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Prepublished online as a Blood First Edition Paper on January 2, 2003; DOI 10.1182/blood-2002-02-0578.
GENE THERAPY
From the Centre National de la Recherche Scientifique
(CNRS) UMR-7087, Biologie et Thérapeutique des Pathologies
Immunitaires, Centre d'Etude et de Recherche en Virologie et en
Immunologie (CERVI), Hôpital La
Pitié-Sâlpétrière, Paris,
France.
Achieving cell-specific expression of a therapeutic transgene by
gene transfer vectors represents a major goal for gene therapy. To
achieve specific expression of a transgene in CD4+ cells,
we have generated lentiviral vectors expressing the enhanced green
fluorescent protein (eGFP) reporter gene
under the control of regulatory sequences derived from the
CD4 gene Achieving specific expression of a therapeutic
transgene is a major hurdle of gene therapy. Given that
CD4+ cells, whether T or non-T cells, are prominent
players in pathologic conditions such as viral infection, autoimmunity,
and cancer, specific expression of therapeutic genes in
CD4+ cells has great potential clinical applications.
CD4+-cell-mediated gene therapy could be achieved with ex
vivo-transduced cells or hematopoietic stem cells (HSCs). Although
transduced CD4+ cells, providing immediate but short-term
immunotherapy, may be valuable for cancer treatment, many other
clinical conditions are likely to require continuous generation of
immune cells expressing therapeutic transgenes. In such cases, HSCs
represent attractive targets for gene therapy, as illustrated by
successful gene therapy of immunodeficient infants with severe combined
immunodeficiency (SCID).1 When HSCs are transduced, it is
often preferable and sometimes mandatory that transgene expression be
limited to a certain subset of their progeny. Indeed, HSCs give rise to
a diverse progeny in which differentiation and function could be
impaired by some transgenes. For HIV gene therapy, transgene expression should be limited to CD4+ cells (for a review, see
Buchschacher and Wong-Staal2). We thus aimed at generating
retroviral vectors driving CD4+-cell-limited
transgene expression.
With retroviral vectors, CD4 specificity can be achieved in at least 2 ways Concerning the second strategy, we previously studied regulatory
sequences of the CD4 gene in vitro6 and in
vivo.7,8 These studies have shown that expression of a
transgene through CD4 gene promoter and enhancer sequences
was restricted to mature T cells.7 Addition of the
CD4 gene-silencing element9 to these regulatory
sequences limited transgene expression to CD4+ T cells in
transgenic mice.8 We then designed a minimal
transcriptional cassette (CD4pmE) that retained T-cell-specificity on
cell lines in vitro10 and used it to successfully
construct T-cell-specific MoMLV retroviral vectors.11 In
other MoMLV-derived vectors in which transgene expression was driven by
the viral long terminal repeat (LTR), the CD4 silencer
conferred some specificity of expression to human CD4+ T
cells in vitro.12 It is thus feasible to design T-
and CD4+ T-cell expression-specific oncoretroviral vectors.
However, oncoretroviral vectors entail 2 major drawbacks when used to
transduce HSCs: (1) expression requires integration into the host DNA
that can only be achieved in dividing cells, and (2) expression is
frequently silenced in vivo.13 Recent advances in
lentiviral vector design (for a review, see Buchschacher and
Wong-Staal14), together with the potential for transducing
quiescent cells such as neurons15 or hematopoietic
cells,16 make lentiviral vectors the vectors of
choice for HSC-based gene therapy.17 In the work presented here, our aim was to develop lentiviral vectors achieving efficient HSC
transduction and subsequent transgene expression limited to their
CD4+-cell progeny. Specific vectors were tested in vivo in
a mouse model of hematopoietic reconstitution and in vitro on human
mature T cells and on CD34+ cells induced to differentiate
into dendritic cells and macrophages.
