With artificial intelligence (AI) and machine learning making headlines, a realm of boundless opportunities unfolds. Could we unleash AI’s potential and merge the forces of random and guided design to engineer advanced AAV vectors? In this journey filled with possibilities and hurdles, we draw upon a wealth of AAV engineering experience, encompassing triumphs and setbacks alike.

Manufacturing sufficient Adeno-Associated Virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. Recent evidence has emerged supporting a functional role of the membrane-associated accessory protein (MAAP) in AAV production and egress. Here, I will present a directed evolution strategy our group developed to engineer novel MAAP variants that conferred increased overall production of multiple recombinant AAV serotypes.

Cirsium Biosciences is a viral vector manufacturing platform developer. We develop viral vector production methods and systems using whole plants as modular, highly scalable, cost effective, and safe bioreactors. Production yields and key quality attributes including infectivity, E:F ratios, and purity are comparable to existing bioreactor-based AAV production methods. Our platform can achieve a 60-70% reduction in production costs and reduces lead time by 70% compared to competing methods.

Enhancing AAV production in mammalian cell culture is crucial for gene therapy advancements. We will focus on optimization of upstream strategies to improve rAAV vector yield and quality. By refining upstream bioprocessing approaches, we aim to achieve higher AAV yields and better quality AAV products. These findings will provide valuable insights to the AAV bioprocessing field, paving the way for more efficient and scalable AAV production in gene therapy applications.

Adeno-associated virus (AAV) has recently emerged as a novel therapeutic modality in gene therapy, yet, there remain major bottlenecks and challenges in the virus manufacturing space. Here, we present a disruptive cell engineering platform to overcome current AAV manufacturing challenges for gene therapy using a directed-evolution strategy via cell fusion, then selecting for HEK-293 cells with enhanced viral production machinery. Engineered pools and clones exhibited up to a 9-fold productivity improvement by transient transfection for AAV1, AAV2, and AAV5 (up to 10¹⁴ vg/mL and 10¹³ vp/mL), with a 2-fold improvement in full-to-empty ratio.

Among many efforts to develop a vaccine against COVID-19, Merck developed a closed industrial-scale, single-use manufacturing process for vaccine candidate V590. For maximum virus productivity, we optimized pH and temperature during virus production in 3L bioreactors. Optimized production conditions were successfully scaled up to a 2000L bioreactor, producing a maximum virus titer of ~1.0e7 plaque forming units/mL. Additional process intensification and simplification were able to further increase virus productivity.

In this presentation, we will discuss how downstream manufacturing can be enhanced for recombinant Adeno-Associated Virus (rAAV) through Single Pass Tangential Flow Ultrafiltration. This innovative approach optimizes the purification process, significantly boosting productivity. Join us as we unravel the transformative impact on rAAV production, showcasing the advantages of our streamlined and intensified method in advancing gene therapy and biomanufacturing.

Preparation of viral vectors poses challenges in terms of stability, concentration, mono-dispersity, and purity. This study compared three protocols using Orf virus vector: steric exclusion chromatography (SXC), SXC combined with centrifugal diafiltration, and sucrose cushion ultracentrifugation. Evaluation parameters included protein removal, size distribution, infectious virus recovery, visual appearance, and electrophoretic mobility at varying pH levels. These quick and user-friendly methods offer potential solutions for efficient viral vector preparation.

With the expanding landscape of gene therapy, there has been a growing need for platform approaches to monitor quality and physicochemical properties of AAV vectors. Emerging analytical methods are often automated, require low sample volumes, and generate high-quality results for various capsid serotypes. Here, I will review recent developments in AAV analytics, highlighting high-throughput methods used to screen and guide selection of lead candidates for research and preclinical studies.

Production of recombinant adeno-associated virus (rAAV) is highly costly and labor intensive. Bottlenecks in manufacturing strongly add to the high price tag of existing rAAV therapeutics. The current production pipeline lasks real-time monitoring tools for rAAV quality attributes which hinders process control and optimization. Here, we developed a microfluidic resonator that rapidly characterizes fundamental biophysical properties of rAAV-producing cells, amenable to real-time monitoring (15 minutes for >3,000 cells). We envision a cell-based approach for monitoring rAAV product attributes with a data-driven model using biophysical properties from rAAV-producing cells.

Potency assays are defined as the quantitative measure of biological activity in a medicinal product. The biological activity in question is unique to each drug product (DP) and inherently linked to a product’s attributes. In cell and gene therapies (CGTs), potency assays need to be capable of adequately accounting for the downstream cellular mechanisms and signaling cascades which constitute the DP’s mechanism of action (MoA). Potency assays in the CGT field are unique to every product and specific to the etiology of the target indication; making it challenging for regulatory bodies to provide generalized guidance and for developers to understand expectations and best practices. However, a decade of CGT regulation and industry knowledge has brought to light consistent technical and regulatory challenges, barriers, and pain points throughout the process of potency assay development.

Self-replicating RNA (srRNA) technology has been enabled for vaccines, requiring lower doses than conventional mRNA due to its amplification in situ. However, their larger size represents a manufacturing challenge. While most srRNAs are derived from the same backbone, we have explored alternate alphaviral vectors to assess their biological utility. We show that with attention to RNA quality, new synthetic srRNA vectors can enable us to build better next-generation therapies.

Analytics of critical quality attributes are key to developing a robust Adeno-Associated Virus (AAV) production process. Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) for AAV is high-throughput, easily implemented, amenable to GMP environment, and requires small sample volume. The study explores SEC-MALS application for process and formulation development analytics of AAVs for capsid content, transgene titer, and capsid titer. Also, capsid content determination with multiple orthogonal methods is compared.

AAV immunogenicity can adversely affect the safety and durability of gene therapy and prevent the possibility of re-dosing. We have developed tolerogenic ImmTOR nanoparticles that induce antigen-specific tolerance to co-administered antigens and have been shown to mitigate anti-drug antibodies against a fungal enzyme in Phase 3 clinical trials. Here we demonstrate the ability of ImmTOR to inhibit humoral and cellular immune responses to AAV, mitigate hepatic inflammation, and enable vector redosing. Furthermore, the combination of ImmTOR with anti-BAFF antibody enables readministration of vector doses up to 5E13 vg/kg.

The work presented here focuses upon the current and emerging technologies available to produce recombinant adeno-associated virus (AAV)-based viral vectors towards treatment of diseases. We will highlight automation enabled serum-free Sanofi’s producer cell lines (PCLs)-based platform for production of gene therapy vectors for delivering safer therapeutics to the patients.