer and more effective therapies against CXCR4-overexpressing tumors, paving the way for precision medicine approaches in oncology.

The overlapping circulation of SARS-CoV-2, Influenza A virus (IAV), and Respiratory Syncytial Virus (RSV) continues to strain global healthcare systems, particularly among vulnerable populations. A single vaccine targeting all three pathogens could streamline immunization efforts and enhance protection, while reducing manufacturing costs. We previously showed that CHO cell–derived enveloped virus-like particles (eVLPs) formed through expression of full-length SARS-CoV-2 spike (S) protein not only exhibit high S density and strong immunogenicity, but also serve as a platform to co-display heterologous antigens such as IAV hemagglutinin (H1) and neuraminidase (N1). Here, we extend this approach by producing a trivalent eVLP candidate that simultaneously displays SARS-CoV-2 S, IAV H1, and the RSV pre-fusogenic fusion (F) protein. These S/H1/F eVLPs were successfully produced using both transient and stable gene expression in CHO cells and were purified via affinity chromatography. The presence of all three antigens on the same particles was confirmed by Western blot and immuno-electron microscopy. Their immunogenicity is currently being evaluated in vivo to assess their potential as a single vaccine against SARS-CoV-2, IAV, and RSV.

Optimization of recombinant adeno-associated virus (rAAV) production is essential for effective gene therapy applications. However, multiple factors affect the rAAV productivity in mammalian cells, and often they interact with each other, making the optimization process highly challenging. In our work, we show how coupling mixture design (MD) with face-centered central composite design (FCCD) is the most suitable design of experiments (DOE) approach, among other common DOE methods, for optimizing rAAV2 productivity and cell viability. moreover, we build on this method and demonstrate that combining MD with FCCD can be used to optimize the percentage of full capsids in rAAV2 upstream preparation. Additionally, we investigate the influence of the gene of interest (GOI) on the optimal conditions for viral particle production and packaging efficiency. By integrating MD and FCCD methodologies, we achieved an improvement of almost 100-fold in Log(Vp) in the case of egfp-expressing rAAV, and a 12-fold increase in bdnf-expressing full rAAV capsids, suggesting that this combined approach is a versatile and effective strategy for optimizing rAAV production processes. These findings emphasize the need for a comprehensive understanding of the factors influencing rAAV production to enhance the efficiency and efficacy of viral vector applications in gene therapy.