Abstract
Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. A disadvantage of this method is the use of a ubiquitous promoter leads not only to genetic modification of the leukocyte subset of interest e.g. T-cells, but also all other subsequent leukocyte progeny of the parent HSCs. To overcome this limitation we tested a bicistronic lentivirus, enabling subset specific modifications. Designed novel lentiviral constructs harbor a global promoter (mPGK) regulating mCherry for HSCs selection and a T-cell specific promoter upstream of eGFP. Two T-cell specific promoters were assessed: the distal Lck—(dLck) and the CD3δ-promoter. Transduced HSCs were FACS sorted by mCherry expression and transferred into sublethally irradiated C57/BL6 mice. Successful transplantation and T-cell specific expression of eGFP was monitored by peripheral blood assessment. Furthermore, recruitment response of lentiviral engineered leukocytes to the site of inflammation was tested in a peritonitis model without functional impairment. Our constructed lentivirus enables fast generation of subset specific leukocyte transgenesis as shown in T-cells in vivo and opens new opportunities to modify other HSCs derived subsets in the future.
Highlights
Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease
Since the global promoter is active in every cell—progenitor, T-cells and non-T-cells—successfully transduced HSCs and all their descendent daughter cells will be mCherry positive, which will allow their enrichment with fluorescence activated cell sorting (FACS) based techniques
Following 7 days post infection, 92% of the cells expressed mCherry and eGFP when utilizing the distal Lckpromoter (dLck)-promoter (Fig. 2A, left) whereas, 67% of the cells were double positive for mCherry and eGFP with lentivirus containing the CD3δ-promoter (Fig. 2A, right)
Summary
Lentiviral modification of hematopoietic stem cells (HSCs) paved the way for in vivo experimentation and therapeutic approaches in patients with genetic disease. To avoid competition of genetic-engineered HSCs with non-modified HSCs, enrichment of lentiviral infected HSCs is necessary prior to reconstitution of the ablated BM For this purpose pharmacological selection models (e.g. puromycin resistance) or physical sorting using fluorescent protein overexpression have been applied in the past[5,6,7]. For these selection processes global promoters with ubiquitous activity are employed, causing the target protein to be expressed in the subpopulation of interest (e.g. T-cell), and in LT-HSCs and all descending daughter cells with the respective global promoter activity. A dual promoter expression cassette with a cell specific second promoter permits definitive lineage specific transgenesis to investigate mechanistic and biological relevance of hematopoietic subsets in inflammatory disease models
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