Table 3.

Percentages of DNA Contaminants in rAAV8-gfp Particles Produced in Mammalian and Insect Cells

Method	Reference Sequence	HEK293	Sf9
SSV-seq	rAAV genome	99.48	98.90
	baculovirus or vector plasmid backbone	0.38	1.03
	rep-cap	0.11	0.05
	producer cell genome	0.04	0.02
qPCR	rAAV genome	99.04	99.40
	baculovirus backbone or vector plasmid backbonea	0.95	0.55
	rep-capb	0.01	0.06
	producer cell genome	< LOQ	< LOQ

The percentages were calculated based on next generation sequencing or qPCR data, relative to each DNA species length. The 3.3-kb rAAV genome is composed of the human cytomegalovirus promoter, the EGFP reporter gene, and the 3′ UTR of the human hemoglobin b gene, flanked by AAV ITR2 from the pSub-201 plasmid.¹⁴¹ The plasmid and baculovirus backbones are 4.8 and 144.4 kb in length, respectively. Recombinant AAV8-gfp vectors were produced in HEK293 mammalian cells by co-transfection of the pDP8 and pFB-gfp plasmids or in Sf9 insect cells by dual-baculovirus infection. After purification by CsCl gradients ultracentrifugation, the rAAV stocks were pretreated with a DNases cocktail before SSV-seq and qPCR analyses.

LOQ, limit of quantification.

^aQuantification of DNA contaminants derived from the baculovirus backbone or the vector plasmid backbone was performed by real-time PCR targeted to the baculoviral DNA polymerase or the amp gene, respectively.

^bFor helper sequences quantification, qPCR was targeted to the rep2 sequence.