Promoter Modification to Reduce AAV Contamination (SJ-19-0003, SJ-23-0028)

St. Jude Reference #SJ-19-0003, SJ-23-0028

Description

SJ-19-0003:
Recombinant adeno-associated virus (rAAV) is commonly used for gene transfer; however, contaminant DNA from AAV producer plasmids can be packaged alongside the intended expression cassette-containing vector genome within rAAV virions. These contaminants represent a potential safety concern for AAV-mediated gene therapy. To avoid this concern when using the P5 promoter researchers inserted a DNA spacer between the P5 RBE and the YY1+1 site to prevent upstream contamination while maintaining vector yield. This modified promoter retains the P5 characteristics of REP-mediated autorepression and Ad5 helper gene mediated induction.

This invention is compatible with all AAV production of all serotypes within a cell transfection-based system and is compatible with insertion into a stable producer cell line to produce AAV particles for clinical or R&D use. Commercial applications of vectors containing this modified P5 promoter include but are not limited to:

• Production of higher quality clinical AAV products by biotech and pharmaceutical companies (clinical trial and market authorized) using a plasmid transfection-based system
• Higher purity AAV preparation for research and development purposes produced by a commercial operation for clients using a plasmid transfection-based system
• Improved purity rAAV preparation for clients using a non-plasmid transfection-based system that uses the P5 promoter to drive REP expression (e.g. stable cell line)

The patent application and a paper (2022) were published, below, with a key finding of the paper being the post infection transcriptional activity of the contaminants from this source. A 2023 literature review discusses the profile, abundance, and post-treatment consequences of nucleic acid impurities within rAAV, and cover strategies that have been developed to improve rAAV purity.

SJ-23-0028:
The researchers also used p5 homologues derived from AAV serotypes other than AAV2 to identify promoters which can be regulated by AAV2 Rep but are encapsidated less efficiently. Alternatively, the researchers directly modified the nicking site on the AAV2 p5 promoter to prevent packaging of unintended nucleic acids during rAAV production. These direct modifications, termed TRS5 or GC DI, disrupt the autologous regulation of the p5 promoter resulting in less large Rep expression and more small Rep expression. This reduces packaging of the p5 promoter directly by point mutations and distant hotspots likely by either competitive inhibition or differential Rep expression. Simple plasmid ratio optimization enables these modified p5 promoters to achieve efficient production of high titer AAV for commercial and research use via plasmid transfection or via stable gene integration of cell lines.

When compared head-to-head to existing technology (the P5HS system), comparable titers with improved purity were achieved with either the TRS5 or GC DI AAV2 p5 mutant. Vectors produced using the modified p5 promoter reduced presence of plasmid sequences in final rAAV preps, and reduced the chance of replication competent AAV.

Keywords

Granted patents or published applications

Pending patent published as WO 2021/242664, and published US 20230203535 (uspto.gov)

Related scientific references

Brimble MA, Cheng P-H, Winston SM, Reeves IL, Souquette A, Spence Y, Zhou J, Wang Y-D, Morton CL, Valentine M, Thomas PG, Nathwani AC, Gray JT, Davidoff AM, “Preventing packaging of translatable P5-associated DNA contaminants in recombinant AAV vector preps,” Molecular Therapy: Methods & Clinical Development (2022), PubMed: https://pubmed.ncbi.nlm.nih.gov/35211640/; doi: https://doi.org/10.1016/j.omtm.2022.01.008.

Brimble, Mark A., Winston, Stephen M., Davidoff, Andrew M., “Stowaways in the cargo: Contaminating nucleic acids in rAAV preparations for gene therapy,” Molecular Therapy (2023), doi: https://doi.org/10.1016/j.ymthe.2023.07.025

An external review of the invention mentioned in section 3.3.1.: https://www.sciencedirect.com/science/article/pii/S2405844023022788?via%3Dihub

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