Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Abstract

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies.

Outcomes Following Gene Therapy in Patients With Severe Wiskott-Aldrich Syndrome

“…TREC content was expressed in copies per 1×10 5 peripheral blood mononuclear cells (PBMCs) (control range value between 150 to 2500 copies/10 5 PBMCs). The vector copy number (VCN) per cell was measured by qPCR detection of the vector’s HIV Psi sequence with normalization against the copy number of the albumin gene, as described elsewhere 22 (for more details, see supplementary methods). Lymphoid, myeloid, naive and memory T cell subpopulations were sorted by flow cytometry, using the corresponding fluorescence-labeled monoclonal antibodies.…”

Sustained Expression of Insulin by a Genetically Engineered Sertoli Cell Line after Allotransplantation in Diabetic BALB/c Mice1

“…However, insulin protein secretion by MSC-EhI-Zs cells was low compared to that in Sertoli cells transduced with an adenoviral vector (1 3 10 À8 lg/cell vs 1.5 3 10 À6 lg/cell, respectively), which could be due to the low transduction efficiency of lentiviral vectors. For adenoviral vectors, multiple copies of the virus are delivered to the cell, whereas only 1-2 copies of the lentiviral genome (carrying transgene of interest) are integrated into the cell [39,40]. Nevertheless, MSC-EhI-Zs cells transplanted as allografts survived and produced insulin mRNA throughout the study (i.e., Day 50 post-transplantation), although, GFP and insulin proteins were detected in only a few of the cells within the sectioned grafts.…”

Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency

“…25,26 The virus integration site assay was carried out as described. 27 Briefly, genomic DNA from lentivirus-transduced cells was digested and ligated with adaptors, and two rounds of nested PCR were performed.…”