Mice
Lentiviral vector construction
Lentiviral vector production and concentration To produce lentiviral vectors, 4 × 106 293T cells were cotransfected using the calcium-phosphate method, as described,20 with 3.5 to 5 µg expression vector encoding the VSV-G envelope (pM .G), 6.5 to 10 µg packaging plasmid
expressing gag, pol, tat, and
rev but lacking the accessory genes nef,
vpr, vpu, and vif (pCMVR8.91),20 and 10 µg gene transfer vector LvPGK,
LvCD4, or LvCD4-Sil. Cells were grown in Iscove modified Dulbecco
medium (IMDM; Biological Industries, Kibbutz Beit Haemek, Israel)
supplemented with 5% fetal calf serum (FCS), penicillin (100 µg/mL),
streptomycin (100 U/mL), 2 mM L-glutamine (Life
Technologies, Gaithersburg, MD). Lentiviral supernatants were collected
at 18, 42, and 66 hours after cotransfection, spun down at
800g, and passed through a 0.45-µm filter before aliquots
were frozen at  80°C. To increase titers further, supernatants were
concentrated by ultrafiltration using either the Ultrafree-15 or the
Centricon Plus-80 filter devices according to the manufacturer's
instructions (Millipore, Bedford, MA). Briefly, 15 mL or 80 mL
supernatant was centrifuged 1 hour at 2000g through
Ultrafree-15 or CentriconPlus-80 apparatus at 20°C. Concentrated
supernatants were separated into aliquots and kept at 80°C until
further use.
Viral titers CD4+ CEMx174 cells were grown in RPMI 1640 10% FCS and antibiotics (Life Technologies). Then 105 cells were transduced for 2 hours at 37°C, 5% CO2 with 0.1 mL to 0.25 mL vector supernatants in the presence of 8 µg/mL polybrene (Sigma, St Louis, MO). Two more milliliters culture medium was added, and the cells were analyzed for enhanced green fluorescent protein (eGFP) expression 48 to 72 hours later. From the different dilutions assayed, only percentages of eGFP falling in the linear curve below 15% were taken into account for the calculation of viral titers. Percentages of eGFP+ cells were corrected by the number of cells infected, the dilution factor, and the volume used for infection to calculate the number of infectious particles per milliliter (ip/mL) supernatant. Titration experiments showed that no lentiviral vectors were present in the flow-through fraction after ultrafiltration (not shown).Murine hematopoietic stem cell purification and transduction Bone marrow from C57Bl/6-Ly5a was flushed from femurs and tibias using a 26-gauge needle in RPMI 1640 (Life Technologies). Lin cells were obtained after incubation with the
cocktail antibodies (CD4, CD8, B220, Mac-1, Gr-1, TER-119; kind gift of
E. Schneider, CNRS URA1461, Paris, France) for 20 minutes at 4°C.
Goat or sheep antirat immunoglobulin magnetic beads (Dynal, Oslo,
Norway) were added to the cells at a ratio of 3 beads per total number
of cells and were incubated under continuous agitation for 30 minutes
at 4°C. The Lin fraction was further enriched for
Sca-1+ cells using the Sca-1 Multisort Kit according to the
manufacturer's instructions (Miltenyi Biotec, Paris, France).
Typically, more than 80% of the Sca-1+ fraction expressed
Sca-1 at a high density (data not shown). Purified
LinSca-1+ cells were cultured for 48 hours in
RPMI 1640 (10% FCS, 50 µM  -mercaptoethanol, penicillin (100 µg/mL), streptomycin (100 U/mL), 2 mM L-glutamine [Life
Technologies]) in the presence of 50 ng/mL murine stem cell factor
(SCF), 20 ng/mL murine interleukin-3 (IL-3; R&D Systems, Abingdon,
United Kingdom), and 100 U/mL human IL-6 (a kind gift from Dr L. Aarden, Amsterdam, The Netherlands) and were transduced in vitro in
24-well plates (Becton Dickinson Falcon; Mountain View, CA) coated with
50 µg/cm2 fibronectin fragments (Retronectin; Takara
Shuzo, Otsu, Japan) according to the manufacturer's instructions. One
hundred fifty microliters concentrated viral supernatants was added for
1 hour at 37°C, followed by spinoculation at 1000g for 2 hours. Cells were washed and resuspended in complete media for further
overnight incubation. Cells were then washed and resuspended once in
phosphate-buffered saline (PBS; Life Technologies). Between
1 × 105 and 2 × 105 Sca-1+
cells were injected intravenously into each mouse. For in vitro differentiation of HSCs, 3 × 104 total bone marrow cells
of reconstituted RAG-2 KO mice were plated in semisolid colony-forming
assay using Methocult media (Stem Cell Technologies, Meylon, France).
