St. Jude Reference #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. Researchers at St. Jude working on rAAV gene therapies for hemophilia analyzed rAAV preps for transfer of clotting factor genes revealed significant contaminant sequences from upstream of the REP78/68 translation-driving P5 promoter on the REP-CAP plasmid. Characterization of these contaminants post-infection showed transfer, persistence, and transcriptional activity in AAV-transduced murine hepatocytes. These contaminants also have the potential to be efficiently translated by the reverse sequence of P5 and splenic T-cells specific to the contaminant product were detected after in-vivo infection, revealing a previously unrecognized potential concern for AAV-mediated gene therapy. Incorporation of contaminants upstream of P5 into rAAV virions was dependent upon the REP-binding element (RBE) and YY1+1 binding site present within P5. The researchers constructed a modified P5 promoter (P5-HS) that has a DNA spacer inserted 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.
Researchers at St. Jude have developed a helper plasmid for rAAV production in which the P5 promoter driving the large REP proteins (REP78 and REP68) has been replaced by a sequence that produces higher titer vector, but does not result in the incorporation of contaminant DNA upstream of the promoter into the AAV virion.
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 this vector could include but are not limited to:
The primary advantage of this invention is that AAV can be produced in a cell transfection system without the incorporation of contaminant DNA upstream of the P5 promoter. The majority of AAV is produced in this manner currently. Levels of contaminant DNA are a release criterion for AAV gene therapy trials, and reduction of levels would improve both the purity of the vector, and the likelihood of a vector being cleared for trial. Recombinant production of alternative viruses that incorporate an AAVREP and AAV P5 promoter to aid the production process, such as recombinant bocavirus vectors, in an R&D or clinical setting could also benefit from this system.
Another very successful design included in the published patent uses a DNA spacer positioned between the P5 Rep bringing site at the YY1 transcription factor binding site to create a Promoter we called 'P5-HS'. This promoter design significantly reduces the contamination present in rAAV preparations and retains equivalent vector production efficiencies to those achieved with the native P5 promoter.
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.
Helper plasmid, AAV, P5 promoter, P97, REP78, REP68
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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