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PLENARY PAPER
From the Division of Immunology and Hematopoiesis,
Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, MD.
Hematopoietic stem cells (HSCs) represent an important target for
the treatment of various blood disorders. As the source of critical
cells within the immune system, genetic modification of HSCs can also
be used to modulate immune responses. The effectiveness of HSC-mediated
gene therapy largely depends on efficient gene delivery into long-term
repopulating progenitors and targeted transgene expression in an
appropriate progeny of the transduced pluripotent HSCs.
Self-inactivating (SIN) lentiviral vectors have been demonstrated to be
capable of transducing mitotically inactive cells, including HSCs, and
accommodating a nonviral promoter to control the transgene expression
in transduced cells. In this study, we constructed 2 SIN lentiviral
vectors, EF.GFP and DR.GFP, to express the green fluorescent protein
(GFP) gene controlled solely by the promoter of either a housekeeping
gene EF-1 Hematopoietic stem cells have the unique capability
of repopulating the entire hematopoietic system, including the immune system, due to their self-renewal and pluripotent differentiation potentials. Therefore, stable gene transfer to these stem cells has
great potential to achieve both long-term and short-term therapeutic effects for the treatment of inherited and acquired hematopoietic disorders.1-6 Currently, retroviral (including
oncoretroviral and lentiviral) vectors remain the only choice to stably
transduce hematopoietic stem/progenitor cells (HSPCs) efficiently,
resulting in permanent integration of the transgene into the host
genome.7-9 However, transgene expression in all progenies
of the transduced HSPCs is often unnecessary and may even be
detrimental in many circumstances.9-10 Thus, targeted
and/or restricted expression of transgenes efficiently and specifically
in one relevant differentiation lineage derived from the transduced
HSPCs may be crucial to achieve maximal therapeutic effectiveness and
to limit potential adverse effects.
Until recently, oncoretroviral vectors (RVs) have been the only
delivery vehicles for HSPCs.1-10 However, RV transduction requires division of the target cells while most of primitive HSPCs are
mitotically quiescent. Although significant progress has been made in
improving conditions for ex vivo HSPC culture and transduction, RV
design and production, and RV-mediated transgene expression,11-16 RVs still possess intrinsic limitations.
For example, a preactivation lasting for 2 to 3 days is required for
efficient RV transduction of HSPCs. Because HSPC engraftment activities can be only maintained in culture for several days currently and extended culture often results in HSPC reduction, a subtle balance of
HSPC maintenance and high-level transduction during ex vivo culture is
critical. Second, the RV long terminal repeat (LTR) is used in almost
all the high-titer RVs as the promoter to transcribe the transgene as
well as the viral genomic RNA.9-10 However, the RV LTR has
been found attributing to the inactive or attenuated transgene
expression in transduced cells after differentiation and/or long-term
culture and engraftment.17-20 Insertion of an additional
promoter into RVs often resulted in "promoter interference" that
reduced promoter activity of the internal promoter and/or the
LTR.21-23 Deletion of the promoter element within the RV
LTR to avoid the promoter interference often resulted in a severe (100-fold) reduction of viral titers.9-10 Except in a few
cases such as the GATA-1 transcriptional factor binding and
self-activation system,24 it has been generally difficult
to construct high-titer RVs in which a transgene is solely controlled
by a non-LTR promoter.