Cells were allowed to grow for 8 to 10 days before optical
examination under ultraviolet (UV) light with a Diaphot microscope
equipped with a mercury-filled UV lamp (Nikon SA,
Champigny-sur-Marne, France).
Transduction of human lymphocytes Peripheral blood mononuclear cells from healthy donors were collected by density-gradient centrifugation using Hystopaque-1077 (Sigma). Cells were either prestimulated for 48 hours with phytohemagglutinin (PHA-P; Sigma) at 5 µg/mL in RPMI 1640 10% FCS with penicillin (100 µg/mL), streptomycin (100 U/mL), 2 mM L-glutamine (Life Technologies), and 100 U/mL human IL-2 (Chiron, Emeryville, CA), or they were cultured only in the presence of 10 ng/mL human IL-7 (R&D Systems) without prestimulation. Cells were transduced with lentiviral vectors in the presence of 8 µg/mL protamine sulfate (Sigma) for 2 rounds of the following cycle of infection: 1 hour at 37°C, 5% CO2, followed by 2-hour spinoculation at 2400g. Cells were analyzed for eGFP expression by flow cytometry from 48 hours to 16 days after infection.Human hematopoietic stem cell purification and transduction Samples that had undergone leukapheresis were obtained after informed consent of the patients and approval by the institutional review board. CD34+ cells were purified from leukapheresis products collected from patients after stem cell mobilization with granulocyte-colony-stimulating factor (G-CSF) and cyclophosphamide using magnetic immunobeads as described elsewhere23 and were frozen at80°C in FCS containing 10% dimethylsulfoxide (DMSO;
Sigma) until further use. After thawing, CD34+ cells were
cultured overnight at 106 cells/mL on 12-well plates
(Costar; Corning SA, Avon, France) in 2 mL IMDM containing 10% FCS,
1% L-glutamine, 1% antibiotics, SCF (300 ng/mL; a gift from Amgen,
Thousand Oaks, CA), Flt3-ligand (Flt3-L; 300 ng/mL, a gift from
Immunex, Seattle, WA), IL-3 (100 U/mL; Genzyme, Cambridge, MA), IL-6
(100 U/mL), insulin-like growth factor (IGF-1; 50 ng/mL; Valbiotech,
Paris, France), and basic fibroblast growth factor (bFGF; 50 ng/mL;
Peprotech, London, United Kingdom). CD34+ cells were then
infected at 1.5 × 105 cells/mL for 48 hours with
different lentiviral supernatants supplemented with the same growth
factor cocktail using an already published protocol23 that
combined cell centrifugation in the presence of 8 µg/mL protamine
sulfate followed by adhesion on wells precoated with Retronectin
(Takara Shuzo). At day 2 after infection, cells were cultured in
24-well culture plates (Costar) at 2.5 × 105 cells/mL
under different conditions to induce differentiation into dendritic
cells (DCs) or macrophages. For DC differentiation, cells were seeded
in IMDM supplemented with 10% FCS, 300 ng/mL SCF, 50 ng/mL Flt3-L, 100 ng/mL granulocyte macrophage-colony-stimulating factor (GM-CSF;
Schering Plough, Levallois-Perret, France), 50 ng/mL IL-4 (a gift from
K. Thielemans, Université libre de Bruxelles, Belgium), 5 ng/mL
tumor necrosis factor- (TNF-; Valbiotech). For macrophage
differentiation, cells were cultured in IMDM containing 10% SVF and 10 U/mL macrophage-colony-stimulating factor (M-CSF; R&D Systems). In
each culture condition, the medium was changed at day 5 after
infection, and cultures ended at day 9 after infection. Cells were then
counted, morphologically characterized, immunophenotyped, and assessed
for eGFP expression.
