Genome Integrity Analysis Of AAV Using Multi-Capillary Gel Electrophoresis

By Tingting Li, Jane Luo, and Sahana Mollah, SCIEX, USA

An AAV is a non-enveloped virus with an icosahedral protein shell capsid of approximately 20 nm in diameter and a single- stranded DNA genome of about 4.7 kb in length. Due to its lack of pathogenicity, low immunogenicity, broad tropism and persistent transgene expression in both proliferating and quiescent cells, AAV is an attractive choice for creating viral vectors for gene therapy for a variety of diseases. The genome of an AAV vector for gene therapy is usually composed of two inverted terminal repeats (ITR), a promoter, a transgene and a poly-A tail. AAV genome integrity analysis is a critical quality test for AAVs because it provides insights into transgene integrity and ensures product safety and efficacy.

Verification of genome size has been traditionally done by denaturing agarose-gel electrophoresis and southern blot.2 Both are time consuming and have limited resolution on size determination. Recently, a capillary electrophoresis with laser induced fluorescent (CE-LIF) detection method was developed for AAV genome integrity. CE-LIF is a well-established, high sensitivity technique for nucleic acid analysis. Currently, AAV genome integrity analysis by CE-LIF is performed one sample at a time using the single-capillary system. Multiplexing the analysis can help decrease the analysis or profiling time for multiple serotypes or AAV vectors with different genome sizes.

In this technical note, it is demonstrated that multiple AAV samples with different serotypes or different genome sizes can be run simultaneously on the multi-capillary BioPhase 8800 system to accelerate the execution of sensitive, AAV genome integrity analysis while retaining excellent resolution, sensitivity and repeatability as seen with the single-capillary PA 800 Plus.