Purespring Therapeutics is pioneering advancements in gene therapy for kidney diseases by integrating cutting-edge technologies in AAV manufacturing. With a focus on precision therapies, Purespring is shaping future trends in gene therapy manufacturing through innovative solutions and scalable platforms. This presentation explores the company’s approach to overcoming challenges in vector manufacturing and highlights the future direction of gene therapy, setting new standards for manufacturing and therapeutic outcomes.

Recombinant adeno-associated virus (rAAV) vectors have been demonstrated as gene delivery vehicles for addressing debilitating chronic diseases and conditions. However, a major challenge lies in developing a cost-efficient, optimised, and scalable manufacturing process to meet the growing global demand for these therapies. Forge Biologics has developed a scalable platform process and performed process improvements through enhanced starting materials and optimised process for high vector yield and product quality.

Recombinant AAVs are crucial for gene therapy due to their safety and lasting transgene expression. This study explores the interplay between AAV promoter activity and helper virus components. We evaluated AAV promoter activity in different mammalian cells and identified the minimal components of the adenovirus E2A and E4 that significantly impacted the overall rAAV productivity and quality of produced virus. These findings aim to improve scalability, advancing gene therapy applications.

Intensified high cell density processes and continuous harvest are innovative strategies that tackle challenges associated with high AAV dose requirements. Here we present the development of a perfusion platform for the generation of cell densities exceeding 20E6 at high viability and transient transfection methods optimised for high cell density, with 10x improved transfection efficiency compared to batch-optimised methodologies, and perfusion culture methods facilitating continuous AAV harvest for improved yield.

• Regulatory environment-today and future concerns.
• Cell line and productivity-triple transfection or stable cell line?
• Early impurity removal-its impact downstream
• Encapsulation-how do we tackle empty, partial, full, and genomic DNA?
• DNA Endonuclease addition-how confident are we that we can remove this CMC concern?

Lentiviral vectors are powerful tools for gene delivery mainly due to their ability to transduce non-dividing cells, large cargo capacity, and stable transgene integration. The standard production process is transient transfection. While suitable for small-scale screening, this method is cumbersome for large-scale production in terms of consistency, stability, reliability, and cost-efficiency. To address these issues, we have developed stable, lentiviral packaging clones for reliable production in oncology and gene therapy.

Recombinant adeno-associated viral vectors (rAAV) continue to expand as a modality, however, manufacturing costs are still a limitation. Switching to stable rAAV-producing cell lines is a strategy to increase robustness and reduce cost of goods. We performed a proof-of-concept study in collaboration among Spark, Cytiva, and Roche using the ELEVECTA producer cell lines and subsequently performed process development to further optimise productivity.

A CRISPR-Cas9 pipeline for genetic engineering of insect Sf9 cells yielding higher editing efficiencies than other existing methods (67% vs. 12%, respectively) was implemented. It was then applied to knock-out caspase initiator Sf-Dronc, as proof-of-concept gene, aimed at alleviating cell apoptosis during a baculovirus expression vector system (BEVS) process. The resulting engineered cell lines were characterised as per their phenotype and production of recombinant adeno-associated viruses (rAAVs).

VectorY develops Vectorised Antibodies (VecTabs) which utilise an AAV vector to deliver the transgene to the target CNS cells where the therapeutic antibody is subsequently produced, and clears the toxic variants of target proteins to improve neuronal health and halt disease progression. The presentation will present AAV production platform "ManuVec" which produces VecTabs from an optimised Baculovirus/insect cell platform in high yields, quality, and potency in a robust manner.

rAAV processes with complex dynamic behaviour requires high experimental effort, and is time consuming and expensive. Digital Twins (DT) that are based on mathematical models can be used for process development and optimisation. A mechanistic model of an rAAV9 production process has been developed and parameterised using in-house experimental data. The validation of the DT models and its application to increase the functional rAAV9 titre will be demonstrated.

Focusing on regulatory challenges, this presentation explores the role of closed processing in Advanced Therapy Medicinal Products (ATMPs). Closed systems, together with standard process platforms and automation-digitalisation, are key for the future of ATMP manufacturing. Through the analysis of the current guidelines, this presentation draws how regulatory frameworks may either support or hinder closed systems adoption. The final goal is to align closed processing with current and future regulations.