Preventing Packaging of Translatable P5-associated DNA Contaminants in Recombinant AAV Vector Preps

Overview

Journal Mol Ther Methods Clin Dev

Publisher Cell Press

Date 2022 Feb 25

PMID 35211640

Authors

Mark A Brimble

Pei-Hsin Cheng

Stephen M Winston

Isaiah L Reeves

Aisha Souquette

Yunyu Spence

Junfang Zhou

Yong-Dong Wang

Christopher L Morton

Marcus Valentine

Paul G Thomas

Amit C Nathwani

John T Gray

Andrew M Davidoff

Affiliations

Soon will be listed here.

Abstract

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used for clinical gene transfer and have shown great potential for the treatment of several monogenic disorders. However, contaminant DNA from producer plasmids can be packaged into rAAV alongside the intended expression cassette-containing vector genome. The consequences of this are unknown. Our analysis of rAAV preps revealed abundant contaminant sequences upstream of the AAV replication (Rep) protein driving promoter, P5, on the Rep-Cap producer plasmid. Characterization of P5-associated contaminants after infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes, in addition to evidence suggestive of integration. These contaminants can also be efficiently translated and immunogenic, revealing previously unrecognized side effects of rAAV-mediated gene transfer. P5-associated contaminant packaging and activity were independent of an inverted terminal repeat (ITR)-flanked vector genome. To prevent incorporation of these potentially harmful sequences, we constructed a modified P5-promoter (P5-HS), inserting a DNA spacer between an Rep binding site and an Rep nicking site in P5. This prevented upstream DNA contamination regardless of transgene or AAV serotype, while maintaining vector yield. Thus, we have constructed an rAAV production plasmid that improves vector purity and can be implemented across clinical rAAV applications. These findings represent new vector safety and production considerations for rAAV gene therapy.

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