Antibodies and flow cytometry The following rat-antimouse-unlabeled monoclonal antibodies (mAbs) were used for cell sorting: CD4 (GK1.5), CD8 (8C6), B220 (6B2), Mac-1, Gr-1, and TER-119. For flow cytometry analysis, the following rat-antimouse or mouse antihuman mAbs were used, labeled with allophycocyanin (APC) or phycoerythrin (PE) (both from PharMingen, San Diego, CA), tricolor (TC; Caltag, Burlingame, CA), quantum red (QR; Sigma), or PE-cyanine 5 (PECy5; Becton Dickinson, Mountain View, CA or Immunotech, Marseilles, France) fluorescent dyes: CD45.1-APC, CD4-QR, CD4-APC, CD4-PE, CD4-TC, CD4-PECy5, CD34-PECy5, CD14-PE, HLA-DR-PE, CD8-PE, CD3-PE, CD11c-PE, NK1.1-PE, Sca-1-PE, and B220-TC. Negative controls were irrelevant isotype-matched mAbs IgG-PECy5 (Immunotech) and IgG-PE (PharMingen). One to 2 million cells per well were pelleted in a 96-well plate (Corning SA) and stained with 20 µL 2.4G2 (FcIII/Fc IIb) antibody for blocking nonspecific binding. After that,
15 to 20 µL properly diluted antibody was added for a 20-minute
incubation at room temperature under gentle agitation. Cells were fixed
with 1% paraformaldehyde (PFA; Sigma) in PBS, and data were acquired
with a FACScalibur (Becton Dickinson) and analyzed using CellQuest 3.3 (Becton Dickinson) or were acquired with an Epics Elite (Beckman
Coulter, Roissy, France) and analyzed with FlowJo (TreeStar,
San Carlos, CA).
Generation and titration of CD4-cell-specific lentiviral vectors Two lentiviral vectors with CD4-cell-specific promoters were designed (Figure 1). The LvCD4 and the LvCD4-Sil vectors were derived from LvPGK,22 a lentiviral vector expressing eGFP under the control of the ubiquitous phosphoglucokinase (PGK) promoter. The silencing element of the CD4 gene was added in the previously described CD4pmE transcriptional cassette10 to generate regulatory sequences specific for CD4-expressing cells. The minimal promoter of the CD4 gene with or without the silencer, the transgene eGFP, and the Wpre sequences were then inserted between the ClaI and Asp718 sites of the LvPGK plasmid to generate the LvCD4 and LvCD4-Sil vectors expressing eGFP as a reporter gene. Infectious lentiviral vectors were titrated by measuring the percentages of eGFP+ in infected CEMx174 T cells. As shown in Table 1, titers of CD4-cell-specific lentiviral vectors exceeded 106 ip/mL. When lentiviral supernatants were concentrated by ultrafiltration, titers increased by 12- to 216-fold, in some cases exceeding 108 ip/mL (Table 1).
In vivo reconstitution of RAG-2-deficient mice with HSCs transduced with eGFP-expressing cell-specific lentiviral vectors To evaluate CD4 specificity on newly generated cells in vivo, LinSca-1+ cells transduced with lentiviral
vectors were injected into RAG-2-deficient mice. These mice are
deprived of T and B cells because of defective genetic rearrangements
at the T-cell receptor (TCR) and B-cell receptor (BCR)
loci,18 but they are fully able to support T- and B-cell
differentiation from healthy mouse bone marrow.24 Furthermore, we chose RAG-2 KO mice as recipient mice to optimize the
detection of eGFP+ cells in donor-derived T cells (earlier
experiments in normal mice showed varying levels of engraftment that
significantly impaired our ability to reliably evaluate the proportions
of eGFP+ cells). Reporter gene expression was quantified in
secondary lymphoid organs, thymus, and bone marrow 2 months after
reconstitution. At this time, the proportion and absolute numbers of T
and B cells in the spleen, lymph nodes, thymus, and bone marrow were
similar to control C57Bl/6 mice (not shown). This indicated that the
transduction protocol used herein did not alter the stem cell
competency of LinSca-1+ cells. All mice
analyzed were reconstituted with a sizable population of
eGFP+ cells, ranging from 4% to 30% of
CD45.1+ (donor-derived) cells in the spleen.
