Murine Retroviral but not Human Cellular Promoters Induce In Vivo Erythroid-specific Deregulation that can be Partially Prevented by Insulators

Abstract

We are developing lentiviral vectors for gene therapy of red blood cell disorders that co-express a transgene in an erythroid-specific manner and the O(6)-methylguanine-DNA-methyltransferase (MGMT) selective gene in a constitutive way. We report that transduction of murine hematopoietic stem cells (HSCs) with a human phosphoglycerate kinase promoter-based vector at low multiplicity of infection (MOI) does not result in a selective in vivo expansion in the presence of alkylating agents. In contrast, by replacing this cellular promoter with the powerful retroviral-derived myeloproliferative sarcoma virus enhancer, negative control region-deleted, dl587rev primer-binding site substituted promoter, the vector allowed efficient chemoprotection of transduced HSCs at low MOI. However, this promoter interacted with the erythroid HS40/ankyrin enhancer/promoter driving green fluorescent protein, leading to an unexpected loss of erythroid specificity. A partial restoration of tissue-specific expression was obtained by interposition of insulator sequences between the expression units. Alternatively, we found that the strong human cellular elongation factor1-alpha promoter allows similar chemoprotection but without any deregulation of the erythroid-specific promoter in the absence of insulators. These data demonstrate that the level of in vivo deregulation induced by a promoter is not correlated with its transcriptional activity.

Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells

“…These results were also confirmed using samples from CML patients sensitive 11 or resistant (12 and 13) to nilotinib ( Figure 8E). …”

“…11 Primers for the short hairpin anti-Syk were designed as followed: forward 5Ј-AGCTTCCGAGCAAATTGTCCTGATAGTTC-AAGAGACTATCAGGACAATTTGCTCTTTTTGGAAG-3Ј, reverse 5Ј-TCGACTTCCAAAAAGAGCAAATTGTCCTGATAGTCTCTTGAACT-ATCAGGACAATTTGCTCGGA-3Ј. Ligation was performed in pTRIP⌬U3EF1␣-DsRed-WPRE-MSC⌬U3 vector and used in with PAX2 and VSVG vectors to transfect HEK293T cells for lentiviral production.…”

The Chicken Hypersensitivity Site 4 Core Insulator Blocks Promoter Interference in Lentiviral Vectors

“…Similarly in our study, insertion of cHS4 increased transgene expression levels from Mp in double promoter vectors in all lineages of mouse peripheral blood cells (Fig. 2) (Robert-Richard et al, 2007).…”

Succès de la thérapie génique d’un modèle murin de porphyrie érythropoïétique congénitale

“…La tendance plus faible des vecteurs lentiviraux (par rapport aux vecteurs onco-rétroviraux murins classiques) à s'intégrer dans des régions potentiellement dangereuses du génome est un argument en faveur de leur utilisation pour les applications cliniques [21]. Dans le cadre de la mise en place d'un protocole clinique, les choix suivants pourraient être retenus pour une amélioration de la sécu-rité du vecteur : (1) utilisation d'un vecteur lentiviral SIN, plus sûr en terme de risque oncogénique que les vecteurs onco-rétroviraux ; (2) suppression des promoteurs viraux potentiellement responsables d'oncogenèse insertionelle [16][17][18]21] dont nous avons souligné récemment la capacité à déréguler l'expression de promoteurs spé-cifiques de tissu [22] ; (3) utilisation d'un promoteur spécifique de la lignée érythroide, limitant le risque de transactivation à la lignée érythrocytaire. Le vecteur lentiviral qui pourrait être utilisé pour l'essai clinique est très proche de celui utilisé pour la thérapie génique du modèle murin.…”