Recently, attention has focused on vectors derived from lentiviruses
(LVs) such as human immunodeficiency virus-1 (HIV-1), which have been
shown to transduce a variety of mitotically inactive cells, including
human HSPCs.25-31 This is consistent with the notion that
LVs can efficiently transduce cells that are in the G1, S, and G2
phases before cell division. In contrast, RV integration can occur only
after breakdown of the nuclear envelope, concomitant with progression
through the M phase of the cell cycle. We and others have found the
pseudotyped LVs can transduce human HSPCs efficiently as assayed in a
surrogate transplantation model using nonobese diabetic/severe combined
immunodeficiency (NOD/SCID) mice.26,29,32-34 Recent
improvements, such as minimizing HIV sequences and eliminating viral
accessory proteins for improved transduction efficiency and
safety,35-36 further enhanced favorability of LVs as
delivery vehicles for HSPCs. Moreover, the self-inactivating (SIN)
modification of LV,37-39 which permanently disables the
viral promoter after integration, enables transgene expression to be controlled solely by an internal promoter. Importantly, the SIN modification of LVs does not reduce viral titers, which is in sharp
contrast to what was observed previously with RV.9-10
Thus, using SIN LVs with a specific promoter, we may achieve targeted transgene expression in a specific lineage of transduced pluripotent HSPCs.
In the present study, we evaluated the ability of SIN LVs to target
genes into antigen-presenting cells (APCs) differentiated in vitro and
in vivo from HSPCs. It is now appreciated that processing and
presentation of antigens by different APC types are the initial events
in determining immune responses. Indeed, major endeavors in engineering
antigen-specific immunotherapy such as vaccines are focused upon the
targeting of antigens to various APC subsets such as dendritic cells
(DCs), which are critical mediators of T-cell
immunity.40-43 We postulated that large numbers of
transduced APCs/DCs could be generated from engrafted HSPCs after
transduction with specific genes to modulate immunity. To achieve
APC-specific transgene expression, we took advantage of the fact that
major histocompatibility complex class II (MHC II) genes are expressed selectively in APCs and highly in DCs after differentiation and maturation. We constructed and evaluated 2 SIN LVs expressing the green
fluorescent protein (GFP) gene controlled by 2 different internal
promoters: DR.GFP, which employs a human MHC II-specific HLA-DR Animals, cell lines, and cytokines
LV construction and virus production
Recombinant LVs were generated using the 3-plasmid system by
cotransfection of 293T cells through calcium phosphate
precipitation.35,38,46 In addition to a transducing vector
(PGK.GFP, EF.GFP or DR.GFP), 2 other vectors were used in viral
production. One, pMD.G, expresses the vesicular stomatitis virus G
envelope protein.46 The other, pCMV Detection of replication-competent retroviruses To rule out the possibility that replication-competent retroviruses (RCRs) could be generated from the DR.GFP or EF.GFP LV, we randomly tested the presence of viral proteins in culture media of stably transduced cells (after continuous culture after transduction) and in sera of NOD/SCID mice engrafted with transduced human cells. The amount of the HIV-1 p24 viral protein, which would exist in sera or culture media and gradually amplify if RCRs arose, was determined using a p24 enzyme-linked immunosorbent assay kit (ZeptoMetrix, Buffalo, NY). Within the detection limit (5 pg per sample) of the assay, all the samples we examined were negative for RCRs in human cells and mouse sera.Antibodies and flow cytometric analysis R-phycoerythrin (PE)-conjugated antihuman HLA-DR (MHC II), CD1a, CD14, CD40, CD80, CD86, biotin-conjugated antimouse CD45 and Ter119, and CyChrome-conjugated antihuman CD45 monoclonal antibodies (mAbs) were purchased from PharMingen (San Diego, CA). PE-conjugated