Endogenous UMIs as quantifiable reporter elements – validation studies & applications in rAAV vectorology

Abstract

In the creation of recombinant adeno-associated viral (rAAV) vectors, terminal DNA elements known ITRs (inverted terminal repeats) of the direct the intracellular synthesis and packaging of nonviral DNA. The need to clonally amplify ITR sequences in one form or another thereby underlies the existence of all rAAV clinical products and research materials worldwide. Their tendency to form strong nonduplex structures raises problems. The genetic precursors to rAAV vectors – typically prokaryotic plasmids – are known to possess heterogenous ITR sequences as a result of replicational instability, the effects of which on vector yield and efficacy are unclear and have not been systematically explored. To shed much-needed light on this decades-old problem, I utilised unique molecular identifiers (UMIs) as reporter elements for different rAAV plasmid preparations, so that massively parallel sequencing could be used to analyse their DNA and RNA derivatives through the course of production and in vivo gene transfer. The range of vector potencies observed, while not calamitous, definitively erases the notion that this problem can be further overlooked.

The success of this unconventional strategy proved to be an equally notable outcome, offering unprecedented insights into population kinetics, and achieving quantitative consistency between biological replicates comparable to q/dPCR measurement replicates of single samples. This triggered concerted efforts to formally investigate the capabilities of UMIs used in this fashion. The probabilistic principles underlying the technique were formalised and empirically validated, confirming precision capabilities akin if not superior to dPCR and qPCR at equivalent levels of stringency. Experiments also revealed a pattern of measurement bias with potentially adverse implications for other areas of count analysis including differential gene expression.