Specificity of eGFP expression in spleen and lymph nodes of RAG-2-deficient mice reconstituted with transduced HSCs Lymph nodes (LNs) and spleens of reconstituted animals were studied to assess the specificity of transgene expression conferred by the CD4 regulatory sequences. Results of a representative experiment showing eGFP expression in CD4+ and CD8+ T cells and in B220+ B cells are shown in Figure 2A. With the LvPGK vector, reporter gene expression was readily detectable in all subsets. With the LvCD4 vector, B cells expressed little eGFP whereas CD4+ and CD8+ T cells exhibited similar proportions of transduced cells. In contrast, with the LvCD4-Sil vector, eGFP expression was dramatically reduced in frequency and in intensity in CD8+ T cells compared with CD4+ T cells. In these experiments, 2 peaks of eGFP+ cells could sometimes be observed with either vector tested.
Although CD4+ T cells are the major cell subset expressing
the CD4 molecule in mice, we also analyzed the specificity of our vectors in NK1.1+ natural killer (NK) cells and
CD4loCD11c+ splenic DCs. To evaluate cell
specificity independently of the variability of HSC transduction
efficiencies, we present our results as an index relative to the
frequency of eGFP+ CD4+ T cells (Figure 2B).
However, it should be stressed that this index only partially reflects
cell specificity because it does not confer the intensity of expression
in positive cells. With the LvPGK vector, indexes were increased by an
approximate factor of 2 in B cells, NK cells, and DCs, but they
were comparable in CD8+ T cells. With the LvCD4 vector, the
indexes were similar in CD8+ T cells, markedly reduced in B
cells and DCs, and moderately reduced in NK cells. Compared with the
LvCD4 vector, reporter gene expression was efficiently silenced with
the LvCD4-Sil vector in CD8+ T cells, as evidenced by an
index reduced by a factor of 4. When 2 peaks of eGFP+ cells
could be observed in CD4+ T cells with the LvCD4-Sil
vector, the strongest peak was efficiently silenced whereas the weakest
peak appeared not to be, which called into question its specificity
(not shown). These specificity index variations were correlated with
variations in the intensity of eGFP expression in positive cells
(Figure 2A). Altogether, CD4+ T cells appeared as the main
lymphocyte subset expressing eGFP after in vivo maturation of
Lin Specificity of eGFP expression in the thymus of RAG-2-deficient mice reconstituted with transduced HSCs We next sought to analyze at which stage of T-cell differentiation in the thymus the transgene would be expressed. Representations of immature CD4CD8 double-negative (DN) and
CD4+CD8+ double-positive (DP) cells and of
mature CD4+CD8 and
CD8+CD4 single-positive (SP) cells in
eGFP and eGFP+ thymocytes are illustrated
from a representative experiment in Figure
3A for the LvCD4 vector and are
summarized in Figure 3B. With the LvPGK vector, the proportions of DN,
DP, CD4-SP, and CD8-SP were similar in eGFP+ and
eGFP cells, in agreement with the ubiquitous nature of
this promoter. In contrast, compared with eGFP cells,
there was an increased proportion of SP cells and a decreased proportion of DP cells among the eGFP+ cells with the LvCD4
vector. Moreover, eGFP expression was clearly correlated with thymocyte
maturation as indicated by the analysis of CD3 expression, which is
absent or low in immature thymocytes and high in mature thymocytes.
With LvCD4, almost all eGFP+ cells expressed high levels of
CD3 (Figure 3A, inset); with the LvPGK vector, there was an even
distribution of CD3-expressing cells among the eGFP+ and
eGFP cells (not shown). This shows that eGFP expression
in DP cells occurs at a late stage of their differentiation.
Noteworthy, with the LvCD4-Sil vector, more than 75% of
eGFP+ thymocytes were CD4-SP cells (Figure 3B), and there
was a 5-fold reduction in the frequency of DP cells among
eGFP+ cells compared with eGFP cells (Figure
3B). As for LvCD4, eGFP expression in the thymus was only achieved in
cells expressing high levels of CD3 (not shown). Approximately 7% of
CD8-SP cells expressed the reporter gene in the thymus with the
LvCD4-Sil vector, but it should be noted that levels were low
(Figure 3C).