antihuman CD13, CD19, and CD83 were obtained from Caltag Laboratories (Burlingame, CA). PE-conjugated antihuman CD34 was purchased from Becton Dickinson (San Jose, CA). PE-conjugated antihuman glycophorin A was obtained from Immunotech (Marseille, France). Fluorescence-activated cell sorter (FACS) analysis was carried out using a FACScan or FACSort (Becton Dickinson).Macrophage and DC differentiation after LV transduction of human blood monocytes Human peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia, Sweden) from healthy donors. Monocytes were enriched by plastic adherence after culturing peripheral blood mononuclear cells (8 × 106 cells/well [9.4 cm2]) with RPMI 1640 medium plus 10% FBS for 2 hours at 37°C. After washing, adherent cells were cultured in the same medium supplemented with M-CSF (25 ng/mL) to induce macrophage differentiation for 7 to 10 days. At days 2 and 3, adherent cells were transduced with EF.GFP or DR.GFP LV twice with 8 µg/mL polybrene. The multiplicity of infection (MOI) was 30 for each round of transduction. Cells were harvested on day 10 and analyzed for expression of GFP and macrophage markers (CD14 and HLA-DR). For DC differentiation, adherent monocytes were cultured in the same medium supplemented with GM-CSF (800 U/mL or 150 ng/mL) and interleukin-4 (IL-4) (1000 U/mL or 20 ng/mL). At days 1 and 2, adherent cells were transduced with DR.GFP or EF.GFP LV (2 mL/well, MOI = 10) with 8 µg/mL polybrene. After 4 to 6 hours of incubation with LV, 2 mL fresh medium containing 2 × GM-CSF and IL-4 was added, and transduction continued overnight. At day 2, transduction was repeated. At day 3, transduced cells were harvested and further cultured in 1 mL fresh medium containing GM-CSF and IL-4. When IL-4 was added, no sign of cell proliferation was observed, in contrast to the case when GM-CSF alone or M-CSF was added to monocyte cultures. At day 6, 1 mL fresh medium containing tumor necrosis factor (TNF)- (final
concentration 50 ng/mL) was added to promote DC maturation. At day 8, total cells were harvested and analyzed for expression of GFP and DC
markers (HLA-DR and CD83) with PE-conjugated mAbs.
To quantitatively determine the specific transgene expression in MHC
II+ cells, we defined the term "specificity factor"
(SF) as the ratio of percentages of GFP+ cells in all the
MHC II+ cells versus that in all the MHC II
 cell populations. SF works best if both MHC
II+ and MHC II cells exist in fair
percentages (Figures 2-4).
LV transduction of human CD34+ cells and DC differentiation Human G-CSF-mobilized peripheral blood or cord blood CD34+ cells were obtained from AllCells (San Mateo, CA). CD34+ cells were purified by immunomagnetic selection (Miltenyi Biotec, Auburn, CA) and then cryopreserved. The purity of CD34+ cells was usually more than 95%. One day before transduction, cryopreserved CB CD34+ cells were thawed and cultured (106 cells/mL) overnight in QBSF-60 medium (Quality Biological, Gaithersburg, MD). Human thrombopoietin (10 ng/mL), stem cell factor/KIT ligand (SCF/KL) (100 ng/mL), and FLT3 ligand (FL) (100 ng/mL) were supplemented (the Tpo/SCF/FL [TSF] medium). In the following days, these CD34+ cells were transduced with EF.GFP or DR.GFP LV (MOI = 10 to 60) once or twice by centrifugation at 2000g for 4 hours at room temperature. The cells with viruses were then diluted with one volume of the TSF medium, cultured overnight, and replaced with fresh TSF medium next morning. Three days after the first LV transduction, transduced CD34+ cells were harvested for FACS analysis and for subsequent in vitro and in vivo studies described below. DC in vitro differentiation was carried out by culturing the transduced CD34+ cells in RPMI 1640 supplemented with 10% FBS, GM-CSF (800 U/mL), IL-4 (200 U/mL), and TNF- (20 ng/mL). The culture medium was replenished every 3 to 4 days. After 14 to 16 days of culture, mature human DCs in suspension
were harvested and examined for GFP and MHC II expression.
