Insertional Transformation of Hematopoietic Cells by Self-inactivating Lentiviral and Gammaretroviral Vectors

Abstract

Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer-promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott-Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention.

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

“…In all, 5Â10 5 HaCaT cells at 25% of confluence were incubated with 5Â10 6 infection units per ml of lentiviral (pRRLsin-18.pptK14-GFP Woodchuck hepatitis virus post-transcriptional regulation element) or g-retroviral (Delta-Moloney leukemia virus-K14-GFP) SIN vectors, pseudotyped with vesicular stomatitis virus glycoprotein G. Cells were incubated with vector supernatants for 6-8 h in the presence of polybrene (8 mg ml À1 ). Transduction efficiencies of X95% were achieved.…”

“…In these vectors (denominated self-inactivating or SIN) transgene expression is driven by cellular promoters, achieving both increased safety and a more physiological regulation of the therapeutic protein expression. 5 Another form of insertional mutagenesis happens when transcription from a provirus inserted in the midst of a gene fails to terminate at the retroviral polyadenylation signals located in the R region of the LTR and continues into the cellular genome, resulting in viral-cellular chimeric fusion transcripts. These read-through transcripts are often processed by splicing events involving both viral and cellular donor and acceptor sites, respectively.…”

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

“…Quantitative PCR was performed using the Applied Biosystems 7300 Real-Time PCR System (Foster City, CA, USA), using the Quantitect SYBR Green kit (Qiagen, Hilden, Germany), as described previously. 8 The IVIM assay was performed as described previously. 54 After retroviral transduction, bone marrow cells were expanded for 14 days, plated onto 96-well plates at a concentration of 100 cells per well and positive wells were counted.…”

“…Extensive studies have shown that the enhancer sequences are the major cause of cell transformation, making the design of viral vectors for life-long therapy approaches even more challenging. [8][9][10] Insulators mark the boundaries of chromatin domains and limit the range of action of enhancers and silencers. 11 They are characterized by at least one of the following properties: Enhancer blocking and/or boundary.…”

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

“…6 Severe side effects resulting from altered gene expression have been observed in several phase I clinical gene therapy trials, including our own study aimed at the correction of X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by a defective superoxide production in phagocytic neutrophils. 7 --9 Since correction of X-CGD requires expression of the therapeutic gp91 phox protein mainly in phagocytic cells, myeloid-specific promoters can be used in combination with self-inactivating vectors to drive expression of the therapeutic transgene.…”