Specificity of eGFP expression in the bone marrow of RAG-2-deficient mice reconstituted with transduced HSCs We assessed eGFP expression in the bone marrow of RAG-2 KO mice reconstituted with HSCs transduced with the lentiviral vectors. In mature T cells in the bone marrow, eGFP expression showed the same patterns of expression as those present in spleen and LNs (data not shown). To assess whether immature cells of the monocytic and erythroid lineages and of granulocytes expressed the transgene, reporter gene expression was studied in Mac-1+, TER119+, and Gr-1+ cells. Expression of the reporter gene with the LvPGK vector was readily detectable in all these populations (Figure 4A). In contrast, no significant expression of eGFP in these subsets was detected with the LvCD4 vector (Figure 4A). Despite this lack of expression, there was a distinct population of eGFP+ cells within the CD45.1+ cells in the same mice (Figure 4B). Similar results were obtained with the LvCD4-Sil vector (not shown). It should be noted that eGFP+ cells with the LvPGK vector were found in CD45.1+ and CD45.1 cells within the small
cells of the bone marrow (Figure 4B).
We also studied the specificity of expression in individual erythroid burst-forming unit (BFU-E) and granulocyte macrophage-colony-forming unit (CFU-GM) colonies derived in vitro from bone marrow of reconstituted RAG-2 KO mice. No eGFP expression could be detected in more than 50 colonies tested for each of the LvCD4 and LvCD4-Sil vectors. In contrast, eGFP-expressing cells were readily detectable with the LvPGK vector in 11 of 50 BFU-E or CFU-GM colonies. Together these results indicated that the regulatory sequences of the CD4 gene were poorly expressed in immature cells of the bone marrow in vitro and in vivo. Specificity of eGFP expression in ex vivo-transduced human lymphocytes To assess whether silencing of the reporter gene would also be successful in human lymphocytes, PHA- or IL-7-activated human lymphocytes were transduced in vitro with the LvCD4 or LvCD4-Sil vectors. As shown in Figure 5, transduction with the LvCD4 vector resulted in similar proportions of eGFP+ cells in PHA-activated CD4+ and CD8+ subsets. In contrast, with the LvCD4-Sil vector, there was a 6-fold reduction in the proportion of eGFP+CD8+ cells compared with eGFP+CD4+ cells. Furthermore, this decrease in the frequency of eGFP+ cells was accompanied by marked reductions in the intensity of eGFP expression in the remaining eGFP+CD8+ cells. Similar results were obtained with T cells from 3 different donors, yielding the following results in CD4+ and CD8+ T cells, respectively: 75.1% ± 12.2% versus 68.1% ± 8.1% for the LvCD4 vector and 63.3% ± 21.1% versus 18.2% ± 10.6% for the LvCD4-Sil vector. When IL-7-treated T cells were infected, a higher proportion of CD4+ cells than CD8+ T cells were transduced with the LvCD4 vector. With the LvCD4-Sil vector, CD4+ T-cell-specificity was evidenced by the concomitant reduction in the proportion and the intensity of eGFP staining. It should be noted that we did not observe the 2 peaks of fluorescence intensity (Figure 2A) in transduced human lymphocytes (Figure 5). The silencing element of the CD4 gene used herein allowed efficient repression of the transgene in PHA- and IL-7-activated human CD8+ T cells.
Specificity of eGFP expression in ex vivo non-T CD4+ cells derived from transduced human CD34+ hematopoietic precursors We next sought to investigate whether expression of the reporter gene would be specific to non-T CD4+ cells. For this, we transduced G-CSF-mobilized CD34+ cells (more than 95% purity) with the different lentiviral vectors and induced them to differentiate in vitro into macrophages or dendritic cells. We focused the analysis of eGFP expression to cells that coexpressed CD4 and the phenotypic markers HLA-DR and CD14, phenotypes characteristic of dendritic cells and macrophages, respectively.25 At day 12 after infection, CD4+ cells represented more than 80% of HLA-DR+ or CD14+ cells (not shown). There were no significant differences in the frequencies of eGFP+ cells between the LvPGK and the specific vectors, although the frequency of eGFP+ cells tended to be lower with the LvCD4-Sil vector, irrespective of the cell type (Table 2). The intensity of eGFP expression with the specific vectors were reduced by a factor of at least 3 compared with the LvPGK vector and only represented minor shifts from negative controls, especially with the LvCD4-Sil vector (Figure 6). We conclude that the specific vectors were expressed in non-T CD4+ cells in these culture conditions at levels well below mature T cells (Figure 5). In marked contrast to CD8+CD4 T cells, non-T
CD4 cells could also express the transgene (not shown),
indicating a lack of specificity in these culture
conditions.