CFC assays resulting in myeloid and erythroid lineage differentiation in methylcellulose Transduced human CD34+ cells were seeded onto 35-mm dishes in triplicate, each containing 450 cells in 1.2 mL methylcellulose media (Marrow-Gro, Quality Biological) supplemented with human stem cell factor (100 ng/mL), IL-3 (10 ng/mL), IL-6 (10 ng/mL), G-CSF (10 ng/mL), GM-CSF (10 ng/mL), and erythropoietin (5 U/mL). After 14 days of culture, total numbers of granculocyte-monocyte colony-forming units (CFU-GMs), mixed colony-forming units (CFU-mix), and erythroid burst-forming units (BFU-Es), as well as GFP+ colonies, were counted under a Nikon light/fluorescence microscope. Additionally, colony-forming cell (CFC)-derived cells were harvested by diluting and washing off methylcellulose with phosphate-buffered saline and then examined for GFP and MHC II expression.Human CB CD34+ stem cell engraftment of NOD/SCID mice The DR.GFP, EF.GFP, or mock-transduced CB CD34+ cells were transplanted into 8- to 10-week-old sublethally irradiated (300 cGy) NOD/SCID mice via tail vein injection (2 × 105 input cells per mouse). Six to 10 weeks after transplantation, the BM and spleens of these engrafted NOD/SCID mice were harvested and analyzed for the presence of human CD45+ cells. GFP expression in MHC II+ and MHC II human cell
populations was examined. Additionally, for further culture and in
vitro differentiation assays of engrafted human cells, murine
(CD45+ and Ter119+) cells were first removed.
Briefly, harvested cells from NOD/SCID BM and spleens were resuspended
in phosphate-buffered saline with 6% FBS at 2 × 107/mL
and incubated with biotinylated antimurine CD45 and Ter119 antibodies
(1 µg/106 cells) at 4°C for 30 minutes. After washing,
the antibody-labeled cells were then incubated with
streptavidin-conjugated magnetic beads (Dynal, Oslo, Norway) for 30 minutes at 4°C (4 beads per cell). The cell mixture was then placed
in the magnetic rack (Dynal) for 5 minutes, and the unbound human cells
were collected.
DC differentiation from the enriched human cells engrafted in NOD/SCID mouse BM was carried out similarly to peripheral blood stem cell (PBSC)-derived DC differentiation described above with the following modification. At day 6 of DC differentiation, the suspension cells were completely removed to further eliminate murine cells in suspension. For CFC assays after engraftment, enriched BM cells were added to methylcellulose media supplemented with human GM-CSF, IL-3, and erythropoietin in each of duplicate 35-mm dishes. After 19 days of culture, numbers of the total and GFP+ colonies were counted, and the cells were harvested for FACS analysis. Alloantigen presentation of differentiated human DCs in mixed leukocyte reactions In vitro-differentiated human DCs either directly from transduced CB CD34+ cells or from reconstituted NOD/SCID BM were irradiated at 3000 cGy and used as APCs in the mixed leukocyte reaction (MLR) T-cell proliferation assay. Briefly, cryopreserved human peripheral lymphocytes (effectors, 2 × 105/well) were seeded in 96-well plates with serially diluted human DCs (stimulators, stimulator:effector ratios 1:10, 1:80, and 1:640, respectively) in triplicate. After 3 days of culture, the cells were pulsed with 1 µCi/well (3.7 × 104 Bq) of [3H]thymidine and harvested 18 to 20 hours later with a Packard Micromate cell harvester (Packard BioScience, Meriden, CT). The [3H]thymidine incorporation was determined through a Packard Matrix 96 direct -counter.
Preferential transgene expression in MHC II+ cells mediated by the DR.GFP vector To achieve restricted transgene expression in APCs that selectively express MHC II molecules such as HLA-DR, we constructed a SIN LV containing the HLA-DR promoter as the sole internal promoter.
Then we compared titers and transgene expression of DR.GFP with LVs in
which transgene expression is controlled by either a human PGK promoter
(PGK.GFP) or EF-1 promoter (EF.GFP). When tested in a variety of
human MHC II+ (such as TF1 and Epstein-Barr
virus-transformed B cells) and MHC II (U937, K562,
HL-60, and EOL) hematopoietic cell lines, the DR.GFP vector directed
GFP expression preferentially (20- to 100-fold) in the MHC
II+ cells with intensities higher than PGK.GFP but slightly
weaker than EF.GFP (data not shown). In contrast, PGK.GFP or EF.GFP
showed equivalent levels of GFP expression in both MHC II+
or MHC II cell lines (the GFP signal by EF.GFP was 3- to
20-fold brighter than that by PGK.GFP as reported
recently30). To assess whether the induction of MHC II
(HLA-DR) expression will result in transgene up-regulation, we treated
the transduced HeLa cells with IFN- after DR.GFP transduction.
