Amplification-Free Analysis Of Lentiviral Vector Genome Integrity On The BioPhase 8800 System

By Jane Luo, Tingting Li, Mario Pulido, Zhichang Yang, Marcia Santos and Sahana Mollah, SCIEX

Lentivirus belongs to a genus of retroviruses that includes the human immunodeficiency virus (HIV). At 80-100 nm in diameter, it has an envelope on the outside and a capsid inside, composed of about 2000 copies of p24. Within the capsid, its genome consists of two single-stranded RNA held together as a dimer through noncovalent interactions near the 5′-end. Over recent years, recombinant LV has become a valuable gene delivery tool in clinical trials for treatment of various diseases. It can deliver transgene as large as 9kb and maintain long-term expression in target cells through integration into host cell genome. It infects dividing and no-dividing cells with broad tropism and low immunogenicity. With the demand of LV growing, so are needs for better characterization of these vectors to ensure their safety and efficacy. Many methods have been established for LV titer determination. However, a technology with the ability to analyze the integrity of RNA genome within LVs is lacking. For example, reverse transcription-quantitative PCR (RT-qPCR) can detect the presence of short fragments on RNA genome, but it lacks the ability to detect mutants or impurities. In addition, both RT and PCR amplification are known to cause variability. In this technical note, an amplification-free genome integrity analysis of LVs by CE-LIF on the BioPhase 8800 system is demonstrated.