Abstract
The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.
Highlights
Gene therapy is the introduction of exogenous nucleic acids into cells or organisms to achieve a therapeutic effect [1,2,3]
The AFIS-Seq method described here is a sensitive strategy for the detection of lentiviral integration sites that does not rely on any form of DNA amplification
Compared with the 3–7 days required for protocols based on linear amplificationmediated PCR (LAM-PCR), the wetlab portion of the AFIS-Seq protocol can be performed in only a few hours, using reagents and equipment readily available in a standard laboratory and analysis can be performed on a basic Windows desktop or laptop computer
Summary
Gene therapy is the introduction of exogenous nucleic acids into cells or organisms to achieve a therapeutic effect [1,2,3]. The expanding knowledge of virology and retroviral vectorology, and the increased understanding of genetic disease, resulted in the first clinical trials of gene transfer into humans [5] These early trials, raised issues regarding the safety of early vectors based on Moloney murine leukemia virus, due to vector toxicity and activation of proto-oncogenes caused by vectormediated insertional mutagenesis [6]. The ability of lentiviruses (LV) to efficiently target and integrate into both dividing and non- or slowly dividing cells (e.g. stem cells and neurons), together with the development of self-inactivating (SIN) vectors, has aided the translation of LV-based therapies into the clinic [8]. This includes multiple LV-based clinical trials targeting hematopoietic stem cells and T cells for a variety of diseases [9] and adoptive T cell therapy (e.g. CAR-T) as a promising oncotherapy approach
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