Corresponding to the up-regulation of MHC II expression by IFN-, GFP
transgene expression was also up-regulated by more than 10-fold in the
transduced HeLa cells (data not shown). Subsequently, we used DR.GFP
and EF.GFP vectors to evaluate gene transfer efficiency and promoter
specificity of these SIN LVs in primary human hematopoietic cells.
Transgene expression in human blood monocyte-derived macrophages and DCs by the DR.GFP SIN vector Adherent monocytes were cultured with M-CSF or GM-CSF plus IL-4 to induce macrophage or DC differentiation, respectively. After 2 rounds of transduction by DR.GFP or EF.GFP SIN LV, transduced cells were cultured in complete media for an additional 6 to 7 days before being analyzed by FACS for GFP and lineage marker expression. For macrophage differentiation, adherent cells were harvested for analysis (Figure 1A). Nearly 100% of cells expressed CD14 (not shown), and most cells expressed moderate levels of MHC II (HLA-DR). After DR.GFP transduction, moderate levels of GFP expression were observed in these MHC II+ (and CD14+) macrophages, particularly in those expressing highest levels of MHC II (Figure 1A). GFP expression by EF.GFP appeared more uniform regardless of the MHC II expression levels.To quantitatively determine the relative specificity of the transgene
expression in the MHC II+ population, we employed the SF
calculation (see "Materials and methods"). The dimensionless SF
computes the percentage of GFP+ cells in MHC
II+ population versus that in the MHC II For DC differentiation, motile cells were collected at day 6 and
further cultured with TNF- Efficient transduction of human CD34+ HSPCs and selective transgene expression in MHC II+ cells by the DR.GFP vector We next examined transgene expression in MHC II+ cells derived from transduced HSPCs by DR.GFP. Cryopreserved CD34+ PBSCs were cultured for 24 hours and transduced once (MOI 10-20). Three days after transduction by DR.GFP or EF.GFP, about 20% of transduced cells in both groups expressed GFP (Figure 2A). More than 95% of the cells expressed MHC II (Figure 2A) as the starting CD34+ cell population. The MHC II expression in most CD34+ HSPCs has been reported previously.48-49 A closer examination of the MHC II (HLA-DR) expression in the transduced CD34+ cells revealed that GFP expression was almost exclusively restricted in the MHC II+ cell population in the DR.GFP transduction group. In contrast, similar percentages of GFP+ cells were detected in both MHC II+ and MHC II
populations in the EF.GFP-transduced group (Figure 2A). The SF of
DR.GFP and EF.GFP in transduced PBSCs was determined to be 3.1 and 0.6, respectively (Figure 2A,D).
