Peripheral Blood Lymphocytes as Target Cells of Retroviral Vector-Mediated Gene Transfer zyx By Fulvio Mavilio, Giuliana Ferrari, Silvan0 Rossini, Nadia Nobili, Chiara Bonini, Giulia Casorati, Catia Traversari, and Claudio Bordignon Peripheral blood lymphocytes zyxwvutsr (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low-affinity nerve growth factor recep- tor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cell lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integra- tion and stability of vector proviruses, and LNGFR expres- sion at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage- specific cell surface markers was performed in transduced zyxwvut ETROVIRAL VECTORS are an efficient and relatively safe tool for the transfer of exogenous DNA into so- matic cells.’ Retroviral vector-mediated gene transfer is cur- rently used in gene therapy protocols for the treatment of inherited and acquired blood diseases and in gene marking and gene therapy protocols for advanced cancer.*,’ Although significant effort is being devoted to optimizing procedures for purification and transduction of human hematopoietic stem cells, peripheral blood lymphocytes (PBLs) are still considered the safest cellular delivery vehicle for human gene therapy. A number of different retroviral vectors have been engi- neered and used for gene transfer into human hematopoietic cells, all based on the Moloney murine leukemia virus (MoMLV) backbone. Usage of different promoters driving expression of the gene of interest, and the position of these sequences with respect to the viral transcription unit, were some of the parameters taken into account in generating alternative vector design^.^.^ Some of these vectors were used to transduce human lymphoid cells under different con- ditions, eg, tumor-infiltrating lymphocytes (TILS),’,’ and CD4+ and CD8’ subsets of both TILSand PBLs.~ Expression zyxwvu R zyxw From the Gene Therapy Program, DIBIT; the Bone Marrow Transplantation Program, Service of Hematology, Istituto Scientijko H. San Raffaele, Milano, Italy. Submitted April zyxwvutsrqp 15, 1993; accepted November 23, 1993. Supported in part by grants from the Italian Association for Can- cer Research (AIRC) and the Ministry of Health (Progetto AIDS). Address reprint requests to Claudio Bordignon, MD, Department of Biology and Biotechnology, Istituto Scientific0 San Raffaele, Via Olgettina, zyxwvutsrqponm 60, 20132 Milano, Italy. The publication costs zyxwvutsrq of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with zyxwvutsrqp 1% U.S.C. section 1734 solely to indicate this fact. zyxwvutsrq 6 1994 by The American Society of Hematology. 0006-4971/94/%307-0014$3.00/0 1988 cell lines, PBLs, and T-cell clones t o study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vec- tors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune rep- ertoire. Gene transfer and expression could be demon- strated also in circulating progenitors of mature T cells. Ex- pression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with op- timal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector-medi- ated genetransferand positive immunoselection of the transduced cells that producesvirtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene- modified T lymphocytes in clinical studies. 6 1994 by The American Society of Hematology. of foreign genes transferred by retroviral vectors in T cells was successfully achieved in cell culture or preclinical mod- els of gene therapy of immune diseases, such as CD18- leukocyte adhesion deficiency (LAD)” and adenosine deam- inase-deficient (ADA-) severe combined immune deficiency (SCID),””4 or acquired immunodeficiency syndrome Although retroviral vectors were shown to trans- duce human lymphoid cells and express the transferred genes in all of these cases, no attempt has yet been made to directly compare the different vector designs for stability, efficiency of gene transfer, and expression of the transduced gene. We have systematically compared four different vector designs for expression of the same reporter gene, ie, the human low-affinity nerve growth factor receptor (LNGFR). This receptor is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression for each vector and each cell target by immunofluorescence analysis, even at the single cell level. Normal human peripheral blood mononuclear cells (PBMCs) and hematopoietic cell lines of myeloid and lymphoid origin were transduced with the four constructs and analyzed for efficiencyand stability of gene transfer and expression of LNGFR. Fluorescence-activatedcell sorter (FACS) analysis of transduced T-cell lines and clones for coexpression of LNGFR and cell surface markers was per- formed to studygene expression into specific T-cell subpop- ulations. Under appropriate infection conditions, all retrovi- ral vectors could transduce a T-cell population representative of the normal immune repertoire, although the level of the transgene expression was strongly dependent on the vector design. MATERIALS AND METHODS Retroviral vectors. Four different retroviral vectors for expres- sion of LNGFR as a reporter gene were generated using the full- coding 1.5-kb Ssr I fragment of the human LNGFR cDNA.” The NSV-N and NTK-N vectors were obtained by cloning the LNGFR cDNA into the unique Hind111 and Bgl I1 sites of the NSV and NTK Blood, Vol 83, No 7 (April l), 1994: pp 1988-1997 Downloaded from http://ashpublications.org/blood/article-pdf/83/7/1988/614161/1988.pdf by guest on 21 February 2023