The present report establishes for the first time that CD4+-cell-specific expression of a transgene can be obtained by using regulatory sequences of the CD4 gene in the context of optimized lentiviral vectors. On reconstitution of RAG-2-deficient mice with transduced HSCs, the CD4-cell-specific vector was efficiently expressed in T cells, not expressed in immature cells of the bone marrow, and minimally expressed in B cells. The addition of the CD4 silencing element in the LvCD4-Sil vector led to further restriction of expression to CD4+ T cells in the thymus, spleen, lymph nodes, and bone marrow of reconstituted animals. The LvCD4 vector was also expressed, but to a lower extent, in CD4loCD11c dendritic cells and NK1.1+ cells. For the latter cells, this could be attributed to high levels of Ets-1,26 a transcription factor that plays a role in CD4 promoter activation.6 Alternatively, it is possible that the NK1.1+ cells expressing the transgene are actually NKT cells, some of which express the CD4 molecule.27 Unfortunately, there were too few of these cells in reconstituted animals to reliably analyze eGFP expression in that subset. Noteworthy, eGFP expression from the LvCD4-Sil vector was reduced in CD4lo splenic dendritic cells and in NK1.1+ cells. This indicates that the effects of the positive and negative CD4 regulatory sequences are different in the context of lentiviral vectors or of the endogenous CD4 gene. As evidenced by the combined frequency and intensity of transgene expression, the specificity achieved herein was comparable to that obtained with erythroid or major histocompatibility complex class II+ cell-specific promoters.28,29 We also found that expression of eGFP in vivo was sometimes divided into 2 or more discrete peaks of fluorescence intensity, independent of the lentiviral vector specificity. When this was the case for the LvCD4-Sil vector, the peak of weaker intensity appeared less sensitive to silencing than the stronger one, questioning its specificity (not shown). The origin of this phenomenon is unclear, but it should be noted that a similar phenomenon has been seen with other lentiviral vectors carrying internal promoters and eGFP as a reporter gene.28,29 Our results, however, emphasize the good level of specificity that can be achieved with internal enhancers/promoters in lentiviral vectors. They are also the first demonstration that a silencer can be functional in the context of lentiviral vectors. Epigenetic silencing of CD4 has been proposed as a mechanism for the repression of cell surface CD4 expression in mature CD8+ T cells.30 Deletion of the silencer in mature CD8+ T cells using Cre recombinase was not accompanied by the reapparition of cell surface CD4 expression.30 Our results on human mature T cells show that eGFP expression was efficiently repressed on infection with the LvCD4-Sil vector carrying an exogenous silencer. This result indicates that the silencer-associated factors responsible for its function31 are operative in this subset. Therefore, the regulation of CD4 expression in mature CD8+ T cells may be tightly controlled by genetic and epigenetic mechanisms. Detailed analysis of transgene expression during T-cell development in
the thymus is important in view of the potential use of these vectors
for the treatment of genetic immunodeficiencies that often result from
blocks in T-cell development at distinct stages of their
differentiation. With the LvCD4 vector, similar transgene expression
was observed in CD4+ and CD8+ SP cells. The
transgene was also expressed in the more mature fraction of the DP
thymocytes We show that the LvCD4 and the LvCD4-Sil vectors were not expressed in the most immature thymocytes or in immature cells of the bone marrow. Similarly, expression was low to undetectable in murine splenic dendritic cells or monocytes. In contrast, our results on human cells show that non-T dendritic and macrophage cells consistently expressed the transgene with the specific vectors. Murine monocytes or splenic dendritic cells expressed no to low levels of the CD4 molecule, whereas human CD14+ and HLA-DR+ cells were uniformly CD4+. Differential levels of endogenous CD4 expression in mice and human cells seem sufficient to explain the results. Of note is that high expression of eGFP was detected on mature
CD4+ T cells with the LvCD4-Sil vector. In contrast, low
levels of eGFP were detected on non-T CD4+ cells, and this
correlated with lower levels of CD4 expression. This would suggest that
the intensity of eGFP expression with the specific vectors parallels
faithfully the endogenous CD4 transcriptional control.
