Notable Reduction in Illegitimate Integration Mediated by a PPT-deleted, Nonintegrating Lentiviral Vector

Abstract

Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, residual, integrase-independent integration raises a concern that may limit the usefulness of this system. Here we present a novel 3' polypurine tract (PPT)-deleted lentiviral vector that demonstrates impaired integration efficiency and, when packaged into integrase-deficient particles, significantly reduced illegitimate integration. Cells transduced with PPT-deleted vectors exhibited predominantly 1-long terminal repeat (LTR) circles and a low level of linear genomes after reverse transcription (RT). Importantly, the PPT-deleted vector exhibited titers and in vitro and in vivo expression levels matching those of conventional nonintegrating lentiviral vectors. This safer nonintegrating lentiviral vector system will support emerging technologies, such as those based on transient expression of zinc-finger nucleases (ZFNs) for gene editing, as well as reprogramming factors for inducing pluripotency.

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

“…This finding suggests that CRISPR/Cas9 does not significantly alter the integration capacity of non-integrating vectors since only slightly lower rates (0.2%-0.5%) of integration were reported previously for non-CRISPR-vectors. 33 The integration frequency of ICLV-CRISPR/Cas9 was found to be $30%, also consistent with our previous observations ( Figure S2). Having established the ability of the IDLV-CRISPR/Cas9 system to mediate robust and sustained gene editing in dividing cells, we next examined target-specificity of the vectors.…”

“…33 Several studies have demonstrated broadly similar levels of illegitimate integration. 27,34,35 IDLVs have garnered significant interest among researchers for precise in vivo analysis of genetic diseases, since they significantly reduce the risk of insertional mutagenesis inherent in integrating delivery platforms.…”

“…33 Briefly, 15 mg vector plasmid, 10 mg psPAX2 packaging plasmid (Addgene #12260 generated in Dr. Didier Trono's lab, EPFL, Switzerland), 5 mg pMD2.G envelope plasmid (Addgene #12259, generated in Dr. Trono's lab), and 2.5 mg pRSVRev plasmid (Addgene #12253, generated in Dr. Trono's lab) were introduced into 293T cells by transfection. To generate IDLVs, a pBK43 (integrase-deficient) packaging cassette was used.…”

“…Vector particles were collected from filtered conditioned medium at 72 hr post transfection. When necessary, the particles were purified using the sucrose-gradient method 33 and concentrated >100-fold by ultracentrifugation (2 hr at 22,000 rpm). Vector and viral stocks were aliquoted and stored at À80 C.…”

Recent Advances and Future Perspectives in Lentiviral Gene Therapy for Hemophilia A and B

“…IDLVs are typically generated by packaging the vector with catalytically inactive human immunodeficiency virus (HIV) integrase [68]. The class I D64V mutation in the integrase catalytic site substantially reduces integration (10 2 -to 10 3 -fold) without compromising other steps in the transduction pathway [56,69]. Hepatocyte-targeted expression using miR-142-3p-regulated IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular (e.g.…”