High-Efficiency Gene Transfer into Rhesus Macaque Primary T Lymphocytes by Combining 32°C Centrifugation and CH-296-Coated Plates: Effect of Gene Transfer Protocol on T Cell Homing Receptor Expression

Abstract

Although steady progress has been made in transducing human T lymphocytes by Moloney murine leukemia virus (Mo-MuLV)-based vectors, few studies have been done to define ex vivo gene transfer protocols to transduce rhesus macaque primary T lymphocytes. Given the fact that simian immunodeficiency virus (SIV) infection in rhesus macaque is a well-characterized model for human immunodeficiency virus (HIV), it is of great interest to develop an efficient protocol to transduce rhesus macaque primary T cells. In this study, we have used MuLV-10A1-pseudotyped retrovirus expressing enhanced green fluorescent protein (EGFP) to evaluate a number of ex vivo gene transfer protocols in rhesus macaque primary T lymphocytes. Our objectives in designing these protocols were (1) to test whether higher efficiency gene transfer could be obtained by combining two previously defined protocols, centrifugation at 32 degrees C and the CH-296-coated plate; and (2) to study the effect of an ex vivo gene transfer protocol on the expression of lymphocyte homing receptors L-selectin and alpha 4 beta 7 and alpha 4 beta 1 integrins. From seven independent experiments we demonstrate by flow cytometry analyses of EGFP expression that whereas centrifugation at 32 degrees C or the fibronectin fragment CH-296-coated plate protocol alone yielded 10-14% transduction efficiency, combining these two protocols resulted in 28.1-51.2% transduction efficiency. EGFP in transduced cells was expressed highly throughout the 14 days of posttransduction expansion. Our results also demonstrate that whereas the transduction procedure per se did not significantly alter the expression of lymphocyte homing receptors, anti-CD3 and anti-CD28 antibody stimulation profoundly reduced the expression of L-selectin. The selective reduction of L-selectin may result in significant in vivo consequences if transduced cells are infused.

A Nonneutralizing Anti-HIV-1 Antibody Turns into a Neutralizing Antibody When Expressed on the Surface of HIV-1-Susceptible Cells: A New Way to Fight HIV

“…After the cells reached confluence, 2-ml fresh complete DMEM was added to each well and cells were incubated at 32°C overnight. Supernatants were harvested, filtered and titrated on HeLa cells as described (28).…”

“…To transduce CEM-ss cells, a 24-well nontissue culture plate was coated with recombinant fibronectin fragment (CH-296) as previously described (28). CEM-ss cells (1 ϫ 10 5 ) and 2.5 ml of recombinant virus-containing supernatants were then added onto the CH-296-coated well.…”

“…Viral DNA was amplified by PCR with a pair of primers (5Ј-GGCTAACTAGGGAACCACTG-3Ј and 5Ј-CTGCTAGAGATTTTCCACACTGAC-3Ј) as described by Zack et al (31) and separated on 2% agarose gels, transferred onto nylon membrane, and probed with 5Ј end-labeled oligonucleotide probe (5Ј-CCGTCTGTT GTGTGACTCTGGTAACTAGAG-3Ј). The internal control ␤-actin DNA was measured as previously described (28).…”

“…Cells then were washed twice with HBSS and cultured in the complete DMEM. eGFP expression was analyzed by FACS in 48 h. eGFP was reported before (28). Fig.…”

Hematopoietic stem cell gene therapy with drug resistance genes: an update

“…[23][24][25][26] The ability to achieve efficient gene transfer to repopulating human HSCs and to maintain appropriate levels of expression in vivo in these cells and their progeny has been poor and the resultant clinical benefits negligible. New vectors and improvements in vector design, that is, lentivirus, foamy virus, and in vitro-packaged SV40 vectors, [27][28][29][30][31][32][33][34][35][36][37] and envelope proteins, 10,27,38-47 use of the CH-296 domain of fibronectin in transduction, [48][49][50][51][52][53][54][55][56] improved cytokine combinations, 52,[56][57][58][59] and changes in transduction methodology, that is, spinoculation and vector preloading, 43,51,56,[59][60][61][62][63][64][65] have improved gene transfer efficiency. 66,67 Improvement in gene transfer technology and the survival advantage of transduced HSCs resulted in the success of HSC gene therapy in patients with X-linked severe combined immune deficiency (X-SCID).…”