However, significant eGFP expression could be detected in
CD4 Finally, we propose that our vectors may be used for direct in vivo injection of specific gene transfer vectors. Indeed, acquired or genetic T-cell immunodeficiencies offer a unique setting in which the transduction of small numbers of HSCs or even T cells could be sufficient for obtaining marked efficacy because of the extraordinary potential of T-cell expansion and of the selective advantages provided by the transgene. Direct in situ injections of CD4+-cell-specific vectors could thus represent the ultimate treatment for HIV infection or SCID such as the one linked to ZAP-70 kinase deficiency.36,37
We thank J. C. Zhao-Emonet, B. Clerc, and C. Baillou for technical assistance, G. Gavory for excellent care of mice, and B. Lucas from Necker Institute (INSERM U345) for the gifts of RAG-2 KO mice. We also thank Drs P. Salmon, D. Trono, L. Naldini, and E. Schneider for kindly providing essential reagents used in this study and Drs J. Cohen, O. Boyer, and B. Salomon for critical reading of the manuscript.
Submitted February 25, 2002; accepted December 23, 2002.
Prepublished online as Blood First Edition Paper, January 2, 2003; DOI 10.1182/blood- 2002-02-0578.
Supported by grants from Agence Nationale pour la Recherche contre le SIDA (ANRS), SiDAction (Ensemble contre le SIDA), Association pour la Recherche sur les Déficits immunitaires Viro-Induits (ARDIVI), and Association Française contre les Myopathies (AFM).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David Klatzmann, CNRS UMR-7087, CERVI, Hôpital La Pitié- Sâlpétrière, 83 Bd de l'Hôpital, 75651 Paris Cedex 13; e-mail: david.klatzmann{at}chups.jussieu.fr.
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Top
Abstract
Introduction
a minimal promoter and the proximal enhancer,
with or without the silencer. Both lentiviral vectors could be produced
at high titers (more than 107 infectious particles per
milliliter) and were used to transduce healthy murine hematopoietic
stem cells (HSCs). On reconstitution of RAG-2-deficient mice with
transduced HSCs, the specific vectors were efficiently expressed in T
cells, minimally expressed in B cells, and not expressed in immature
cells of the bone marrow. Addition of the CD4
gene-silencing element in the vector regulatory sequences led to
further restriction of eGFP expression into CD4+ T cells in
reconstituted mice and in ex vivo-transduced human T cells. Non-T
CD4+ dendritic and macrophage cells derived from human
CD34+ cells in vitro expressed the transgene of the
specific vectors, albeit at lower levels than CD4+ T cells.
Altogether, we have generated lentiviral vectors that allow specific
targeting of transgene expression to CD4+ cells after
differentiation of transduced mice HSCs and human mature T cells.
Ultimately, these vectors may prove useful for in situ injections for
in vivo gene therapy of HIV infection or genetic immunodeficiencies.
(Blood. 2003;101:3416-3423)
,
packaging signal; GA-RRE, truncated gag sequence with the
rev-responsive element; cppT (central polypurine tract of
HIV); CD4pmE, human CD4 minimal promoter/murine enhancer cassette (590 bp); Sil, human CD4 silencer (484 bp); Wpre, posttranscriptional
cis-acting regulatory element of the woodchuck hepatitis
virus (587 bp); LTR-SIN, self-inactivating 3' LTR (deleted of 400 bp in
the U3 region). Enzymatic sites used for cloning the minimal promoter
are indicated together with the eGFP and the Wpre
(ClaI/Asp718).
.05) and (P 
