A Human Immunodeficiency Virus Type 1polGene-Derived Sequence (cPPT/CTS) Increases the Efficiency of Transduction of Human Nondividing Monocytes and T Lymphocytes by Lentiviral Vectors

Abstract

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

Lentiviral Manipulation of Gene Expression in Human Adult and Embryonic Stem Cells

“…Previous studies have shown that the incorporation of a central polypurine tract (cPPT) 16 and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) 17 can increase transgene expression levels. By incorporating these elements into the lentiviral backbone (Fig.…”

Silencing of HIV-1 Gene Expression by Two Types of siRNA Expression Systems

“…In To construct the lentiviral vectors (CS-env-shRNA, CS-m-env-shRNA), the generated U6 hairpin vectors were digested with EcoR I and Nhe I and then cloned into the same site in the lentiviral transfer vector (CS-CDF-CG-PRE) (Naldini et al, 1996;Manganini et al, 2002;Rubinson et al, 2003;Stewart et al, 2003). …”

“…For the control vector, a G was mutated to a C in the siRNA sequence. We also constructed two different strand vectors, antisense (env-A) and sense (env-S), which were under the control of the U6 promoter (Figure 1) (Naldini et al, 1996;Manganini et al, 2002;Rubinson et al, 2003;Stewart et al, 2003), to generate the CS-env-shRNA and control transfer vectors (CS-m-envshRNA). The sequences and orientations of the constructed vector inserts were confirmed by nucleotide sequencing.…”

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors

“…[10][11][12][13][14][15][16][17][18][19][20][21][22][23] Viral vector particles were generated by cotransfection of the VSV containing plasmid pMD-G, the packaging construct pCMVdR8.74 and the indicated transfer constructs into 293T cells, followed by determination of concentration of particles by ultracentrifugation, as previously described. 65 Viral titers were evaluated by infecting a known number of REH cells with different volumes of viral vector preparations in a 200 ml volume. 65,66 B-cell precursor ALL cells were collected from 14 children at diagnosis, purified using Ficoll-HyPaque (Pharmacia LKB, Uppsala, Sweden) and frozen until use.…”

“…65 Viral titers were evaluated by infecting a known number of REH cells with different volumes of viral vector preparations in a 200 ml volume. 65,66 B-cell precursor ALL cells were collected from 14 children at diagnosis, purified using Ficoll-HyPaque (Pharmacia LKB, Uppsala, Sweden) and frozen until use. In all cases, more than 80% of blast infiltration was present and more than 90% of the blasts expressed CD19 antigen and lacked surface Ig (sIg).…”