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UMass Chan research explores differences in AAV vector manufacture, shows clear winner for method

Horae Gene Therapy Center paper published in Human Gene Therapy describes innovative technique for evaluating manufacture of vectors for gene therapy

By Mark L. Shelton

UMass Chan Medical School Communications

May 20, 2022

A paper from the Horae Gene Therapy Center at UMass Chan Medical School may settle a longstanding debate about how to best manufacture adeno-associated virus (AAV) vectors for gene therapy. The paper, published online in the journal Human Gene Therapy, describes using AAV genome population sequencing (AAV-GPseq) to explore the genomes of vectors produced by two common production pipelines. One is based on human kidney cells; the other on insect cells. One approach was a clear winner.

“My colleagues in the Horae Gene Therapy Center used a sophisticated next-gen sequencing approach to create a level of detail of vector genome characterization that was previously unattainable, showing there is a there is a hands-down winner in this study: the vector produced in mammalian cells is far superior to that produced in the insect cells,” said Terence R. Flotte, MD, the Celia and Isaac Haidak Professor, dean of the T.H. Chan School of Medicine and provost and executive deputy chancellor at UMass Chan Medical School. “The percentage of genome fragments and unresolved terminal repeat sequences in the insect cell vector is unacceptably high in my opinion. This is important work and should influence the field.”

Phillip W. L. Tai, PhD

Phillip W. L. Tai, PhD, assistant professor of microbiology & physiological systems and corresponding author on the study said, “Adeno-associated virus vectors have been at the forefront of effective and safe platforms for gene therapy. Here, we describe new means of quantifying vector genome heterogeneity using next-generation sequencing methods and we were able to show that different production platforms generate distinctive types and abundances of non-unit length genomes. These findings demonstrate that there is much to be learned about how to produce safe and efficient gene therapy vectors. Despite the promise for these types of biotherapies, the development of new technologies for quality control pipelines is essential to ensure that breakthrough medicines are safe.”

In the past two decades, AAV vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy and researchers around the world have made a number of refinements in the process, including choices of cell lines used in vector production. But the presence of empty AAV capsids and particles containing incomplete or inaccurate vector genomes has long been a subject of concern, since dosages of the therapeutic material contained in the vectors must be calculated precisely. Empty capsids or incomplete payloads can alter these calculations.

Several methods exist to separate empty capsids from full particles but no single technique can produce vectors that are free of empty or partial capsids and the exact genome compositions of full, intermediate and empty capsids remain largely unknown.

In this study, Dr. Tai and colleagues used AAV-GPseq sequencing to explore the compositions of vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells and baculovirus expression vectors in Spodoptera frugiperda insect cells. The results show that vectors originating from the same construct design that were manufactured by the insect cell system produced a higher degree of truncated and unresolved species than those generated by the human kidney cell production.

“These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated,” said Tai.

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