To further examine DR.GFP-mediated gene delivery into progenitor cells
and the transgene expression in differentiated progeny with various
levels of MHC II expression, the transduced human PBSCs were
differentiated in culture into either DCs or erythroid/myeloid lineages. After 16 days of DC differentiation, more than 95% of the
suspension cells exhibited the DC phenotype (ie, morphologically rich
in surface dendrites, high levels of surface expression of MHC II,
CD86, CD40, CD83, and CD1a) (Figure 2B). Further examination of GFP
expression in DCs derived from the DR.GFP-transduced PBSCs demonstrated
substantial specificity in MHC II+ cells (SF = 12.6,
Figure 2B,D). In contrast, GFP-expressing cells derived from
EF.GFP-transduced PBSCs were equally allocated between the MHC
II+ and MHC II DR.GFP- and EF.GFP-transduced PBSCs were also assayed in
methylcellulose for CFCs, resulting in erythroid/myeloid
differentiation. Mock-, DR.GFP-, and EF.GFP-transduced PBSCs all formed
BFU-E, CFC-GM, and CFU-mix colonies, and colony numbers in 3 groups
were similar, ranging from 185 to 207 (total). We found that 42% of the total colonies in the EF.GFP group displayed strong GFP signal by
fluorescence microscopy. However, 25% of the colonies in the DR.GFP-transduced group were GFP+ and their GFP
fluorescence intensities were much weaker, indicative of transgene
down-regulation in differentiated erythorid/myeloid progeny. To further
examine the correlation of GFP and MHC II expression in CFC-derived
progeny, the cells were harvested from the methylcellulose assay
(called non-DCs) and analyzed via FACS. In these mixed populations
consisting of monocytes, granulocytes, and erythroid cells, about 20%
of the cells expressed cell surface MHC II (Figure 2C).
GFP+ cells were found only in MHC II+ cells in
the DR.GFP transduction group (non-DC, SF = 26, Figure 2C).
GFP+ cells in the DR.GFP group were restricted to cells
expressing the highest level of CD13/CD33 differentiation marker
(Figure 2C), and the intensity of GFP signals was lower than in
differentiated DCs. These GFP+/MHC II+ cells
are likely to be monocytes and macrophages present in the CFC assay.
The other major cell type present in the CFC assay is erythroid cells
(glycophorin A+), which are MHC II Preferential transgene expression in MHC II+ human cells engrafted in NOD/SCID mice with DR.GFP-transduced CD34+ cells We next tested the specificity of the HLA-DR promoter in the
DR.GFP vector to direct transgene expression in human cells in vivo
using the NOD/SCID mouse model. We first used human cord blood stem
cells (CBSCs) because they have higher engraftment capacity in
this mouse model than PBSCs or BM CD34+
cells.12-13,16,50-52 Cyropreserved CB CD34+
cells were cultured overnight and transduced twice with DR.GFP or
EF.GFP at the MOI of 60 in the next 2 days. Two days after the last
transduction, GFP and CD34 expression of the transduced cells was
examined by FACS analysis. Under our culture and transduction conditions, more than 90% of cells still maintained CD34 (Figure 3A) and MHC II expression (data not
shown), and about 50% of the transduced CBSCs expressed GFP 2 to 3 days after transduction (Figure 3A). These cells were then used for
NOD/SCID transplantation as well as for in vitro differentiation assays.
After 14 days of in vitro differentiation in methylcellulose, numbers
of total colonies and GFP+ colonies were determined. Figure
3B is a representative illustration of colonies derived from mock-,
DR.GFP-, and EF.GFP-transduced CBSCs and shows percentages of
GFP+ colonies. Subsequent FACS analysis showed similar
patterns of preferential transgene expression in MHC II+
cells by DR.GFP (data not shown), as we observed with PBSCs (Figure 2C). The reduction of GFP expression in MHC II To examine transgene expression in engrafted human cells from CBSCs
after transduction, NOD/SCID mice were terminated 6 to 10 weeks after
transplantation. Human (CD45+) cell engraftment and GFP
expression in mouse BM and spleens were analyzed. Among the 15 animals
that underwent transplantation, up to 20% human cells were
detected in the NOD/SCID BM and about 1% to 2% in spleens (Table
1), consistent with previous results published by us and others.12,13,15,16,29,32-34 Figure 3C illustrates a representative FACS analysis of NOD/SCID BM at 10 weeks
after transplantation. These animals had about 20% human CD45+ cells in the BM (Figure 3C, top panels), and the
engrafted human cells were further analyzed for the expression of GFP
and human-specific surface markers such as CD34, CD19, and CD13 as well
as MHC II (Figure 3C). In the animals engrafted with DR.GFP- or
EF.GFP-transduced CBSCs, 41% to 88% of the human cells expressed GFP
at high levels (Table 1 and Figure 3C). Ten weeks after
transplantation, about 20% to 30% engrafted human cells in mouse BM
retained the CD34+ phenotypes, about 60% expressed a human
B cell marker (CD19), and about 5% expressed a marker for mature
myeloid cells (CD13). Consistent with previous
reports,51,52 more than 90% of the human cells in the BM
of NOD/SCID mice of both groups were MHC II+ at 10 weeks
after transplantation, including CD19+ B lymphocytes (about
60%), CD34+ cells (about 30%), and fewer
CD13+ cells (Figure 3C). Cells expressing high-level GFP
were found in all the expected human cell subsets in the NOD/SCID
engraftment model, particularly in mice engrafted with
EF.GFP-transduced CBSCs (Figure 3C and Table 1). Notably, these
GFP+ cells found in vivo in the DR.GFP transduction group
were exclusively in the MHC II+ population (SF = 4.8)
but equally distributed between CD34+ and
CD34
To further examine the gene transduction into pluripotent human HSPCs
engrafted in the BM of NOD/SCID mice, human cells were further cultured
under different conditions favoring either DC or myeloid/erythroid
lineage differentiation. We found that human cells isolated from BM or
spleens after engraftment were capable of differentiation into mature
DCs. Most (> 80%) of the differentiated cells displayed a DC
phenotype after being cultured with human GM-CSF, IL-4, and TNF- Similarly, in the CFC assay resulting in myeloid/erythroid
differentiation, we found that about 50% of colonies in the EF.GFP group were GFP+, whereas less than 10% of the colonies in
the DR.GFP group were GFP+. We also examined the
correlation of GFP and MHC II expression in CFC-derived
erythroid/myeloid (non-DC) cells after engraftment, as in the CFC
progeny before engraftment (Figures 2C and 3B). Once again, GFP
expression was limited only to the MHC II+ populations in
the DR.GFP transduction group (SF = 9.5). In contrast, in the EF.GFP
group, GFP was expressed in both MHC II+ and MHC
II Based on our in vivo and in vitro data, we conclude that engrafting and
pluripotent human HSPCs have been transduced by both DR.GFP and EF.GFP
vectors. Under the conditions employed (transduction at days 1 and 2),
50% SCID repopulating cells, DCs, as well as CFC progenitors were
transduced, as evident in the EF.GFP transduction group. In contrast to
the EF-1 Human DCs derived from the BM and spleen of engrafted NOD/SCID mice were potent APCs in stimulating T-cell proliferation To further confirm the proper antigen presentation function of the transduced DCs after engraftment, the immune stimulatory potency of these cells was examined in the MLR assay with allogeneic human T lymphocytes subsequent to transplantation and ex vivo differentiation/maturation. As shown in Figure 5, these human DCs stimulated strong allogeneic-MLR responses even at stimulator:effector (T cells) ratios as low as 1:640. The stimulatory capacity of in vivo-derived DCs after transplantation was compatible to that of transduced DCs derived from untransplanted human CBSCs (Figure 5). Furthermore, there were no obvious differences in the stimulatory effects of DCs derived from mock- and LV-transduced cells.
This study addressed 2 important aspects of stem cell
gene therapy: efficient transgene delivery into pluripotent HSPCs and restricted transgene expression in a selected differentiation lineage
of transduced HSPCs. We demonstrated efficient gene delivery into
NOD/SCID repopulating human HSPCs by a short transduction protocol, as
measured by GFP transgene expression in multiple lineages derived from
transduced HSPCs. The high-level gene delivery and persistent
expression was evident by the fact that 41% to 88% of human cells
(including lymphoid and myeloid lineages) engrafted in NOD/SCID mice
were GFP+ (Table 1). Similar transduction efficiencies were
observed in human progenitor cells capable of generating
erythroid/myeloid colonies and DCs, before and after transplantation.
