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Scaling Down for Big Impact: Streamlined High-throughput Recombinant Adeno-associated Virus Production

In recent years, recombinant adeno-associated viral (rAAV) vectors have emerged as a leading platform for gene therapy applications. However, the ...

Roche Diagnostics GmbH

Owing to the advances in transgenic animal technology and the advent of the next-generation sequencing era, over 120 genes causing hereditary hearing loss have been identified by now. In parallel, the field of human gene therapy continues to make exciting and rapid progress, culminating in the recent approval of several ex vivo and in vivo applications. Despite these encouraging developments and the growing interest in causative treatments for hearing disorders, gene therapeutic interventions in the inner ear remain in their infancy and await clinical translation. This review focuses on the adeno-associated virus (AAV), which nowadays represents one of the safest and most promising vectors in gene therapy. We first provide an overview of AAV biology and outline the principles of therapeutic gene transfer with recombinant AAV vectors, before pointing out major challenges and solutions for clinical translation including vector manufacturing and species translatability. Finally, we highlight seminal technologies for engineering and selection of next-generation "designer" AAV capsids, and illustrate their power and potential with recent examples of their application for inner ear gene transfer in animals.

Breaking the sound barrier: Towards next-generation AAV vectors for gene therapy of hearing disorders.

Parvoviruses are a diverse family of small, non-enveloped DNA viruses that infect a wide variety of species, tissues and cell types. For over half a century, their intriguing biology and pathophysiology has fueled intensive research aimed at dissecting the underlying viral and cellular mechanisms. Concurrently, their broad host specificity (tropism) has motivated efforts to develop parvoviruses as gene delivery vectors for human cancer or gene therapy applications. While the sum of preclinical and clinical data consistently demonstrates the great potential of these vectors, these findings also illustrate the importance of enhancing and restricting in vivo transgene expression in desired cell types. To this end, major progress has been made especially with vectors based on Adeno-associated virus (AAV), whose capsid is highly amenable to bioengineering, repurposing and expansion of its natural tropism. Here, we provide an overview of the state-of-the-art approaches to create new AAV variants with higher specificity and efficiency of gene transfer in on-target cells. We first review traditional and novel directed evolution approaches, including high-throughput screening of AAV capsid libraries. Next, we discuss programmable receptor-mediated targeting with a focus on two recent technologies that utilize high-affinity binders. Finally, we highlight one of the latest stratagems for rational AAV vector characterization and optimization, namely, machine learning, which promises to facilitate and accelerate the identification of next-generation, safe and precise gene delivery vehicles.

Fantastic AAV Gene Therapy Vectors and How to Find Them-Random Diversification, Rational Design and Machine Learning.

 To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption.  An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection.  The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies.  The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1-13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing  gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency.

Article	Year
Breaking the sound barrier: Towards next-generation AAV vectors for gene therapy of hearing disorders. Hearing research\| PubMed ID: 33268240	2022
Fantastic AAV Gene Therapy Vectors and How to Find Them-Random Diversification, Rational Design and Machine Learning. Pathogens (Basel, Switzerland)\| PubMed ID: 35890005	2022
Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control. Bioanalysis\| PubMed ID: 38497775	2024
Improved Recombinant Adeno-Associated Viral Vector Production via Molecular Evolution of the Viral Rep Protein. International journal of molecular sciences\| PubMed ID: 39941089	2025

Julia Fakhiri

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