Comprehensive genomic access to vector integration in clinical gene therapy

Abstract

Retroviral vectors have induced subtle clonal skewing in many gene therapy patients and severe clonal proliferation and leukemia in some of them, emphasizing the need for comprehensive integration site analyses to assess the biosafety and genomic pharmacokinetics of vectors and clonal fate of gene-modified cells in vivo. Integration site analyses such as linear amplification-mediated PCR (LAM-PCR) require a restriction digest generating unevenly small fragments of the genome. Here we show that each restriction motif allows for identification of only a fraction of all genomic integrants, hampering the understanding and prediction of biological consequences after vector insertion. We developed a model to define genomic access to the viral integration site that provides optimal restriction motif combinations and minimizes the percentage of nonaccessible insertion loci. We introduce a new nonrestrictive LAM-PCR approach that has superior capabilities for comprehensive unbiased integration site retrieval in preclinical and clinical samples independent of restriction motifs and amplification inefficiency.

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…47 rAAV integration also appears to be enhanced in the absence of p53 via uncertain mechanisms, although this might be mediated by p53 interactions with a functioning Rep protein, 48 which is not encoded in the rAAV vectors used in the current study. The absence of malignancies after subretinal administration of a range of high-titre vectors in an animal model that is defective in a major apoptosis pathway, possesses a heightened susceptibility to intraocular oncogenesis and that may possibly also permit higher levels of rAAV integration, suggests that any vector-chromosomal insertional events arising from subretinal vector delivery appear insufficient to induce uncontrolled clonal proliferation.…”

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

“…M, 100 bp ladder; MC, monoclonal; OC, oligoclonal; PC, polyclonal. This figure has been modified from 2,9 . Table 1.…”

“…The sensitivity and robustness of this method arises from the initial preamplification of the vector-genome junctions and magnetic selection of amplified PCR products. Like the alternative methods mentioned, LAM-PCR relies on the use of restriction enzymes, introducing a bias into retrieval capacity of IS [9][10][11] . Thus, only a subset of the IS repertoire (the integrome) can be detected in one reaction.…”

“…Thus, only a subset of the IS repertoire (the integrome) can be detected in one reaction. This bias is minimized by the parallel analysis of a given sample using optimal combinations of restriction enzymes 9 . Recently, a variant of the technology termed non-restrictive LAM-PCR (nrLAM-PCR) has been developed that circumvents the use of restriction enzymes and allows unbiased genome-wide analysis of a sample in a single reaction 9,12 .…”

Cytoreductive conditioning intensity predicts clonal diversity in ADA-SCID retroviral gene therapy patients

“…An adaptation of nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) was used to produce integration site sequencing libraries for Illumina sequencing. 5,[15][16][17][18][19][20][21] …”