title: High-throughput genomic mapping of vector integration sites in gene therapy studies.
abstract: Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods. Here, we describe an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. We also discuss methods to assess dominant/overrepresented gene-modified clones in preclinical and clinical models.
authors: Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, WA, USA.
authors: Brian C
authors: BC
authors: Beard
authors: Jennifer E
authors: JE
authors: Adair
authors: Grant D
authors: GD
authors: Trobridge
authors: Hans-Peter
authors: HP
authors: Kiem
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D017422:Sequence Analysis, DNA; D016662:Virus Integration / Q000235:genetics*
chemical_list: D004247:DNA