Was cDNA sequences modulate transgene expression of was promoter-driven lentiviral vectors.
Abstract The development of vectors that express a therapeutic transgene efficiently and specifically in hematopoietic cells (HCs) is an important goal for gene therapy of hematological disorders. We have previously shown that a 500-bp fragment from the proximal Was gene promoter in a lentiviral vector (LV) was sufficient to achieve more than 100-fold higher levels of Wiskott-Aldrich syndrome protein in HCs than in nonhematopoietic cells (non-HCs). We show now that this differential was reduced up to 10 times when the enhanced green fluorescent protein gene (eGFP) was expressed instead of Was in the same LV backbone. Insertion of Was cDNA sequences downstream of eGFP in these LVs had a negative effect on transgene expression. This effect varied in different cell types but, overall, Was cDNA sequences increased the hematopoietic specificity of Was promoter-driven LV. We have characterized the minimal fragment required to increase hematopoietic specificity and have demonstrated that the mechanism involves Was promoter regulation and RNA processing. In addition, we have shown that Was cDNA sequences interfere with the enhancer activity of the woodchuck posttranscriptional regulatory element. These results represent the first data showing the role of Was intragenic sequences in gene regulation.
Toscano
Miguel G
MG
Immunology and Cell Biology Department, Institute of Parasitology and Biomedicine López Neyra-CSIC, Parque Tecnológico Ciencias de la Salud, Granada, Spain.
Benabdellah
Karim
K
Muñoz
Pilar
P
Frecha
Cecilia
C
Cobo
Marién
M
Martín
Francisco
F
D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D005453:Fluorescence; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006402:Hematologic Diseases / Q000235:genetics / Q000628:therapy*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins