Alpharetroviral Self-inactivating Vectors: Long-term Transgene Expression in Murine Hematopoietic Cells and Low Genotoxicity

Abstract

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

“…Infections in the presence of 0.2 M JQ1 or 0.01% DMSO were performed as described above. Linkermediated PCRs (LM-PCRs) were performed as previously described with minor modifications (73,75). …”

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

“…The classical LV integration pattern, which is generally accepted to be safer than that of murine leukemia virus-based vectors in hCD34 1 cells, is conserved in H/F-LV transduction in the absence of cytokine stimulation (G 0 CD34 1 cells). 3,4,[51][52][53]76 A recent study determined the VSV-G-LV IS pattern in thymidinetreated CD34 1 cells inducing a blockage into the G 0 /G 1a phase of the cell cycle. 77 Of interest, they revealed ISs primarily in RefSeq genes, in agreement with our results.…”

Development of gene therapy for blood disorders: an update

“…22 This favorable integration pattern has been shown to result in lower genotoxicity compared with lentiviral and g-retroviral vectors. 20 A split-packaging design for self-inactivating a-retroviral vectors has been developed and shown to be capable of generating high-titer vector particles. 19 The therapeutic potential for this class of vectors has been shown by functional correction of chronic granulomatous disease (CGD) in a cell line as well as within transduced human cells that engraft in the mouse model.…”

“…18 The a-retroviral vectors are being considered for therapeutic gene transfer as well. [19][20][21] Such vectors integrate more uniformly within the genome, particularly within intergenic regions compared with g-retroviral vectors, which are found near transcriptional start sites and cytosine guanine dinucleotide islands, and lentiviral vectors, which tend to integrate within genes. 22 This favorable integration pattern has been shown to result in lower genotoxicity compared with lentiviral and g-retroviral vectors.…”