Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements

Abstract

Current gene therapeutic protocols directed towards the vectors encoding a truncated human low-affinity nerve treatment of inherited disorders (eg ADA-SCID) and viral growth factor receptor (⌬LNGFR) as a gene transfer infections (eg AIDS), as well as adoptive immunotherapy marker, we obtained transduction frequencies of more than approaches are based on the use of genetically modified 70% of CD3 + cells after two cycles of infection. Our protolymphocytes. Since only insufficient transduction of T cells col is based on the use of FBS-free media for both the is obtained using existing techniques, the development of production of retrovirus-containing supernatant and the culmore efficient gene transfer protocols into these cells is of tivation of the primary T cells. Since the protocol presented great importance. We present here a protocol for the highly here works just as efficiently under large-scale conditions, efficient transduction of human primary T cells at high denit may be easily adapted to clinical needs and 'good manusities (1 × 10 6 /ml) by retroviral infection. Using retroviral facturing practice' (GMP) standards.

Self-inactivating retroviral vectors with improved RNA processing

“…T-lymphocytes were activated (using anti-CD3 and anti-CD28 antibodies) and cultured in X-Vivo10 containing 8% autologous serum (in the presence of 300 U/ml IL-2) essentially as described. 49 The first transduction was carried with the five vectors SF91, SF91P, SinSF91, SinSF91P and, for control, SF11EGFPrev 24 and with noninfectious supernatant (mock control) in the morning of day 4 using our centrifugation protocol. 49 A second infection cycle was performed in the evening of the same day for the low titer constructs SinSF91 and SinSF91P.…”

A novel ‘sort-suicide’ fusion gene vector for T cell manipulation

“…22,23 After titration on human fibroblasts (~2 × 10 6 infectious particles/ml), we infected Jurkat and primary T cells at an MOI of about 1 as described. 24,25 In some experiments, an analogous retroviral vector encoding enhanced green fluorescent protein (eGFP) 23 was used in parallel as a control.…”

Recombinant Sendai virus vectors for activated T lymphocytes

“…[5][6][7][8][30][31][32][33] However, in our systems, excellent gene transfer could be achieved by the simple addition of SeV vector solution without specific reagents or centrifugation. Moreover, optimal SeV-mediated gene transfer to activated T cells could be performed during a relatively brief exposure time (less than 30 min, Figure 4d), with representative results seen in nasal mucosa, 20 in the vasculature, 21 in retinal tissue, 24 as well as in human umbilical cord bloodderived CD34-positive cells.…”

“…Although an encouraging human study using a retrovirus demonstrated the success of T-cell-directed gene therapy against severely combined immunodeficiency disease (SCID) owing to adenosine deaminase deficiency (ADA), 1,3 it has been difficult to use this strategy in cases of other diseases, because the gene transfer efficiency is usually a critical factor limiting the outcome. Therefore, current efforts are now focused on the development of pseudotyped vectors 4 and transduction techniques [5][6][7][8] for the use of retroviral vectors for clinical applications.…”