A Capillary Electrophoresis Sequencing Method for the Identification of Mutations in the Inverted Terminal Repeats of Adeno-Associated Virus

Abstract

Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5' ITR (T>G, nt 2) and the downstream MscI site of the 3' ITR (del. nt 4672-4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR-chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.

Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

“…S5 A and B). Since the high GC content of the palindromic AAV ITRs is known to be difficult to sequence through, we developed a method that keeps the ITRs sufficiently denatured so as to reproducibly obtain complete sequence reads (40). Sequence analysis from edited CD34 + , K562, and HepG2 cells revealed that targeted insertion was precise, with the chromosomal sequences being contiguous with HA sequences, followed by the SA/T2A and the GFP ORF.…”

Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated Viral Vector

“…This might be partly due to non-HDR integration of AAV donor with ITR (Inverted Terminal Repeat), whose secondary structure hampers PCR. However, we could not determine vector-derived sequences, even by PCR with 7-deaza-dGTP, which improves PCR for AAV-ITR (data not shown) ( Mroske et al., 2012 ). We could not rule out intra-vector deletion of concatemeric insertion either ( Nowrouzi et al., 2012 ).…”

Adeno-associated virus in the liver: natural history and consequences in tumour development

“…Junctions of the circular AAV were amplified using two couples of primers surrounding the ITRs (online supplementary table 2) in 2.5% glycerol and 5% dimethyl sulfoxide. PCR products were sequenced by Sanger after ExoSAP-IT (Applied Biosystem) purification 41…”