As evident with the transduction group by the EF.GFP vector, the
transduction efficiency of pluripotent human HSPCs engrafted in
NOD/SCID mice The more unique and significant aspect of this study is the
demonstration of restricted transgene expression in a specific lineage
derived from transduced pluripotent HSPCs. We specifically focused on
APCs/DCs in this study because of our interest in expressing genes
selectively and highly in APCs/DCs. We constructed a SIN LV (DR.GFP)
containing the MHC II-specific promoter from the human HLA-DR By transducing pluripotent HSPCs, we can obtain genetically modified DC
progenitors that are capable of engrafting into NOD/SCID mouse BM,
differentiating into mature DCs, and potently stimulating human T cells
(Figure 5). Our study has provided new insights on human APCs/DCs
differentiated from engrafting HSPCs. For example, we found that DC
progenitors can engraft and survive in mouse BM and spleens, because
functional human DCs can be generated subsequently from engrafted human
cells. However, we did not find human MHC II+ cells with
the mature DC phenotype (CD83+, CD40+, and
CD86high) either in mouse BM or spleens. Our results are
consistent with a recent report that human DC differentiation is
blocked prior to the CD83+ stage in NOD/SCID mice that
underwent transplantation.54 It is unclear, however,
whether there was true (partial) DC differentiation from human HSPCs in
NOD/SCID mice or whether simply a fraction of engrafted human HSPCs
maintained their pluripotency, including the ability to differentiate
in subsequent DC cultures. The latter hypothesis is supported by the
fact that about 30% of engrafted human cells retained the
CD34+ phenotype (Figure 3C) 10 weeks after engraftment.
Future experiments with the NOD/SCID model with the addition of human
fetal thymus and/or human cytokines may allow us to examine the
function, as well as transgene expression, in the human APC/DC progeny
matured in vivo after HSPC transduction and transplantation. In
parallel, we have also started to test the DR.GFP LV to transduce mouse HSPCs using immunocompetent mice. We observed similarly preferential and high-level transgene expression in mouse APCs/DCs in reconstituted mice after HSPC transplantation by the DR.GFP LV and another SIN LV
containing a model antigen driven by the HLA-DR In summary, this study demonstrated the feasibility of targeted gene expression in specific differentiation lineage derived in vivo from transduced pluripotent stem cells. The methodology provides a basis for future targeted gene therapy applications to reduce potential adverse effects of broad transgene expression and increase therapeutic effectiveness mediated by gene transduction of HSPCs and other types of stem cells.
We thank Dr Didier Trono for providing the pRLLhPGK.GFP Sin-18 lentiviral vector and 2 packaging and envelope-expressing vectors, Dr Curt Civin and Ms Rene Smith for providing NOD/SCID mice, Dr Dae-Chul Joeng for Wright-Giemsa staining, Ms Leslie Meszler for technical assistance of microscopic photographing, and gifts from the Topercer family and Mrs Doris Needle to D.P. We gratefully appreciate the intellectual discussion and suggestions by Drs Peter Gao and Xianzheng Zhou. We also thank Drs X. Zhou, Katherine Whartenby, and Enrico Novelli for critical reading of the manuscript.
Submitted April 12, 2001; accepted August 31, 2001.
Supported in part by a National Institutes of Health grant (P30-CA06973 to L.C. and D.P.). Y.C. is supported by a Cancer Research Institute/Libby Bartnick Memorial Fellowship. L.C. is supported by the Alexander and Margaret Stewart Trust Scholarship. D.P. is a Janney Foundation Scholar.
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: Linzhao Cheng, Div of Immunology and Hematopoiesis, Johns Hopkins Oncology Center, Bunting-Blaustein Cancer Research Bldg, Rm 208, 1650 Orleans St, Baltimore, MD 21231; e-mail: lcheng{at}welch.jhu.edu.
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Top
Abstract
Introduction
R8.91, expresses the
HIV-1 gag/pol, tat, and rev genes required for
efficient LV production.
80°C.
evaluated by persistent transgene expression in multiple
lineages